Saccharomyces cerevisiae, class II gene promoters have been divided into two subclasses, TFIID- and SAGA-dominated promoters or TFIID-dependent and coactivator-redundant promoters, depending on the experimental methods used to measure mRNA levels. A prior study demonstrated that Spt3, a TBP-delivering subunit of SAGA, functionally regulates the PGK1 promoter via two mechanisms: by stimulating TATA box-dependent transcriptional activity and conferring Taf1/TFIID independence. However, only the former could be restored by plasmid-borne SPT3. In the present study, we sought to determine why ectopically expressed SPT3 is unable to restore Taf1/TFIID independence to the PGK1 promoter, identifying that this function was dependent on the construction protocol for the SPT3 taf1 strain. Specifically, simultaneous functional loss of Spt3 and Taf1 during strain construction was a prerequisite to render the PGK1 promoter Taf1/TFIID-dependent in this strain. Intriguingly, genetic approaches revealed that an as-yet unidentified trans-acting factor reprogrammed the transcriptional mode of the PGK1 promoter from the Taf1/TFIID-independent state to the Taf1/TFIID-dependent state. This factor was generated in the haploid SPT3 taf1 strain in an Hsp104-dependent manner and inherited meiotically in a non-Mendelian fashion. Furthermore, RNA-seq analyses demonstrated that this factor likely affects the transcription mode of not only the PGK1 promoter, but also of many other class II gene promoters. Collectively, these findings suggest that a prion or biomolecular condensate is generated in a Hsp104-dependent manner upon simultaneous functional loss of TFIID and SAGA, and could alter the roles of these transcription complexes on a wide variety of class II gene promoters without altering their primary sequences. Therefore, these findings could provide the first evidence that TFIID dependence of class II gene transcription can be altered epigenetically, at least in Saccharomyces cerevisiae.In  To initiate transcription in eukaryotes, TATA-binding protein (TBP) first binds to the TATA box or TATA-like element , and subClass II genes can be divided into two subclasses, TFIID- or SAGA-dominated genes, depending on whether steady-state mRNA levels are impaired more robustly by TFIID or SAGA mutations, respectively . A recenin vivo  or the taf1-N568Δ [YTK16993/16999/17029/17035] gene in a LEU2-based low-copy vector, only YTK17025/17029/17031/17035 carry an additional deletion of the chromosomal SPT3-coding region. Furthermore, the strains carry a reporter VTC1 gene on the chromosome that is driven by a TATA box-containing [YTK16989/16993/17025/17029] or TATA-less [YTK16995/16999/17031/17035] PGK1 promoter.YTK16989/16993/16995/16999/17025/17029/17031/17035 have been described previously  (TK13846-TK13856/pFA6a-13Myc-His3MX6  (TK13848-TK13849/BY4741), were fist amplified by PCR, and were then fused with TK13236-TK13849 to generate a 2.2-kb fragment that was used for transformation of YTK16989/16995/16993/16999, respectively.To generate YTK18751/18766/18778/18789, the four sub-fragments containing V5-tagged SPT3 promoter [-489 to -1 bp] (TK13988-TK13989/BY4741), TDH3 promoter [-673 to -24 bp] (TK13990-TK13991/BY4741), or V5-tagged SPT3 + URA3 (TK13987-TK13849/YTK18751), were first amplified by PCR, and then fused with TK13988-TK13849 to generate a 4.0-kb fragment that was used for transformation of YTK16989/16995/16993/16999, respectively.To generate YTK20400/20413/20415/20425, the three sub-fragments containing the BalI-linearized pM8169 (V5-tagged SPT3/pAUR101).YTK19261/19265/19267/19272 were generated from YTK17025/17031/17029/17035, respectively, by transformation with SPT3 + ADH1 terminator + URA3 (TK13750-TK13751/YTK18751), was first amplified by PCR, and then used for transformation of YTK17025/17029/17031/17035, respectively.To generate YTK19100/19102/19105/19108, the 3.3-kb fragment containing V5-tagged VTC1 promoter [-320 to -1 bp] + loxP-SpHIS5-loxP [PGK1 promoter [-598 to -1 bp]+ VTC1 ORF [+1 to +390 bp] + VTC1 terminator [+391 to +603 bp] (TK7872-TK11973/pM8176 or pM8177), was first amplified by PCR, and was then used to transform YTK11411  (TK14415-TK14416/BY4741), His3MX6 (TK4003-TK4006/pFA6a-13Myc-His3MX6 [SPT3 terminator [+1015 to +1287 bp](TK14417-TK14418/BY4741) were first amplified by PCR, and then fused with TK14415-TK14418 to generate a 2.7-kb fragment that was used for transformation of YTK19244/19246, respectively.To generate YTK19301/19303, the three sub-fragments containing the -His3MX6  or SPT3 StuI-linearized pM8165.YTK19317/19319 were generated from YTK19301/19303, respectively, by transformation with SPT3 promoter [-300 to -1 bp] + His3MX6 + SPT3 terminator [+1015 to +1287 bp] was amplified by PCR (TK14415-TK14418/ YTK19301) and used for transformation of YTK19281/19283, respectively.To generate YTK19325/19327, the 2.7-kb fragment containing the TAF1/pRS315).YTK19349/19351/19357/19359 were generated from YTK19317/19319/19325/19327, respectively, by transformation with pYN1, and then by segregating out pM4201 (HA-tagged taf1-N568Δ/pRS315)  + His3MX6 + SPT3 terminator [+1015 to +1287 bp] was amplified by PCR (TK14415-TK14418/ YTK19301), and used for transformation of YTK19587/19590, respectively.To create YTK19613/19614, the 2.7-kb fragment containing the StuI-linearized pM8165.YTK19623/19624 were generated from YTK19613/19614, respectively, by transformation with YTK19663/19665 were generated from YTK19623/19624, respectively, by transformation with pM4201, and then by shuffling out pYN1 on 5FOA-containing media.YTK19626/19628/19664/19666 were generated from YTK19613/19614/19623/19624, respectively, by transformation with pM8201, and then by shuffling out pYN1 on 5FOA-containing media.StuI-linearized pM8165.YTK19716/19717 were generated from YTK19626/19628, respectively, by transformation with YTK19489/19492/19317/19319/19489/19492/19401/19402/19401/19402 were crossed with YTK19664/19666/19663/19665/19716/19717/19664/19666/19716/19717, respectively, to generate YTK19746/19747/19748/19749/ 19763/19764/19765/19766/ 19767/19768, respectively.YTK19746 and YTK19747 were sporulated and dissected to generate YTK19803/19804/19805/19806/19807/19808/19809/19810/19811/19812/19813/19814 and YTK19815/19816/19817/19818/19819/19820/19821/19822/19823/19824/19825/19826.HSP104 promoter [-358 to -1 bp] (TK14581-TK14582/BY4741), hphMX4 (TK14574-TK14575/pAG32 [KanMX4 linker + HSP104 terminator [+2728 to +3326 bp] (TK4716-TK14584/Y01514), were first amplified by PCR, and then were fused with TK14581-TK14584 to generate a 2.7-kb fragment that was used for transformation of YTK19551/19553/19587/19590.To generate YTK19981/19982/19983/19984, the three sub-fragments containing the SPT3 promoter [-300 to -1 bp] + His3MX6 + SPT3 terminator [+1015 to +1287 bp]was amplified by PCR (TK14415-TK14418/YTK19301), and used for transformation of YTK19981/19982/19983/19984, respectively.To generate YTK19989/19990/19991/19992, the 2.7-kb fragment containing the StuI-linearized pM8165.YTK20017/20018/20019/20020 were generated from YTK19989/19990/19991/19992, respectively, by transformation with YTK20037/20039/20041/20043 were generated from YTK20017/20018/20019/20020, respectively, by transformation with pM4201, and then by shuffling out pYN1 on 5FOA-containing media.YTK20038/20040/20042/20044 were generated from YTK20017/20018/20019/20020, respectively, by transformation with pM8201, and subsequently by shuffling out pYN1 on 5FOA-containing media.YTK20045/20046/20047/20048 were generated from YTK19989/19990/19991/19992, respectively, by transformation with pM8201, and then by shuffling out pYN1 on 5FOA-containing media.StuI-linearized pM8165.YTK20049/20050/20051/20052 were generated from YTK20045/20046/20047/20048, respectively, by transformation with SPT3 promoter + SPT3 ORF [-667 to +1011 bp](TK13229-TK13212/BY4741), V5 tag , were fist amplified by PCR, and then fused with TK13229-TK13214 to generate a 2.2-kb DNA fragment. This 2.2-kb fragment was digested with EcoRI and SpeI, and then ligated into the EcoRI/SpeI site of pRS426, generating pM7932.To create pM7932-expressing V5-tagged Spt3 in yeast cells, the three sub-fragments containing the f pM7318 ), or SPTTDH3 promoter [-673 to -24] + V5-tagged SPT3 ORF + ADH1 terminator [+1036 to +1235 bp] (TK14327-TK14328/YTK20400) was amplified by PCR, digested with SphI and XbaI, and then ligated into the SphI/XbaI site of pAUR101 (TaKaRa), generating pM8165.The 2.2-kb fragment containing the SPT3 promoter [-667 to -1 bp] + V5-tagged SPT3 ORF + SPT3 terminator [+1015 to +1227 bp] (TK13985-TK13986/pM7932) was amplified by PCR, digested with SphI and XbaI, and then ligated into the SphI/XbaI site of pAUR101, generating pM8169.The 2.2-kb fragment containing the PGK1 promoter [-598 to -1 bp]+ VTC1 ORF [+1 to +190 bp](TK14391-TK14386/YTK16989 [TATA] or YTK16995 [GAGA]) or VTC1 ORF [+168 to +390 bp]+ VTC1 terminator [+391 to +690 bp](TK14385-TK2454/YTK16989 or YTK16995) were first amplified by PCR, and then fused with TK14391-TK2454 to generate a 1.3-kb DNA fragment, respectively. These 1.3-kb fragments ([TATA] or [GAGA]) were digested with BamHI and XbaI, and then ligated into the BamHI/XbaI site of pRS316  + VTC1 [+1 to +690 bp](TK14391-TK2454/YTK16989 [TATA] or YTK16995 [GAGA]) were amplified by PCR, digested with BamHI and XbaI, and then ligated into the BamHI/XbaI site of pRS316, generating pM8172/8173, respectively.The 1.3-kb fragments containing the VTC1 [-764 to -1 bp] (TK14411-TK14389/BY4741) or loxP-SpHIS5-loxP , taf1-N568Δ , TAF1 hsp104Δ (YTK20037), taf1-N568Δ hsp104Δ (YTK20038 [protocol #2] and YTK20049 [protocol #3]) strains cultured in the presence or absence of 3 mM Gdn-HCl were examined using RNA-seq analysis as described previously, with minor modifications  for #1,  for #2, and  for #3 . This supports the conclusion that simultaneous functional loss of Spt3 and Taf1 during strain construction is a prerequisite for rendering PGK1 transcription Taf1/TFIID-dependent in the SPT3 taf1-N568Δ strain.Consistent with above finding, RT-qPCR revealed that PGK1 transcription was Taf1/TFIID-dependent in the SPT3 taf1-N568Δ strain specifically when it was generated by protocol #3  functioned in cis in a manner tightly associated with the originally reprogrammed PGK1 promoter, or rather in trans in a more diffusible manner, for example even if the PGK1 promoter was later introduced into cells.The observation that tocol #3 , in whicVTC1# allele carrying eight silent point mutations in the middle of the open reading frame (ORF) , but not in strains generated by protocol #2 , regardless of which reporter alleles they harbored.We thus generated a me (ORF) , such thl RT-PCR . The SPTmeasured . PGK1 trPGK1 transcription in diploid strains harboring both VTC1 and VTC1# alleles generated by crossing two of several haploid SPT3 taf1-N568Δ strains tested in PGK1 transcription of both reporter alleles and presumably both endogenous PGK1 alleles, while the latter two could not be discriminated by RT-qPCR, was Taf1/TFIID-dependent in the diploid strains when at least one mother haploid strain was generated by protocol #3 . Thus, PGK1 transcription remained Taf1/TFIID-independent only in the diploid strain in which both mother haploid strains were generated by protocol #2 . These observations indicated that promoter reprogramming factor(s) produced in mother haploid strains generated by protocol #3  functioned in trans to alter the transcription mode of a PGK1 promoter that had not yet been reprogrammed.We subsequently measured PGK1 promoter was likely to be an epigenetic event, as all the tested SPT3 taf1-N568Δ strains carried essentially the same genetic backgrounds, irrespective of whether PGK1 transcription was Taf1/TFIID-independent or Taf1/TFIID-dependent. Thus, we subsequently examined the inheritance of this unexpectedly acquired trait from mother diploid strains , to render the PGK1 promoter Taf1/TFIID-dependent.The reprogramming phenomenon observed for the  strains . This trPGK1 transcription was examined in haploid or diploid strains used in PGK1 transcription in the VTC1-carrying haploid strain constructed by protocol #3 . However, and surprisingly, Gdn-HCl treatment increased Taf1/TFIID dependence of PGK1 transcription in the VTC1-carrying haploid strain constructed by protocol #2 . Furthermore, in the VTC1#-carrying haploid strains, Taf1/TFIID dependence or Taf1/TFIID independence of PGK1 transcription was not significantly affected by Gdn-HCl treatment, regardless of whether the strains were constructed by protocol #2 or #3 . Contrastingly, in the diploid strains obtained from one or more haploid progenitors generated by protocol #3, Gdn-HCl treatment decreased Taf1/TFIID dependence of PGK1 transcription, which was more evident for endogenous PGK1 transcription than reporter gene transcription . Importantly, PGK1 transcription in the diploid strain obtained by crossing two haploid strains, both of which were constructed by protocol #2, was nearly affected by Gdn-HCl treatment . Collectively, these observations suggest that the promoter reprogramming factor(s) could have prion-like properties, but the effect of Gdn-HCl-mediated HSP104 inhibition on their function was complex.To determine if these epigenetic promoter reprogramming factor(s) had prion-like properties, PGK1 promoter, we next sought to examine more directly the effect of Hsp104 by genetic approaches utilizing the hsp104Δ mutation. For this purpose, test haploid strains were constructed by protocol #2 or #3 after disruption of the HSP104 gene , indicating that Taf1/TFIID dependence could be conferred to the PGK1 promoter specifically by protocol #3 only when Hsp104 function was intact. Because the hsp104Δ mutation did not affect PGK1 transcription  could reprogram the transcription mode of other class II genes as well as how extensively this reprogramming could be affected by Gdn-HCl treatment or the hsp104Δ mutation, we conducted genome-wide RNA-seq analyses for the SPT3 TAF1 (wild type [WT] as a control) and SPT3 taf1-N568Δ strains constructed by protocol #2 or #3 (hereafter designated as #2 strains or #3 strains) in the presence or absence of Gdn-HCl treatment or the hsp104Δ mutation.The above analyses indicated that promoter reprogramming factor(s) that switch the transcription mode of the hsp104Δ mutation when they were incubated at 37°C  data revealed that the gene expression profiles of the #2 and #3 strains were significantly different from that of the WT strain at 37°C  and even at 37°C  or 25°C  at 37°C . These oPGK1 is a CR gene , #2 , and # 3 . The two TAF1 and three taf1 strains with the same genetic backgrounds are shaded for clarification. (B) RT-qPCR analyses to measure PGK1 (top panel) or VTC1 (bottom panel) mRNA levels in the ten strains carrying the VTC1 reporter driven by the PGK1 promoter in which the TATA box was intact  or substituted with the GAGA sequence , as indicated below the bottom panel. Lane 1–4 strains were generated by protocol #1, lane 5–8 strains were generated by protocol #2, and lane 9–10 strains were generated by protocol #3, as indicated in A. The strain used are YTK19325 (lane 1), YTK19405 (lane 2), YTK19327 (lane 3), YTK19406 (lane 4), YTK19317 (lane 5), YTK19401 (lane 6), YTK19319 (lane 7), YTK19402 (lane 8), YTK19489 (lane 9), and YTK19492 (lane 10). Cultivation and data presentation were conducted as described in (TIF)Click here for additional data file.S2 FigVTC1-specific TK13936-TK13937  or VTC1#-specific TK14458-TK13937 . These cDNA and primer pairs are the same as those used in lanes 1 and 7 of PCR was conducted using cDNA derived from YTK19317  or YTK19663  as a template, and primer pairs were (TIF)Click here for additional data file.S3 FigPGK1 (top panel) or VTC1/VTC1# (bottom panel) in the 32 haploid strains carrying VTC1  or the VTC1# reporter  driven by the PGK1 promoter in which the TATA box was intact  or substituted with the GAGA sequence , as indicated below the bottom panel. The strains used in lanes 1–8 were the same as those used in lanes 1, 2, 3, 4, 7, 8, 9, and 10 of RT-qPCR analysis to measure mRNA levels of (TIF)Click here for additional data file.S4 Fig(A) RT-qPCR analysis to measure mRNA levels of PGK1 (top panel) or VTC1/VTC1# (bottom panel) in the twelve haploid strains carrying VTC1 (lanes 1–6) or the VTC1# reporter (lanes 7–12) driven by the PGK1 promoter in which the TATA was intact (odd-numbered lanes) or substituted with the GAGA sequence (even-numbered lanes), as indicated below the bottom panel. These strains are the same as those used in (B) RT-qPCR analysis to measure mRNA levels of PGK1 (top panel) or VTC1/VTC1# (bottom panel) in the ten diploid strains carrying both of the VTC1 and VTC1# reporters driven by the PGK1 promoter in which the TATA box was intact  or substituted with the GAGA sequence , as indicated below the bottom panel. The strains are the same as those used in (TIF)Click here for additional data file.S5 Fig(A) Northern blot analysis of VTC1 (middle panels), PGK1 (top panels), or SCR1  RNA levels in the twelve strains used in Figs (B) Raw expression data in A were quantified and normalized to SCR1. Values for each transcript derived from PGK1 or VTC1 are summarized in the upper or lower panel, respectively. In each panel, data are presented relative to the value obtained for the strain indicated on the left.(TIF)Click here for additional data file.S6 Figtaf1 or hsp104Δ mutations were averaged and subjected to volcano plot analyses as shown in TPM data of RNA-seq analyses obtained from two biological replicates of six strains carrying or not carrying (TIF)Click here for additional data file.S7 Figtaf1 or hsp104Δ mutations after cultivation under the conditions indicated in the bottom panel were subjected to hierarchical clustering analysis and visualized as a heat map. The same data were analyzed by volcano plot in Duplicate TPM data from six strains carrying or not carrying (TIF)Click here for additional data file.S8 FigSPT3 and VTC1 genes are driven by the TDH3 and PGK1 promoters, respectively, in the strains subjected to RNA-seq analyses. (A) The metabolic pathways from glucose to ethanol (glycolysis and ethanol fermentation) are summarized with the names of gene products (open rectangles) catalyzing the corresponding reactions, if they are present in the cluster shown in A.The cluster comprising the 21 genes and located at the top of the heatmap in (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


AML-IRF7−/− cells showed increased proliferation and elevated leukemia stem cell (LSC) levels. Overexpression of IRF7 in AML cells decreased cell proliferation and LSC levels. Furthermore, overexpression of transforming growth-interacting factor 1 (TGIF1) rescued the enhanced proliferation and high LSC levels caused by IRF7 deficiency. Moreover, upregulation of vascular cell adhesion molecule 1 (VCAM1), which correlated with high LSC levels, was detected in AML-IRF7−/− cells. In addition, blocking VCAM1-very late antigen 4 (VLA-4) axis delayed disease progression and attenuated intracerebral invasion of AML cells. Therefore, our findings uncover the intrinsic effects of IRF7 in AML and provide a potential strategy to control central nervous system myeloid leukemia.Interferon regulatory factor 7 (IRF7) is widely studied in inflammatory models. Its effects on malignant progression have been documented mainly from the perspective of the microenvironment. However, its role in leukemia has not been established. Here we used MLL-AF9-induced acute myeloid leukemia (AML) mouse models with IRF7 knockout or overexpression and xenograft mouse models to explore the intrinsic effects of IRF7 in AML. AML-IRF7 Acute myeloid leukemia (AML) is characterized by the accumulation of a large number of immature myeloid cells  and diffThe interferon regulatory factor (IRF) family contains 9 members (IRF1 to IRF9), which play pivotal roles in both innate and adaptive immunity . They al−/− hematopoietic stem cells (HSCs) are less proliferative, more quiescent, and have higher repopulation potential [Transforming growth-interacting factor 1 (TGIF1) belongs to the three-amino acid loop extension family of homeodomain proteins and is primarily described as a transcriptional repressor in the transforming growth factor-β (TGF-β) signaling pathway . TGIF1 potential . The expotential  and overotential , 22. It otential . The regMalignant cells capable of crossing the BBB and forming brain metastases acquire a more malignant phenotype . In leukIn this study, MLL-AF9-induced mouse AML models with IRF7 knockout or overexpression and xenograft mouse models were used to explore the intrinsic effects of IRF7 in AML. The deficiency of IRF7 in AML cells accelerates disease progression by promoting cell proliferation and elevating LSC levels. These adverse phenotypes are mediated by the downregulation of TGIF1. Furthermore, knockout of IRF7 causes intracerebral invasion of AML cells. Blocking VCAM1-VLA-4 axis delayed disease progression and attenuated intracerebral invasion of AML cells.−/− mice , 1337 upregulated and 1402 downregulated, were detected between AML-IRF7−/−-c-kit+ and AML-WT-c-kit+ cells  were enriched in AML-IRF7−/−-c-kit+ cells. Six of these genes were finally selected and verified by qRT-PCR  was detected in AML-WT mice  level are two well-established adverse factors in malignancies. They seem to be mutually exclusive since LSCs/TSCs are considered quiescent cells , 37. Howth Smad3 , which ath Smad3 . Furtherth Smad3 , which ith Smad3 . Therefo−/− AML is a good myeloid CNSL mouse model to explore therapeutic strategies. We found that the VCAM1-VLA-4 axis, specifically VCAM1(AML cells)-VLA-4 , was crucial in this pathologic process since a dramatic increase of VCAM1 but not VLA-4 was detected in AML-IRF7−/− cells while endothelial cells expressed VLA-4. Notably, the mechanism is different from those reported in the literature, i.e., VLA-4 (leukocytes or lymphoblastic leukemia cells)-VCAM1  [−/− cells. Importantly, blocking VCAM1-VLA-4 axis attenuated intracerebral invasion and prolonged the survival of AML mice, which might be a potential strategy for the treatment of myeloid CNSL. The mechanism for the upregulation of VCAM1 in AML-IRF7−/− cells has not been established. However, it is interesting that the positive correlation between LSC level and VCAM1 expression was also reported in some AML models [CNSL is associated with poor prognosis in both lymphocytic and myeloid leukemia , 47. CNSl cells) . Other al cells) , and moll cells) , CCLs, Cl cells) , may alsL models . FurtherL models . FurtherCollectively, we demonstrate that loss of IRF7 in AML cells accelerates disease progression by simultaneously promoting cell proliferation, elevating LSC levels, and causing intracerebral invasion. Downregulation of TGIF1 in AML cells accounts for enhanced proliferation and increased LSC levels. Upregulation of VCAM1 in AML cells accounts for intracerebral invasion. Therefore, IRF7 is an intrinsic suppressive factor in AML progression and blocking VCAM1-VLA-4 axis may be a potential strategy for controlling myeloid CNSL.+ cells were sorted 2 days later and designated THP1sc, THP1sh1, THP1sh2, Kasumi-1sc, Kasumi-1sh1, and Kasumi-1sh2.In knockdown experiments, THP1 and Kasumi-1 cells were infected with pLV-hIRF7sc, pLV-hIRF7sh1, and pLV-hIRF7sh2 lentiviruses. The GFP+RFP+ cells were sorted 2 days later and named THP1-IRF7sh1-PCDH, THP1-IRF7sh1-TGIF1, Kasumi-1-IRF7sh1-PCDH and Kasumi-1-IRF7sh1-TGIF1.To explore the effect of TGIF1, THP1sh1 and Kasumi-1sh1 cells were infected with blank or PCDH-EF1α-hTGIF1-mRFP lentivirus. The GFPnull (NSG) and nude mice were provided by the Animal Center of the Institute of Hematology and Blood Diseases Hospital, CAMS & PUMC. IRF7−/− mice (C57BL/6 J background) were constructed by replacing the exon 2 and 3 of Irf7 gene with a PGK-beta-geo cassette [C57BL/6 J, NOD/LtSz-scid IL2rγcassette . They we+ cells from WT or IRF7−/− mice were infected with MSCV-MLL-AF9-GFP retrovirus before transplantation into mice by intravenous tail injection  software.GFP5 AML-IRF7−/− cells on day 0. The mice were administered firategrast (20 mg/kg/day) daily by oral gavage from day 0 to the end of their lives. The PB GFP+ cells were monitored on days 5, 10, and 15. The survival of mice was recorded. For pathologic analysis of tissue sections, the mice were sacrificed when PB GFP+ cells reached approximately 20%. Standard hematoxylin-eosin (HE) staining was performed.The VLA-4 inhibitor firategrast  was used to block the VCAM1-VLA-4 interaction . Mice we-, AML-WT-c-kit+, and AML-IRF7−/−-c-kit+ cells were sorted by flow cytometry. Microarrays were carried out by Shanghai Majorbio Biopharm Technology (China) following standard protocols. The data are available in the National Center for Biotechnology Information Gene Expression Omnibus database under accession number GSE178560. Data for all genes were subjected to gene set enrichment analyses (GSEAs) to investigate the biological functions or pathways. Standard analyses were also performed on the online Majorbio Cloud Platform. Fold change (FC) ≥ 2.0 and p value < 0.05 were used as the cutoff for screening the differentially expressed genes (DEGs). The DEGs between AML-IRF7−/−-c-kit+ and AML-WT-c-kit+ cells are shown in a volcano plot.The AML-WT-c-kitt test was used for comparisons between two groups, whereas one-way ANOVA was used for comparisons among multiple groups. Kaplan–Meier estimates were used for survival curves. ELDA was used for the limiting-dilution transplantation experiments. P < 0.05 was considered statistically significant. Statistically, we have compared similar variances between the groups as well.All experiments were repeated two to three times in accordance with the indicated numbers of mice. The sample size of each experiment is determined based on experience or previously published papers. The investigator was not blinded during experiment or evaluating the result. The SPSS 16.0 software package  and GraphPad Prism 5.0  were used. Shapiro-Wilks method was used for distribution test. When parameters followed Gaussian distribution, unpaired Student’s Additional experimental procedures are provided in the Supplementary Experimental Methods.supplemental figuresupplemental materials


Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling.To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment.Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/β-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8.We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.The online version contains supplementary material available at 10.1186/s41232-023-00258-6. Canonical Wnt signaling controls the stem cell properties of self-renewal and differentiation and is involved in embryonic development and tissue regeneration. During early vertebrate development, canonical Wnt signaling is required for mesoderm formation and it participates in anterior-posterior patterning, specifying the posterior mesodermal fates , 2. FurtWnt3a-induced signaling cascade is initiated by binding of Wnt3a to the single-transmembrane (TM) protein low-density-lipoprotein receptor-related protein 6 (LRP6) and the seven-transmembrane protein frizzled 8 (FZD8) . The staWe previously developed proliferation-inducing and migration-inducing chimeric antigen receptors (piCARs and miCARs), which we also called growth signalobodies and migration signalobodies, respectively –22. In mIn the present study, we, for the first time, developed GPCR-based CARs, which are able to activate endogenous Wnt3a signaling in mouse ES cells. We constructed a pair of chimeras, in which scFv of anti-FL antibody was tethered to the TM/cytoplasmic domains of LRP6 or FZD8. These chimeras are able to bind to BSA-FL and are expected to form BSA-FL/chimeric LRP6/chimeric FZD8 complexes mimicking the Wnt3a/LRP6/FZD8 complexes. We named this chimeric pair Wnt3a-diCAR and investigated its capability to transduce Wnt3a signaling in response to BSA-FL stimulation. Because BSA-FL is a stable molecule with no toxicity and it costs much less than recombinant Wnt3a, regulation of Wnt3a signaling by the Wnt3a-diCAR may contribute to highly efficient and economical myocardial creation from pluripotent stem cells.BstBI and EcoRV and subcloned into pLenti-CAG-IRES-Puro and pLenti-CAG-IRES-Blast, resulting in pLenti-CAG-scFv-IRES-Puro and pLenti-CAG-scFv-IRES-Blast, respectively. The cDNAs encoding mouse LRP6 and FZD8 were obtained from total RNA of mouse heart by RT-PCR using SuperScript III Reverse Transcriptase (Life Technologies) and PrimeSTAR HS DNA polymerase (TAKARA Bio). The primers used for plasmid construction and RT-PCR are listed on Supplementary Table EcoRV-digested pLenti-CAG-scFv-IRES-Puro, and that of FZD8L was inserted into EcoRV-digested pLenti-CAG-scFv-IRES-Blast to create pLenti-CAG-scLRP6L-IRES-Puro, pLenti-CAG-scLRP6M-IRES-Puro, and pLenti-CAG-scFZD8L-IRES-Blast, respectively. To construct pLenti-CAG-scLRP6S- IRES-Puro and pLenti-CAG-scFZD8S-IRES-Blast, fragments of LRP6S and FZD8S were digested with AgeI, and subcloned into pLenti-CAG-scFv-IRES-Puro and pLenti-CAG-scFv-IRES-Blast digested with BspEI, respectively.To construct the plasmids for the lentivirus vector preparation, pLenti-CAG-EGFP , 29 was A mouse ES cell line, CGR8, derived from 129/Ola was maintained on gelatin-coated dishes in DMEM -based ES cell culture medium containing 10% fetal bovine serum, 1 mM sodium pyruvate, MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, GlutaMAX , nucleosides (Chemicon), penicillin/streptomycin , and 1000 U/ml LIF (Chemicon). For generating stable cell lines, ES cells were transduced with lentivirus vectors encoding the transgene and antibiotic-resistant gene. The cells were subsequently selected by antibiotics to create the antibiotic-resistant stable ES cell line co-expressing scLRP6S and either scFZD8S (ES/SLR6S/scFZD8S cell line) or scFZD8L (ES/SLR6S/scFZD8L cell line). Mouse ES cells were also transduced with a pair of mock vectors, pLenti-CAG-IRES-Puro and pLenti-CAG-IRES-Blast, as a negative control cell line (ES/mock). ES cell differentiation experiments were performed through embryoid body (EB) formation, as previously described , 30. In 4 cells/well) for 24 h and subsequently transfected with a TOPflash firefly luciferase reporter plasmid [Cells were cultured on a gelatinized 48-well plate . Protein samples were then reduced and boiled. Subsequently, equal amounts of protein were applied into each lane and separated in an 8% polyacrylamide tris glycine SDS gel, then transferred to a nitrocellulose membrane. The membranes were blocked in 2% skim milk-containing TBS supplemented with 0.1% Tween-20 for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Following a washing step, the membranes were incubated with HRP-conjugated secondary antibodies. Signals were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific), and acquired with Image Quant LAS 4000 mini . Antibodies used in this study were rabbit polyclonal anti-Ror2 antibodies (BD Biosciences), mouse monoclonal anti-β-actin antibodies (Sigma-Aldrich), anti-rabbit IgG-HRP goat antibodies (Jackson), and anti-mouse IgG-HRP antibodies (Jackson).Based on the standard protocol of immunofluorescence labeling as previously described , 30, EBsTotal RNA was isolated using a TRIzol reagent (Life Technologies), and sense-strand cDNA samples were prepared using Ambion WT Expression Kit (Life Technologies). The samples were hybridized in GeneChip Mouse Gene 1.0 ST Array (Affymetrix) following the manufacturer’s instructions. Hybridized arrays were imaged using GeneChip Scanner (Affymetrix), and the data were analyzed by an Expression Console software (Affymetrix) for differential expression among the examined conditions. The similarity of the gene expression profiles among the datasets was measured by Pearson’s correlation coefficient analysis. Hierarchical clustering was performed for heatmap visualization using Cluster 3.0 software. GO enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) software.Gapdh. Gene-specific primers used for quantitative PCR are listed on Supplementary Table Total RNA was isolated using the TRIzol reagent, and cDNA was synthesized by High Capacity cDNA Reverse Transcription Kit. StepOnePlus Real-time PCR System (Life Technologies) and SYBR Premix Ex Taq II (Takara Bio) were used for quantitative PCR. Gene expression levels of target genes were normalized to the levels of ES/mock and ES/SLR6S/scFZD8L stably expressing the TOPflash reporter gene were created and stimulated with Wnt3a, BSA-FL, or vehicle from day 2 to day 3 of differentiation. The resulting EBs were transferred into low attachment dishes, and luciferase activities in 50 live EBs were measured by a Kronos Dio luminometer (ATTO) every 10 min from day 3 to 8 of differentiation. All measured values were corrected to estimated cell numbers at the same time points. The cell numbers from days 3 to 8 were calculated by Gompertz curve or Logistic curve using actual cell numbers of 15 to 20 EBs counted on days 3, 5, 6, and 8.+ cells were quantified with FACS Aria III cell sorter and FlowJo software (BD Biosciences).EBs from ES/mock or ES/scLRP6S/scFZD8L stimulated with vehicle or 1 μg/ml BSA-FL were dissociated with 0.25% trypsin/EDTA on day 10 of differentiation. Cells were fixed, permeabilized, and then immunolabeled with anti-cardiac troponin T (cTnT) mouse monoclonal antibodies (abcam). Anti-mouse IgG antibodies conjugated with Alexa Fluor 488 (Life Technologies) were used as secondary antibody. After cells were washed and filtrated through a cell strainer, cTnTEBs of ES/scLRP6S/scFZD8L were stimulated with 1 μg/ml BSA-FL from day 2 to day 3 and transferred into gelatinized dish on day 6 of differentiation. On day 17, the cells were stained with Fluo-8 AM calcium indicator (AAT Bioquest) according to the manufacturer’s instructions. Time-lapse images of calcium oscillation were acquired using BZ-X710 fluorescence microscope and BZ-X software (Keyence). Signal intensities were quantified using ImageJ software.t test was used to compare the mean between two groups. Tukey’s multiple comparison test was performed to analyze the differences among the results of three groups. All data are expressed as mean ± SD.For statistical analysis, Student’s To activate Wnt3a signaling via diCARs, we designed and constructed antibody/LRP6 chimeras and antibody/FZD8 chimeras Fig. A, B. TheTo examine their signal transduction properties, several combinations of scLRP6 and scFZD8 constructs were transiently transfected into mouse ES cells. Activities of TCF/β-catenin-dependent transcription, a convergent downstream target of Wnt3a signaling, were subsequently measured by a luciferase reporter assay Fig. C. We useTo examine the functional role of the Wnt3a-diCAR in ES cells, we transduced them with a pair of lentiviral vectors. We established the stable ES cell line co-expressing scLRP6S and either scFZD8S or scFZD8L, named ES/scLRP6S/scFZD8S or ES/scLRP6S/scFZD8L, respectively. A pair of mock vectors, which do not carry the diCAR genes, was also used to generate the negative control cell line (ES/mock). Then, we performed the TCF/β-catenin-dependent luciferase reporter assay to determine whether these ES cell lines exhibit Wnt3a signal activation in response to BSA-FL stimulation. ES/scLRP6S/scFZD8S showed only a modest TCF/β-catenin transactivation in response to BSA-FL, and ES/mock displayed no response Fig. S. HoweverUsing DNA microarray and quantitative RT-PCR analyses, we next examined BSA-FL-induced changes of the mRNA expression profile of ES/mock and ES/scLRP6S/scFZD8L. BSA-FL treatment and Wnt3a treatment in ES/scLRP6S/scFZD8L resulted in similar mRNA expression profile, while global mRNA expression levels of BSA-FL-treated ES/mock and vehicle-treated cells were similar Fig. C. As forWnt/β-catenin signaling is involved in mesodermal formation and cardiogenesis. Thus, we examined the role of BSA-FL-dependent Wnt3a signal activation in in vitro differentiation of ES cells. ES/mock and ES/scLRP6S/scFZD8L were subjected to EB formation using a hanging drop method Fig. . The effTo further examine the preferential cell differentiation accelerated by BSA-FL in ES/scLRP6S/scFZD8L, we evaluated the expression levels of mesodermal, ectodermal, and endodermal marker genes in the differentiated EBs in the mid-phase (day 6) and late-phase (day 13) of differentiation. The expression levels of mesodermal progenitor markers   and two types of chimeric FZD8 (scFZD8S and scFZD8L). Among the combinations of chimeric LRP6 and FZD8, scLRP6S/scFZD8S, and scLRP6S/scFZD8L appeared appropriate for establishing the stable ES cell lines, in which canonical Wnt signal is activated in response to the surrogate ligand, BSA-FL. Compared to the transient transfection Fig. C, the stWe also examined the effects of single chimeric LRP6 or chimeric FZD8 on ligand-activated Wnt signal transduction. Each of the LRP6 chimeras activated the TCF/ β-catenin-dependent transcription without the ligands, while chimeric FZD8 alone showed almost no signal activation even with BSA-FL Fig. S. AccordiDue to the hydrophobic property of Wnt3a, which is caused by its post-translational palmitoylation, the cost of production of recombinant Wnt proteins is very high, therefore hindering their further applications. Chemical Wnt agonists such as CHIR99021 are also expensive than BSA-FL. Recent advances of Wnt studies on adult stem cells have revealed pivotal roles of canonical Wnt signaling in tissue maintenance of hair follicles, mammary glands, hematopoietic systems, and gastrointestinal organs [Accumulating evidence revealed that gene mutations in Wnt pathway components are frequently identified in cancer cells, and aberrant Wnt signal activation is considered as an oncogenic driver in several types of cancer , 43. ThuConsidering clinical applications, it would be necessary to establish suitable lines of human pluripotent stem cells that sufficiently express Wnt3a-diCARs and highly respond to surrogate ligands, whereas we confirmed that BSA-FL-induced canonical Wnt signaling can be activated in several lines of Wnt3a-diCAR-expressing mouse ES cells Fig. S. FurtherIn the present study, we, for the first time, developed GPCR-based CARs. The Wnt3a-diCAR successfully activates endogenous Wnt3a signaling in response to BSA-FL, a surrogate ligand, which is chemically stable and inexpensive to produce. As one example of the utilities, we show that the Wnt3a-diCAR would be available to generate a sufficient number of cardiomyocytes differentiated from pluripotent stem cells with low costs. In addition, the Wnt3a-diCAR might be a valuable tool to analyze the Wnt signaling pathway. Further studies contributing to better control of the canonical Wnt signaling would facilitate the application of this technology in the clinic for regenerative medicine.Additional file 1: Fig. S1. TCF/β-catenin-dependent transcriptional activities of mouse ES cells stably expressing scLRP6S and scFZD8S. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8S following stimulation with BSA-FL are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean of triplicates and standard deviation from one representative experiment. Fig. S2. TCF/β-catenin-dependent transcriptional activities in mouse ES cells stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8L cells following stimulation with CHIR99021 (a WNT activator) or BSA-FL at indicated concentrations are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation. Fig. S3. TCF/β-catenin-dependent transcriptional activities of mouse embryonic fibroblasts (MEFs) expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in MEFs expressing mock or scLRP6S/scFZD8L following stimulation with BSA-FL at the indicated concentrations are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation from triplicate measurements. Fig. S4. Western blotting for non-canonical Wnt activation in scLRP6S and scFZD8L-expressing cells. Protein expression levels of Ror2 and β-actin in mock- or scLRP6S/scFZD8L-expressing ES cells (A) or NIH3T3 cells (B) treated with Wnt5a, Wnt3a or BSA-FL are shown. Fig. S5. BSA-FL diffuses into EBs. Representative fluorescence microscopy images in EB sections from ES/mock and ES/scLRP6S/scFZD8L cells treated with vehicle or BSA-FL. Nuclei are counterstained with DAPI showing BSA-FL deposition possibly in the plasma membrane. The right panels are enlarged images of the boxed area of the BSA-FL-treated EBs. Scale bars = 50 μm. Fig. S6. Incidence of spontaneously beating EBs derived from three independent ES cell lines expressing scLRP6S and scFZD8L. The incidence of spontaneously beating EBs stimulated by 0.1 μg/ml BSA-FL, 50 ng/ml Wnt3a, or vehicle for 24 h from day 2 of differentiation is shown. Time course of the incidence of spontaneously beating EBs differentiated from ES/scLRP6S/scFZD8L stimulated by 0.1 μg/ml BSA-FL, 50 ng/ml Wnt3a, or vehicle for 24 h from day 2 after EB formation. Fig. S7. BSA-FL enhances cardiac differentiation of EBs expressing scLRP6S and scFZD8L. Representative flow cytometry plots of cardiac troponin T  vs. phycoerythrin (PE) for differentiated ES/mock and ES/scLRP6S/scFZD8L treated with vehicle, Wnt3a or BSA-FL. Fig. S8. TCF/β-catenin-dependent transcriptional activities in mouse ES cells stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8L cells following BSA-FL treatment with or without IWP-4 or XAV939, WNT inhibitors, are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation. Fig. S9. TCF/β-catenin-dependent transcriptional activities in mouse ES cells expressing single chimeric receptors. TCF/β-catenin-dependent transcriptional activities measured by luciferase reporter assay in mouse ES cells transiently transfected with plasmids encoding various chimeric LRP6 or FZD8. The values in mock-transfected cells treated with vehicle were set to 1. The data represent the mean of duplicates and standard deviation from one representative experiment. Fig. S10. Activity of BSA-FL and Wnt3a after incubation at 37 °C. TCF-dependent transcriptional activities were measured by luciferase reporter assay in mouse ES cells transiently transfected with scLRP6S and scFZD8L. The cells were stimulated with 0.1, 1, or 10 μg/ml BSA-FL or 50 ng/ml Wnt3a that were incubated at 37 °C for 0, 1, 2, 4, or 7 days. Luciferase activities of samples stimulated by each ligand with no incubation were set to 100. Fig. S11. TCF/β-catenin-dependent transcriptional activities in three independent mouse ES cell lines stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/scLRP6S/scFZD8L following indicated BSA-FL treatment are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The data represent the mean and standard deviation. Table S1. List of primer sequences used for plasmid construction. Table S2. List of primer sequences used for real-time qPCR.Additional file 2: Movie S1. Calcium oscillations in differentiated ES/scLRP6S/scFZD8L. Calcium oscillations, monitored by using Fluo-8 AM calcium indicator, were observed on day 17 of differentiation in ES/scLRP6S/scFZD8L stimulated with 1 μg/ml BSA-FL from day 2 to day 3.


Cancer-derived exosomal miRNAs are implicated in tumorigenesis and development of lung adenocarcinoma (LUAD). The objective of this study is to unravel the biological function of exosomal miR-197-3p in LUAD metastasis. qRT-PCR showed that elevated miR-197-3p in LUAD tissues was positively correlated with LUAD metastasis. CCK-8, tube formation, transwell and wound healing assays revealed that exosomal miR-197-3p from LUAD cells promoted the proliferation, angiogenesis and migration of HUVECs in vitro. LUAD cells-derived exosomal miR-197-3p also facilitated tumor growth and angiogenesis in LUAD cells-derived tumor xenograft model. TIMP2 and TIMP3 were identified as target genes of miR-197-3p in HUVECs by bioinformatics analysis and luciferase reporter assay. Functional studies illustrated that exosomal miR-197-3p promoted angiogenesis and migration via targeting TIMP2 and TIMP3 in HUVECs. In vivo data further supported that exosomal miR-197-3p promoted lung metastasis via TIMP2/3-mediated angiogenesis. In conclusion, LUAD cells-derived exosomal miR-197-3p conferred angiogenesis via targeting TIMP2/3 in LUAD metastasis. Lung cancer remains the leading cause of cancer mortality . Lung adMetastasis is a multi-step process in lung cancer, including tumor growth, cell migration, invasion, and angiogenesis. These processes occur in overlap or simultaneously, ultimately leading to distant metastasis . To promCommunication between tumor cells and tumor microenvironment (TME) is critical for cancer development and progression. TME has effects on tumor cells by its composition, cell proportion, and activation state . The celHerein, we demonstrated that miR-197-3p expression was upregulated in serum exosomes of LUAD patients and LUAD tissues. Exosomes transported miR-197-3p from LUAD cells to human umbilical vein endothelial cells (HUVECs), thereby enhancing proliferation, angiogenesis and migration of HUVECs. Exosomal miR-197-3p also promoted tumor growth and angiogenesis in vivo. TIMP2 and TIMP3 were identified as functional targets of miR-197-3p in HUVECs. Exosomal miR-197-3p/TIMP2/3 axis contributed to angiogenesis and metastasis in vitro and in vivo. Our findings suggest that circulating miR-197-3p may be a promising target for anti-angiogenic treatment of LUAD.n = 30; metastatic LUAD, n = 32) from 2016 to 2019 at Xiangya Hospital of Central South University . The fresh whole-blood samples from LUAD patients  and healthy donors (n = 25) were also collected for exosome purification. Informed consents were obtained from all recruiters. This study was approved by the Ethics Committee of Xiangya Hospital of Central South University.A cohort of 62 LUAD tumor tissues and adjacent normal counterparts were collected from LUAD patients , HUVECs, LUAD cell lines including SK-LU-1, H358, H1650, H2030, H1975, A549, HCC827, PC9, H2009 and H1299 cells were purchased from Cell Bank of Type Culture Collection, Chinses Academy of Science , without contamination with mycoplasma. Cells authenticated before use were cultured in DMEM containing 10% FBS  at 37 °C. For overexpression experiments, the coding sequence (CDS) of human TIMP2 or TIMP3 was cloned into pcDNA3.1 vector (Invitrogen). Cells were transfected with miR-197-3p mimics, inhibitor, pcDNA3.1-TIMP2, pcDNA3.1-TIMP3 or their corresponding negative controls using Lipofectamine 3000 . For in vivo study, the stable transfection of overexpression or inhibition of miR-197-3p in A549 cells was performed using lentivirus . Lenti-mCherry containing a miR-197-3p overexpressing sequence, a miR-197-3p inhibiting segment or their negative controls were purchased from GeneChem, namely mimics NC, miR-197-3p mimics, inhibitor NC and miR-197-3p inhibitor. For the exosome treatment, exosomes were isolated from conditioned medium of LUAD cells and incubated with HUVECs as previous described [For hematoxylin and eosin (H&E) staining, the mouse lung tissues were fixed with 4% paraformaldehyde for 24 h. The paraffin-embedded tissues were cut in 5 µm thick sections. H&E staining was employed to assess the alterations in morphology in these tissues using an optical microscope, and different fields were subsequently photographed.For immunohistochemistry (IHC) analysis, paraffin-embedded tumor and lung tissues were deparaffinized and rehydrated as described . After ag to remove cells. After filtration through 0.22 μm filter, exosomes were precipitated using ExoQuick TC Exosome Precipitation Solution . Exosomes were purified from serum of patients with LUAD using Total Exosome Isolation Kit (from serum) according to the manufacturer’s instructions (Invitrogen). For transmission electron microscopy (TEM), exosomes were fixed with 2% paraformaldehyde and absorbed onto formvar-coated grids (200 mesh). The grids were then stained with 2% uranyl acetate and allowed to air dry. The samples were observed using TEM .Exosomes were purified from A549 or H1299 cells-derived conditioned medium as previously described . In brie5). Cells were harvested at 24 h for immunofluorescence analysis. Images were acquired by an Olympus confocal microscope .Exosome uptake assay was performed using PKH26 Red Fluorescent Cell Linker Kit  according to the manufacturer’s instruction. PKH26-labeled exosomes (10 μg) were resuspended in 100 μL PBS, and incubated with HUVECs (1 × 103) were plated in 96-well plates at 24 h prior to exosome treatment. HUVECs were incubated with different exosomes derived from A549 or H1299 cells. At designated time points, CCK-8 solution was added into each well, and incubated at 37 °C for 1 h. Absorbance at 450 nm was measured using a microplate reader .Cell proliferation was monitored by using CCK-8 kit . Briefly, HUVECs or LUAD cells .5) and treated with indicated exosomes derived from LUAD cells. After 24 h, tubes were photographed using an inverted microscope (Olympus).In vitro angiogenesis was assessed by tube formation assay as previously described . Briefly5) were incubated with PBS or exosomes (10 μg) for 24 h. Cells were then harvested and cultured in endothelial cell EGM-2 medium  containing 20% methocel in 96-well suspension plates (Corning). Spheroids were formed at 37 °C overnight, and suspended with EGM-2 medium containing collagen type I . After 1.5 h incubation, DMEM medium containing 10% FBS was added into each well. After 48 h, sprouts were photographed using an Olympus confocal microscope (Olympus).3D spheroid sprouting assay was conducted as previously shown . Briefly5) were then collected and seeded in 6-well plates. The scratch was generated using a sterile 200 μL pipette tip as described [HUVECs or LUAD cells were incubated with PBS or indicated exosomes (10 μg) for 24 h. Cells (1 × 105) were then harvested and cultured in serum-free medium in 24-well transwell upper chambers (Corning) without Matrigel coating. The lower chambers were filled with complete medium. After 24 h, the migrated HUVECs were fixed with methanol and stained with 0.1% crystal violet solution (Sigma-Aldrich). The stained cells from five random visual fields were then counted using a light microscope.HUVECs or LUAD cells were incubated with PBS or indicated exosomes (10 μg) for 24 h. Cells (1 × 10https://starbase.sysu.edu.cn/index.php), TargetScan (https://www.targetscan.org/vert_72/) and miRTarBase (https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php). Among these three tools, StarBase was used to predict the binding sites between miR-197-3p and 3’UTR of both TIMP2 and TIMP3. The 3’UTR of TIMP2 or TIMP3 were cloned into psiCHECKTM-2 vector . Co-transfection of TIMP2 or TIMP3 3’UTR construct and miR-197-3p mimics or mimics NC were conducted using Lipofectamine 3000 (Invitrogen). Luciferase activity was assessed using Dual Luciferase Reporter Assay System (Promega).The putative binding sites between miR-197-3p and TIMP2/TIMP3 3’UTR were predicted using three bioinformatics programs, namely StarBase (Caenorhabditis elegans miR-39 (cel-miR-39) was used as an exogenous control for detect gene expression in exosomes. Results were analyzed using 2−ΔΔCT method. The primers used in this study were listed in Supplementary Table Total RNA was isolated from cells, tissues or exosomes using Trizol reagent (Invitrogen) or SeraMix Exosome RNA Purification Kit (System Biosciences), respectively. Reverse transcription was conducted using PrimeScript RT Reagent Kit . Quantitative real-time PCR was conducted using SYBR Green PCR Master Mix (Applied Biosystems). The expression levels of genes were normalized to that of GAPDH or U6. Synthetic Protein lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific), and protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). After blocking, the blot was incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody. Chemiluminescence signal was visualized using SuperSignal Pico PLUS chemiluminescent Substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: anti-GM130 ; anti-CD54 ; anti-Annexin V ; anti-CD9 ; anti-TIMP2 ; anti-TIMP3 ; anti-GAPDH ; anti-VEGFR2 ; anti-ERK ; anti-p-ERK ; anti-MMP2 ; anti-MT1-MMP  antibodies were purchased from Cell Signaling technologies .n = 8 per group, 6-week-old) were purchased from SJA Laboratory Animal Co. Ltd . All protocols were approved by Xiangya Hospital of Central South University. A549 cells (5 × 106) were subcutaneously injected into the flank of mice. Mice were randomly divided into five groups: PBS, mimics NC-Exo, miR-197-3p mimics-Exo, inhibitor NC-Exo and miR-197-3p inhibitor-Exo. After 12 days, exosomes (10 μg) were injected into the xenograft tumor every 4 days as previously described [2 × length. Mice were sacrificed, and tumors were weighted and subjected to IHC analysis after five injections.Male BALB/c nude mice , which were randomly divided into four groups: mimics NC-Exo, miR-197-3p mimics-Exo, miR-197-3p mimics-Exo+TIMP2, and miR-197-3p mimics-Exo+TIMP3. For in vivo study, the CDS of TIMP2 or TIMP3 was cloned into pCDH-CMV-MCS-EF1-copGFP lentiviral vector (GeneChem). A549 cells (1 × 107) pre-treated with exosomes or/and TIMP2/TIMP3 lentiviral particles were injected into the lateral tail vein into BALB/c nude mice for observation of lung metastasis. 4 weeks after injection, mice were anesthetized and lungs were dissected for subsequent analyses. All procedures have got the approval of Animal Use and Care Committee of Xiangya Hospital of Central South University. The investigator was blinded to the group allocation during the experiment.The function of miR-197-3p was examined in vivo using experimental model of lung cancer metastasis with male BALB/c nude mice  followed by Tukey’s post hoc test. Pearson’s correlation analysis was used to assess the degree of linear relationship between miR-197-3p and CD31, TIMP2, or TIMP3. P < 0.05 was considered statistically significant.Statistical analyses were conducted using the SPSS 20.0 software. Kaplan–Meier method was utilized for analyzing the survival curves, which was then evaluated the significance using a log-rank test. All error bars are displayed as the mean ± standard deviation (SD) derived from three independent experiments. The data were normally distributed, and variance was similar among the groups that are being statistically compared. Statistical analysis was performed using two-tailed Student’s Previous study demonstrated that miR-197-3p was highly expressed in LUAD tissues . In ordeEmerging evidence shows the correlation between exosomal miRNAs and metastasis in various cancers . We nextTo study the effects of exosomal miR-197-3p on umbilical vein endothelial cells, we investigated whether LUAD cells transported exosomal miR-197-3p to HUVECs. We first examined miR-197-3p levels in different LUAD cells by qRT-PCR. Compared with normal lung epithelial cells (NLEC) or HUVECs, miR-197-3p was highly expressed in LUAD cells, including SK-LU-1, H1650, H2030, H1975, A549, HCC827, PC9, H2009, and H1299 cells Fig. . A549 anTo further explore the function of exosomal miR-197-3p in HUVECs, we next assessed cell proliferation by CCK-8 assay. LUAD cells were transfected with mimics NC, miR-197-3p mimics, inhibitor NC or miR-197-3p inhibitor. Exosomes were then isolated from the conditioned medium of transfected LUAD cells, and HUVECs were subjected to designate treatment with A594 or H1299 cells-derived exosomes for 0, 24, 48, and 72 h. As shown in Fig. In order to validate the role of exosomal miR-197-3p in vivo, tumor xenograft model was established. A549 cells-xenograft bearing mice were subjected to designate treatment of exosomes every 4 days. As shown in Fig. We further explored the miR-197-3p target genes using bioinformatics programs. TIMP2 and TIMP3 were predicted as putative miR-197-3p target genes, and the putative binding sites between 3’UTR of TIMP2 or TIMP3 were shown in Fig. Moreover, we studied the downstream signaling and function of miR-197-3p/TIMP2 and miR-197-3p/TIMP3 axes in HUVECs. Western blot revealed that miR-197-3p mimics decreased TIMP3 expression, thereby inducing VEGFR2 expression and activating ERK signaling in HUVECs. These effects were reversed in miR-197-3p mimics+TIMP3 group, compared with control group Fig. . InteresTo test whether A549 or H1299 cells-derived exosomal miR-197-3p exerted similar effects in HUVECs, exosomes were purified from conditioned medium of transfected A549 or H1299 cells. Consistently, miR-197-3p mimics-Exo-mediated changes of TIMP3 and VEGFR2, as well as ERK activation were attenuated by TIMP3 overexpression Fig. . WesternWe next validated the effect of exosomal miR-197-3p on metastasis in LUAD lung metastasis model. The mice were injected with A549 cells after designate treatment of exosomes and TIMP2/3 lentiviral particles through tail vein. As shown in Fig. Circulating exosomes extracted from serum of healthy donors and LUAD patients with or without metastasis were collected for assessing the level of miR-197-3p, and the results showed that exosomal miR-197-3p rose in the serum of LUAD patients and positively correlated with metastasis Fig. . We furtIn this study, we demonstrated that miR-197-3p was elevated in both LUAD tissues and their serum exosomes, and the upregulation of miR-197-3p positively correlated with metastasis in LUAD patients. LUAD cells-derived exosomal miR-197-3p exerted oncogenic roles in vitro and in vivo. Mechanistic studies further revealed that exosomal miR-197-3p/TIMP2/3 axis was implicated in angiogenesis and metastasis in vitro and in vivo. Our findings shed novel insights on the biological role of exosomal miR-197-3p in LUAD.Cancer-derived exosomes are involved in various biological processes, including tumor development and progression. Given the non-invasive properties of blood-based biomarkers, exosomes are recognized as promising diagnostic and prognostic biomarkers in lung cancer . In addiAngiogenesis plays an essential role in tumor progression and metastasis. Intratumoral hypoxia induces angiogenesis by modulating pro- and anti-angiogenic factors. It is well established that the pro-angiogenic factor VEGFA and its receptor VEGFR2 are principally responsible for tumor angiogenesis , 31, 32.To further delineate the mechanism by which exosomal miR-197-3p regulated angiogenesis in LUAD, mechanistic studies were conducted. Since the known target genes of miR-197-3p are responsible for cell apoptosis in NSCLC , 29, we In conclusion, LUAD cells-derived exosomal miR-197-3p conferred angiogenesis via targeting TIMP2/3 in LUAD metastasis. Therefore, exosomal miR-197-3p level might be a promising prognostic biomarker and therapeutic target for anti-angiogenic treatment of LUAD.Fig. S1Fig. S2Supplementary figures and tableOriginal Data FileRe-aj-checklist


The result showed that dioscin pre-treatment counters MPP+-mediated autophagic flux impairment and alleviates MPP+-induced apoptosis by downregulating activated caspase-3 and BCL2 associated X, apoptosis regulator (Bax) expression while increasing B-cell lymphoma 2 (Bcl-2) expression. In addition, dioscin pre-treatment was found to increase neurotrophic factors and tyrosine hydroxylase expression, suggesting that dioscin could ameliorate MPP+-induced degeneration in dopaminergic neurons and benefit the PD model. To conclude, we showed dioscin’s neuroprotective activity in neuronal SH-SY5Y cells might be partly related to its autophagy induction and suppression of the mitochondrial apoptosis pathway.Autophagy is a cellular homeostatic process by which cells degrade and recycle their malfunctioned contents, and impairment in this process could lead to Parkinson’s disease (PD) pathogenesis. Dioscin, a steroidal saponin, has induced autophagy in several cell lines and animal models. The role of dioscin-mediated autophagy in PD remains to be investigated. Therefore, this study aims to investigate the hypothesis that dioscin-regulated autophagy and autophagy-related (ATG) proteins could protect neuronal cells in PD via reducing apoptosis and enhancing neurogenesis. In this study, the 1-methyl-4-phenylpyridinium ion (MPP Parkinson’s disease (PD) is one of the most prevalent age-related neurodegenerative diseases characterised by the loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc), the formation of intraneuronal proteinaceous inclusion and Lewy bodies ,2. Altho+) is an active metabolite form of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [+ translocates through the DAergic neurons by the dopamine transporter (DAT), leading to the inhibition of mitochondrial complex I and ATP synthesis and increases in reactive oxygen species (ROS), which, in turn, causes neuronal degeneration [1-methyl-4-phenylpyridinium ion (MPPe (MPTP) , which i+ has been shown to increase the microtubule-associated protein light-chain 3 (LC3)-II/LC3-I expressions, a major marker of autophagy, in SH-SY5Y cells [+ reduces the LC3 expression, as chronic exposure to MPP+ impairs autophagosome [Autophagy is a cellular homeostatic process by which intracellular components, including damaged organelles, misfolded or aggregated proteins, and intracellular pathogens, degrade into the cytoplasm of the cell ,8. In th5Y cells , but prohagosome  by lowerhagosome .Diosocorea nipponica Makino and Diosocorea rizhoma, and it is widely used to synthesise hormonal drugs [1-42-induced neurotoxicity in an immortalised mouse hippocampal neuronal precursor cells (HT-22 cells) [Dioscin is a steroidal saponin , abundanal drugs . Dioscinal drugs ,18 and aal drugs . Previoual drugs  and alleal drugs . This stal drugs , improveal drugs  and spatal drugs . In rece2 cells) .+ precursor MPTP treatment causes partial deficiency of autophagy-related 5 (ATG5) protein [Although the molecular mechanism of autophagic involvement in neurogenesis via regulating neurotrophic factors is not fully revealed, recent studies suggest that MPP protein  in PD an protein . Further protein . ATG5 is protein ; ATG5 up protein . We, the+ (250–2000 μM) in SH-SY5Y cells and incubated them for 24 h to select the optimal dose of MPP+. The cell survivability was dose-dependently reduced by MPP+ treatment (+ is 1 mM (the dose used for further experiments). Dioscin dose-dependently, except at 800 ng/mL [+-induced apoptosis on SH-SY5Y , Bcl-2 associated X protein (Bax), caspase-3, and activated caspase-3 (cleaved caspase-3/clvCas3). Dioscin treatment dose-dependently reduced Bax expression A but inc+-induces PD protein aggregation and causes neuronal loss [cAMP response element-binding protein  is a transcription factor that could modulate the expression of various other transcription factors like cFos, which in extension regulates brain-derived neurotrophic factor (BDNF) translation. As MPPnal loss , we testnal loss . The resTyrosine hydroxylase (TH) is the key enzyme for DAergic synthesis in the brain. Reduction in TH level causes downregulation of DAergic synthesis, which is key for motor dysfunction syndrome in PD. As the TH level is a positive sign of DAergic synthesis and neurogenesis, our findings support the hypothesis that dioscin promotes DAergic neuronal survival B. In sum+ inhibits autophagosome formation (phagophore phase) by inhibiting ATG5 [+-mediated ATG5 impairment in SH-SY5Y cell lysates  inhibited ATG5, indicating downregulation of autophagic flux formation, which was well supported by the up-regulation of LC3-adaptor protein SQSTM1/p62 (p62)  in SH-SY5Y. The result showed significant (#p < 0.05) impairment in MPP+ and CQ treated group, and dioscin at the high dose alone increased p62 degradation, but no significant changes were observed in the other two-dose  A. p62 isaded p62 A, indicatwo-dose B. A simitwo-dose B.+ for 24 h showed impaired conversion into LC3II, indicating impaired autophagosome formation  treatment enhanced autophagic fusion  from mitochondria to plasma, forms apoptosome and leads to activation of caspase-3 [It has been shown that PD pathogenesis involves apoptosis and caspase activation that causes DAergic cell degeneration ,37. MPP+aspase-3 . Additio+ treatment impairs Ca2+ influx, leading to Akt/mTOR pathway inactivation in SH-SY5Y cells that causes downregulation of BDNF and pCREB and degenerates TH-positive neurons [+-treated cellular PD model.Autophagy plays a part in the integrated pro-survival signalling, including AKT/CREB and defects in autophagy lead cells toward necrotic death . In this neurons ,46,47. D+-induced cell apoptosis in the SH-SY5Y human neuroblastoma cells line. Further investigation revealed that dioscin upregulates neurotrophic factors and dopamine precursor TH. The autophagy pathway plays a significant role in dioscin-mediated neuroprotection on SH-SY5Y neuroblastoma cells, but several limitations of this study need to be overcome by future studies, including in MPTP-intoxicated, trans-genic and autophagy impaired (ATG5-/-) animals. In addition to these validations, pharmacokinetic properties of dioscin, such as bioavailability, blood-brain barrier permeation and therapeutic window, need to be studied.To conclude, dioscin ameliorated autophagic flux impairment that might be involved in its neuroprotective effect against MPP+), chloroquine diphosphate , dimethyl sulfoxide (DMSO) and 3--2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich . Foetal bovine serum (FBS) , phosphate-buffered saline (PBS) and Dulbecco’s modified Eagle’s medium (DMEM/F12) were purchased from Gibco-BRL Technologies . RIPA buffer (10x) was purchased from Millipore , and protease and phosphatase inhibitors were obtained from Roche . All other chemicals utilised in this research were of analytical grade and were purchased otherwise noted Sigma-Aldrich.Dioscin (≥98% purity) was purchased from Chem Faces , 1-methyl-4-phenylpyridinium (MPPv/v) inactivated FBS in an incubator, condition maintained at 37 °C temperature and 5% CO2. The cells were trypsinised (0.05% trypsin-EDTA) after reaching 80–90% confluence for sub-culture, and the media was replaced every 2 days interval. Each experiment was conducted at least three times from three consecutive passages for statistical analysis.Human neuroblastoma SH-SY5Y cells were obtained from the American Type Culture Collection , as mentioned earlier . The neu+ (1 mM) for 24 h to establish the PD model. During the experiment, different doses of dioscin (100–400 ng/mL) were added to SH-SY5Y cells 2 h before the addition of MPP+ (1 mM), and the cells were maintained at 37 °C with 5% CO2 conditioned incubator. For the study of autophagy, CQ (40 µM) were incubated 1 h before MPP+ treatment.The SH-SY5Y cells were exposed to MPP5 cells/mL in a 96-well plate to assess viability using the previously described MTT assay [+ (0.25–2 mM) or MPP+ and dioscin (50–800 ng/mL), MPP+ and dioscin for another 24 h. Later, 20 µL of MTT (5 mg/mL) was added to each well, and the plate was incubated for an additional 3 h at 37 °C and 5% CO2. The medium was then carefully removed using a suction pump, and 200 µL of DMSO was added to each well and shaken for 30 mins to dissolve formazan crystals. The absorption was taken at 552 nm using a microplate reader .The SH-SY5Y cells were seeded at 2.5 × 10TT assay ,49. BrieTM protein assay kit  according to manufacturer instruction. Equal amounts of protein (20 µg) were separated electrophoretically using 8~15% sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene-difluoride (PVDF) membranes . The membranes were incubated at room temperature for 1 h with 3% bovine serum albumin in Tris-buffered saline (containing 0.1% Tween 20 buffer) to prevent nonspecific binding. Subsequently, the blots were incubated overnight at 4 °C with specific primary antibodies, including anti-Caspase3 , anti-clvCaspase3 , anti-pCREB , anti-TH , anti-BDNF , anti-SQSTM1/p62 , anti-ATG5 , anti-Bax , anti-Bcl-2  and anti-LC3  at 1:1000, and anti-β-actin  at 1:5000 concentration. Next, each blot was incubated at room temperature with either anti-mouse or anti-rabbit  secondary antibody. The blots were visualised with an enhanced chemiluminescence detection system  as per the recommended protocol.The cells were washed twice with cold PBS and subjected to lyse using lysis buffer (1× RIPA lysis buffer containing protease- and phosphatase inhibitor (1:1) cocktail). Whole mixtures were centrifuged at 14,000 rpm at 4 °C for 15 min, and the supernatants were collected carefully without disturbing pellets. The total protein obtained from cell lysates was quantified using DCThe immunocytochemistry was performed as described previously . Brieflyp-value was set <0.05 to be considered statistically significant.Statistical analyses were performed using GraphPad Prism  software. Data represented as means ± SEM (standard error mean) of three independent experiments. All data were analysed using one-way ANOVA followed by Sidak’s multiple comparisons to determine the significance; the


ADCK2 transcripts in NSCLC correlate with poor overall survival and poor anti-PD-1/PD-L1 therapy response. ADCK2 is overexpressed in local human NSCLC tissues and various primary and established NSCLC cells. In NSCLC cells, ADCK2 shRNA or CRISPR/Cas9 knockout remarkably suppressed cell viability, proliferation, cell cycle progression, cell mobility, and provoked cell apoptosis. Moreover, ADCK2 depletion disrupted mitochondrial functions in NSCLC cells, causing cytochrome C release, mitochondrial depolarization, DNA damage and ATP reduction. Contrarily, ectopic ADCK2 overexpression promoted NSCLC cell growth. Further studies revealed that ADCK2 depletion inactivated Akt-mTOR signaling in primary NSCLC cells. NSCLC xenograft growth in nude mice was significantly hindered after ADCK2 silencing or knockout. ADCK2 depletion, apoptosis induction and oxidative injury as well as ATP reduction and Akt-mTOR inactivation were detected in ADCK2-silenced or ADCK2-knockout NSCLC xenograft tissues. Together overexpressed ADCK2 is important for the growth of NSCLC cells, representing an important therapeutic molecular oncotarget.The aarF domain containing kinase 2 (ADCK2) is a mitochondria-locating protein, important for fatty acid metabolism and coenzyme Q biosynthesis. The bioinformatics results show that elevated Non-small cell lung cancer (NSCLC) represents the most prevalent lung cancer, and it accounts for 85-90% of all lung cancers Mitochondria are vital for ATP production and macromolecules biosynthesis in eukaryotic cells. Recent studies have also proposed that mitochondrial bioenergetics and signaling are essential for tumorigenesis and cancer progression The aarF domain containing kinase 2 (ADCK2) locates at mitochondria with its function largely unknown. It is important for mitochondrial metabolism and bioenergetics The anti-ADCK2 antibody was obtained from OriGene . All other antibodies were provided by Cell Signaling Technologies . The sequences and the associated viral constructs were all obtained from Genechem Co. . The fluorescence dyes, z-DEVD-cho and z-VAD-cho were from Biyuntian .A549 and NCI-H1944 cell lines, and BEAS-2 epithelial cells were described previously As described previously The verified ADCK2 shRNA or the ADCK2 cDNA was cloned into a GV248 vector (from Li's group ADCK2 KO and single stable ADCK2 KO NSCLC cells were obtained.Using Lipofectamine 3000 (Invitrogen) protocol, a LentiCas9-puro construct, provided by Genechem, was transduced to cultured NSCLC cells for 36h, and stable cells were selected through selection by puromycin. A Lenti-CRISPR/Cas9-ADCK2-KO construct  was synthesized by Genechem. The construct and lentivirus packaging and envelope constructs were transfected into HEK293T cells. The virus-containing medium was then collected after 24h and virus was filtered and was utilized to infect Cas9-expressing NSCLC cells. After 96h, cells were verified of NSCLC cells or the lung epithelial cells were seeded at optimal cell density and cultured for indicated time periods. CCK-8, colony formation assay, “Transwell” assays, nuclear EdU staining, cell immunofluorescence staining, and the single strand DNA (ssDNA) ELISA were described early The adenoviral constitutively-active Akt1  construct, as reported All laboratory athymic nude mice  were described early in vitro experiments were always repeated five times. “n.s.” indicates non-statistical differences (P > 0.05).Data were always presented as mean ± standard deviation. Statistical analyses were carried out as described ADCK2 mRNA transcript number in NSCLC tissues was higher when compared to that in lung epithelial tissues . ADCK2 mRNA overexpression in NSCLC was associated with poor overall survival (OS) (P = 0.00093) and poor progression-free survival  .TCGA (The Cancer Genome Atlas)-LUAD/LUSC cohorts revealed that P value < 25%) collection enrichment. KEGG pathway analysis further revealed that DEGs of basal cell carcinoma, cell cycle and DNA replication cascades were enriched in the ADCK2-high NSCLC . Whereas the gene set of complement and coagulation cascades as well as nitrogen metabolism showed enrichment in the ADCK2-low NSCLC .ADCK2 TCGA-LUAD/LUSC cohorts were further explored and differentially expressed genes (DEGs) were retrieved in NSCLC tissues. Gene Set Enrichment Analysis (GSEA) revealed a significant difference . Specifically, ADCK2-high expression is significantly correlated with less enrichment of CD8T cells, eosinophils and master cells . The immunophenoscore (IPS) measures the tumor immunogenicity. To access the potential relationship between ADCK2 expression and possible immunotherapy response, IPS were retrieved from TCGA-LUAD/LUSC. There are significant differences between the high ADCK2 gene expression group and the low ADCK2 gene expression group in all four immunotherapy regimens , and ADCK2-low predicts better anti-PD-1/PD-L1 therapy response . Together, the bioinformatics analysis results show that increased ADCK2 transcripts in NSCLC correlates with poor overall survival and poor anti-PD-1/PD-L1 therapy response.Anti-PD-1/PD-L1 therapies are being applied for NSCLC patients ADCK2 mRNA expression was dramatically elevated when compared to its expression in the adjacent normal lung tissues (“N”) . ADCK2 protein was upregulated in six representative NSCLC tissues (tissues were derived from “Patient #1 to #6”) . After combining the 20 sets tissue specimen blotting data, results demonstrated that elevation of the ADCK2 protein in the NSCLC tissues was significant . The tissue immunofluorescence staining at the junction of cancer and normal tissues of two representative NSCLC patients (Patient-7#/-13#) showed that ADCK2 expression (green fluorescence) in cancer cells was higher than it in epithelial cells  . The immunohistochemistry results in Figure 2E confirmed ADCK2 overexpression in tumors (“T”) of the representative NSCLC patient  primary human NSCLC patients with cancer resection surgery were enrolled. The fresh tumor tissues were obtained. In NSCLC tumor tissues (“T”) ADCK2 mRNA  and protein  levels in the NSCLC cells were higher than those in primary human lung epithelial cells (“pEpi” 2G further confirmed that ADCK2 expression (in red fluorescence) in primary NSCLC cells was higher than its expression in pEpi, the latter was labeled with the epithelial marker EpCAM . Therefore, ADCK2 is upregulated in local NSCLC tissues.We next examined ADCK2 expression in different NSCLC cells, including A549/NCI-H1944 established cell lines and primary patient-derived NSCLC cells (namely “pCan-1”/“pCan-2”/“pCan-3”). s “pEpi” , 31. MorADCK2 mRNA decreased over 90% in ADCK2-sh and ADCK2-ko pCan-1 cells . ADCK2 protein expression was depleted as well . Figure 3C demonstrated that ADCK2 silencing/KO decreased viability (CCK-8 OD) of the pCan-1 cells. Moreover, the pCan-1 cell colony number was significantly reduced in ADCK2-sh and ADCK2-ko pCan-1 cells . The increased percentage of Trypan blue-positive staining indicated that ADCK2 silencing or KO induced death of pCan-1 cells . These results implied that ADCK2 depletion led to potent cytotoxicity to pCan-1 cells.The lentiviral particles encoding the ADCK2 shRNA were transfected to the pCan-1 cells. The stable pCan-1 cells, “ADCK2-sh”, were established after selection. Also, a CRISPR/Cas9-ADCK2-KO construct was stably transduced to the Cas9-expressing pCan-1 cells to establish single stable “ADCK2-ko” cells. The lentiviral scramble control nonsense shRNA (“shScr”) and the CRISPR/Cas9 empty construct (“koC”) were tranduced to the control cells (“shScr+koC”). vs. DAPI) was significantly decreased in ADCK2-sh and ADCK2-ko pCan-1 cells , indicating proliferation inhibition by ADCK2 depletion. In addition, ADCK2 depletion in pCan-1 cells disrupted cell cycle progression, leading to G1-phase increasing but S-phase reduction . ADCK2 silencing/KO in pCan-1 cells potently inhibited cell migration . The shScr+koC treatment did not significantly alter ADCK2 expression  or pCan-1 cellular functions .Nuclear EdU staining assays were employed to test cell proliferation, and EdU-stained nuclei ratio . In the tested NSCLC cells, ADCK2 shRNA reduced CCK-8 viability , decreased EdU-stained nuclei ratio ,  and slowed in vitro cell migration .Two primary NSCLC cells, “pCan2” and “pCan3” 3M) was unable to significantly reduce CCK-8 viability  and EdU incorporation , supporting a cancer cell-specific activity by ADCK2 depletion.In the pEpi primary lung epithelial cells, ADCK2 silencing by the same lentiviral shRNA . Further supporting caspase activation, we showed cleavages of caspase-3/-9 and PARP were robustly induced in pCan-1 cells with ADCK2 shRNA/KO , where the Histone-bound DNA contents were augmented . Expression of total and cleaved GSDME (Gasdermin E), the indicator of pyroptosis activation 4C).ADCK2 shRNA or KO induced cytotoxicity in NSCLC cells, we thus analyzed whether apoptosis was induced. Caspase-3 and caspase-9 activities were dramatically augmented in ADCK2-sh and ADCK2-ko pCan-1 cells . shScr+koC treatment was unable to stimulate caspase-apoptosis activation in pCan-1 cells . Significantly, ADCK2-sh- and ADCK2-ko-caused viability decrease  and cell death  were ameliorated by the caspase inhibitors z-DEVD-cho and z-VAD-cho. Therefore, apoptosis induction should be the primary cause of ADCK2 silencing-caused NSCLC cell death.Confirming apoptosis induction, the ratio of TUNEL-positively stained nuclei was increased in pCan-1 cells after ADCK2 silencing or KO  and increased the ratio of TUNEL-stained nuclei , supporting apoptosis induction. Interestingly, in normal “pEpi” cells, ADCK2-sh treatment failed to significantly increase nuclear TUNEL ratio/apoptosis , again indicating the cancer cell specific effect by ADCK2 depletion.In primary NSCLC cells (“pCan2” and “pCan3”) and A549 cells, ADCK2 silencing, by ADCK2-sh, significantly augmented the activity of caspase-3 . Moreover, the increased lipid peroxidation, or TBAR activity enhancement, was observed . The ssDNA contents were elevated in ADCK2-sh and ADCK2-ko pCan-1 cells, indicating augmented DNA breaks . Moreover, ATM and ATR phosphorylation was significantly increased in ADCK2-depleted pCan-1 NSCLC cells . The mitochondrial complex I activity was significantly decreased in ADCK1-silenced/depleted cells . ATP contents were reduced as well in ADCK2-depleted pCan-1 cells . shScr+koC treatment did not significantly alter mitochondrial functions in pCan-1 cells .ADCK2 is a mitochondrial protein with the aarF domain, we therefore tested whether ADCK2 knockdown/depletion could disrupt normal functions of mitochondria. As shown in Figure 5H), and augmented DNA breaks , but reduced mitochondrial complex I activity  and decreased ATP contents . These results implied that ADCK2 depletion in NSCLC cells disrupted normal functions of mitochondria.In primary “pCan2” and “pCan3” cells and established A549 cells, ADCK2 shRNA similarly resulted in JC-1 green monomer increasing and mitochondrial depolarization . As demonstrated, in oe-ADCK2 pCan-1 cells, the cellular ATP contents were significantly increased . Ectopic ADCK2 overexpression promoted pCan-1 proliferation, and the ratio of the EdU-stained nuclei was increased . Moreover, pCan-1 in vitro cell migration was enhanced as well after ADCK2 overexpression .Next lentiviral particles with ADCK2-expressing construct were added to pCan-1 cells and stable cell selections, namely oe-ADCK2-StL1 and oe-ADCK2-StL2, were formed through selection. ADCK2 mRNA and protein expression in ADCK2-sh pCan-1 cells . Significantly, ADCK2-sh-induced mitochondrial complex I activity inhibition  and ATP reduction  were reversed by oe-ADCK2.The rescue experiments were carried out. pCan-1 cells were stably infected with ADCK2 shRNA lentivirus (“ADCK2-sh”) or together with the ADCK2-expressing construct lentivirus (“+oe-ADCK2”). As shown, oe-ADCK2 restored ADCK2 mRNA expression . Consequently, the number of viable NSCLC cells was dramatically increased . Interestingly, in “pEpi” normal cells, ADCK2 overexpression using the same construct  was unable to alter CCK-8 viability  and cell proliferation . These results again supported the selected effect of ADCK2 in cancerous cells.In other primary NSCLC cells (“pCan2” and “pCan3”) and established A549 cells, stable transfection of the ADCK2-expressing lentiviral construct (“oe-ADCK2”) similarly increased 7A), suggesting that ADCK2 depletion indeed hindered Akt-mTOR activation in primary NSCLC cells. Conversely, ADCK2 overexpression further enhanced Akt-mTOR activation in pCan-1 cells , as phosphorylation Akt and S6K1 was augmented in oe-ADCK2-StL1 and oe-ADCK2-StL2 pCan-1 cells .Considering that ATP production and energy are vital for activation of Akt-mTOR signaling 7C), but did not bring back ADCK2 protein expression . Significantly, shADCK2-caused anti-proliferative activity  and apoptosis  were ameliorated by caAkt1. Thus Akt-mTOR inactivation should be one primary mechanism of ADCK2 depletion-induced activity against NSCLC cells.To explore the relationship between ADCK2 depletion-induced anti-NSCLC cell activity and Akt-mTOR inactivation, a constitutively-active Akt1 . Figure 8A demonstrated that the growth of the ADCK2-silenced pCan-1 xenografts and the ADCK2 KO pCan-1 xenografts was dramatically slower than that of shScr+koC NSCLC xenografts. At Day-35, NSCLC xenografts were isolated very carefully and individually weighted. pCan-1 xenografts with ADCK2 silencing or KO were dramatically lighter than shScr+koC xenografts . The mice body weights showed no significant difference among the three experimental groups .Lastly, ADCK2-silenced pCan-1 cells (“ADCK2-sh”), the ADCK2 KO pCan-1 cells (“ADCK2-ko”) as well as the control “shScr+koC” pCan-1 cells were injectedADCK2 mRNA/protein expression was decreased significantly in the ADCK2-sh-xenograft tumors and the ADCK2-ko-xenograft tumors . Notably, caspase-3 and PARP cleavages were significantly augmented in ADCK2-depleted tumors , indicating apoptosis induction. The cleaved-GSDME expression was unchanged . In addition, levels of the cytosol cytochrome C were increased in NSCLC xenograft tumors bearing ADCK2 shRNA or ADCK2 KO construct . Moreover, the increased TBAR activity in ADCK2-sh- and ADCK2-ko-xenograft tumor tissues implied oxidative injury and mitochondrial injury . Further studies showed that ATP contents were decreased after ADCK2 silencing or KO in pCan-1 xenograft tissues . Moreover, phosphorylation of Akt-S6K1 were remarkably decreased in ADCK2-depleted tumors . Thus, ADCK2 depletion decreased ATP contents and inhibited Akt-mTOR activation in pCan-1 xenografts.At experimental Day-10, one xenograft per group was separated and analyzed.  ADCK2 transcripts in NSCLC correlate with poor overall survival and poor anti-PD-1/PD-L1 therapy response. Moreover, ADCK2 overexpression was detected in local NSCLC tissues and different NSCLC cells. While its expression in lung epithelial tissues and cells is relatively low.The prognosis of the recurrent, metastatic, therapy-resistant and other advanced NSCLC is still poor. The five-year survival has not been significantly improved recently Few studies have reported ADCK2's potential function in cancer cells. A functional viability profile study in breast cancer has identified ADCK2 as a key gene for breast cancer cell survival in vitro cell migration. The growth of NSCLC xenografts in nude mice was significantly hindered after ADCK2 knockdown or KO.The work herein supported that ADCK2 exerted significant pro-cancerous activity. In NSCLC cells, ADCK2 shRNA or KO robustly suppressed malignant behaviors, and provoked cell apoptosis. Contrarily, ectopic ADCK2 overexpression augmented NSCLC cell growth and accelerated NSCLC, like other common tumors, exhibits elevated mitochondrial metabolism, including glycolysis and glucose oxidation ADCK2 is required for lipid transport into mitochondria, and following fatty acid β-oxidation and CoQ biosynthesis Akt-mTOR aberrant activation contributes significantly to tumorigenesis and cancer progression in NSCLC Overexpressed ADCK2 is important for the growth of NSCLC cells, representing an important therapeutic molecular oncotarget.Supplementary figure.Click here for additional data file.


E. coli population is dominated by diverse sequence types with varied geographic distributions, warranting ongoing genomic surveillance.The  Escherichia coli in 36 countries during 2015–2017. Common carbapenemases were oxacillinase (OXA) 181 (23%), New Delhi metallo-β-lactamase (NDM) 5 (20%), OXA-48 (17%), Klebsiella pneumoniae carbapenemase 2 (15%), and NDM-1 (10%). We identified 5 dominant sequence types (STs); 4 were global , and 1 (ST1284) was limited to Turkey. OXA-181 was frequent in Jordan (because of the ST410-B4/H24RxC subclade) and Turkey (because of ST1284). We found nearly identical IncX3-blaOXA-181 plasmids among 11 STs from 12 countries. NDM-5 was frequent in Egypt, Thailand (linked with ST410-B4/H24RxC and ST167-B subclades), and Vietnam (because of ST448). OXA-48 was common in Turkey (linked with ST11260). Global K. pneumoniae carbapenemases were linked with ST131 C1/H30 subclade and NDM-1 with various STs. The global carbapenemase E. coli population is dominated by diverse STs with different characteristics and varied geographic distributions, requiring ongoing genomic surveillance.We describe the global molecular epidemiology of 229 carbapenemase-producing  Carbapenems are effective options available for treating serious infections caused by multidrug-resistant (MDR) Enterobacterales bacteria (Klebsiella pneumoniae carbapenemases (KPCs), imipenemases (IMPs), Verona integron–encoded metallo-β-lactamases (VIMs), New Delhi metallo-β-lactamases (NDMs), and oxacillinase (OXA) 48–like enzymes. Escherichia coli is the second most common carbapenemase-producing Enterobacterales species  reservoir for antimicrobial resistance (AMR) genes , other β-lactamases, AMR genes, and virulence factors.Comprehensive epidemiology data about carbapenemase-producing E. coli isolates collected from 2 global surveillance programs (SMART and INFORM) during 2015–2017  tested nonsusceptible to >1 of the carbapenems. Most (229 [83%]) were positive for either blaKPC, blaOXA-48-like, blaNDM, blaVIM, or blaIMP and were included in this study. The remaining 46 were negative for blaKPC, blaVIM, blaNDM, blaOXA-48-like, blaIMP, and blaGES.We obtained ethics approval for this study through the University of Calgary Conjoint Health Research Ethics Board . We included 229 clinical, nonrepeat 015–2017 . IsolateE. coli carbapenemase population  to short-read WGS by using NovoSeq  with 151 × 2 paired-end reads (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine AMR genes, plasmid replicons, and virulence genes against the following databases or typing schemes: National Center for Biotechnology Information Bacterial Antimicrobial Resistance Reference Gene Database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047), ResFinder (mlst 2.19 (https://github.com/tseemann/mlst). We identified ST410 and ST131 clades and subclades as described previously (We subjected the carbapenemase-producing https://github.com/tseemann/snippy). We filtered single-nucleotide polymorphisms (SNPs) among prophages, repeated sequences, or insertion sequences as previously described  by using snippy  were found in Greece, Spain, Mexico, and Egypt; E. coli with IMP-59 were obtained from Australia  and 3 minor STs  among this collection. The next most common STs did not fulfill the definition of a dominant ST: ST38 (n = 10 [4%]), ST354 (n = 7 [3%]), ST361 (n = 9 [4%]), ST648 (n = 8 [4%]), and ST11260 (n = 6 [3%]).H24Rx (n = 10) and B4/H24RxC (n = 35) (ST410 was the most common ST (n = 45/229 [20%]) and was positive for KPC-2 (7%), NDM-5 (27%), NDM-7 (2%), OXA-48 (4%), OXA-181 (47%), and VIM-23 [4%])  Table 1.H41 (n = 2) and subclades C1_nonM27 (n = 10), C1_M27 (n = 4), and C2 (n = 10). We also note the global distribution of different minor STs  and their clades (ST131 was the second most common ST (n = 26/229 [12%]) and was positive for KPC-2 (n = 8), KPC-3 (n = 7), KPC-18 (n = 1), NDM-1 (n = 5), NDM-5 (n = 1), OXA-48 (n = 2), OXA-181 (n = 1), and OXA-232 (n = 1). One NDM-1 isolate was also positive for VIM-1. ST131 belonged to clade A/r clades .E. coli STs (aac(6')-Ib-cr, and sul1 were common among isolates.We determined quinolone resistance–determining regions mutations, β-lactamases (noncarbapenemases), aminoglycoside modifying enzymes, and plasmid replicon types among the different coli STs  Table 1.fimH (100%), fyuA (55%), traT (64%), and iss (52%). Some virulence factors were associated with certain STs: papA (81%), iha (77%), sat (81%), fyuA (100%) usp (100%), ompT (100%), and malX (100%) with ST131, and astA (100%) and iutA (100%) with ST1284. ST131 had the highest overall number of virulence genes (n = 11), and ST410 had the lowest number of virulence genes (n = 2) (We assessed the presence of 37 putative virulence factors among the different dominant STs  Table 2. (n = 2)  Table 2.blaOXA-48 and blaVIMs. We obtained results for 20/22 of blaNDM-1, 2/2 of blaNDM-4, 46/48 of blaNDM-5, 1/1 of blaNDM-6, 4/5 of blaNDM-7, 34/35 of blaKPC-2, 14/14 of blaKPC-3, 1/1 of blaKPC-18, 1/41 blaOXA-48, 55/55 of blaOXA-181, 3/3 of blaOXA-244, and 7/7 of blaOXA-232.Because of the limitations of short-read sequencing . Nineteen were associated with non-Tn4401 mobile elements (NTMKPC) (blaKPC-3 genes were associated with Tn4401a (n = 9), Tn4401b (n = 3), and Tn4401d (n = 2). The blaKPC-18 was located on a novel Tn4401 variant (186 bp deletion). The blaNDMs were located on truncated Tn125 elements, and the blaNDM upstream regions showed substantial diversities with various IS element insertions . Among blaOXA-232 genes were located on the same 6.1 kb colKp3 plasmids (pOXA-232) (blaKPC-3 isolates with previously sequenced plasmids in GenBank showed that most (n = 9) were harbored within IncFIBQil plasmids (blaKPC-3 was within the IncFIA plasmid pBK30683 (blaKPC-3 was in the IncI2 plasmid pBK15692 (blaOXA-181 (n = 55) were situated within Tn2013 harbored on the identical IncX3 plasmids with 99%–100% similarities to plasmid p72_X3_OXA181  during 2015–2017. Isolates with multiple AMR genes dominated the population. The most common carbapenemase group was the OXA-48-like carbapenemases (44%), followed by NDMs (32%), KPCs (21%), VIMs (2%), and IMPs (1%). OXA-48-like carbapenemases were numerous in Egypt, Jordan, and Turkey; NDMs were numerous in Egypt, Thailand, and Vietnam, and KPCs were numerous in Colombia, Italy, and the United States.A World Health Organization report showed the lack of adequate surveillance programs in many parts of the world, especially from lower- and middle-income countries (LMICs) . Phylogroup B2 isolates were common, and phylogroup A was dominant in Asia. Global ST131 with blaKPCs was the most common ST, followed by ST648 with blaOXA-48-like and ST405 with blaNDMs.A recent survey of global carbapenemase-producing K. pneumoniae and Enterobacter cloacae complex with carbapenemases obtained from the same surveillance programs (K. pneumoniae population was dominated by ST258 with KPC-2 from Greece and KPC-3 from the United States (E. cloacae complex isolates (various STs) were dominated by VIM-1 from Greece and Italy , NDM-5 (20%), OXA-48 (17%), KPC-2 (15%), and NDM-1 (10%). This result was different from carbapenemase-producing E. coli with blaOXA-48 is linked to various STs . Clade B is divided into subclades B1/H24, B2/H24R, B3/H24Rx, and B4/H24RxC . K. pneumoniae ST307 with p72_X3_OXA181 was previously responsible for large outbreaks in South Africa , and Turkey (linked with ST1284). The ST410-B4/H24RxC subclade with blaOXA-181 was also found in Thailand and South Korea. The ST410-B3/H24Rx subclade with blaOXA-181 was present in South Africa and Kuwait.OXA-181 is linked with certain E. coli with blaNDM-1 was not linked with a specific ST and was evenly distributed among the different countries.Molecular-based surveillance studies have shown that NDMs are often the most common carbapenemase in certain regions  , Thailand (linked with ST410 [B4/H24RxC] and ST167 [B2]), and Vietnam (linked with ST448). ST167 belongs to phylogroup A and is an emerging carbapenemase clone associated with blaNDM-5 , Egypt (subclades B1 and B2), and Thailand (subclade B2).E. coli with blaKPC is associated with ST131 . ST131 with blaKPC was dominated by the C1_nonM27 subclade. This dominance is different from that observed by Johnson et al. study .Among this study’s strengths is that it included a large global collection of recent isolates representing multiple LMICs. We characterized all isolates using short-read WGS and provided novel information regarding the geographic distribution and MDR determinants of dominant STs and their respective clades and subclades . The geographic distribution of isolates with NDM-1 and NDM-5 was different . Similar differences were described for isolates with OXA-48 (various STs) and OXA-181 (linked with ST410B4/H24Rx). Future genomic surveys should use methodologies that characterize individual carbapenemases.We showed that the underlying molecular epidemiology within the same carbapenemase groups were very different . This finding suggests that highly similar IncX3 plasmids were mainly responsible for the global distribution of OXA-181 genes, the most common carbapenemase in this collection. The control of such IncX3 plasmids should be a public health priority.We also showed that global E. coli, and a follow-up study using long-read sequencing is under way. Several countries included only few isolates . Ongoing genomic surveillance to characterize individual carbapenemases will assist in designing management and prevention strategies to help curtail the spread of AMR bacteria.In summary, the global carbapenemase-producing Escherichia coli, 2015–2017.Additional information about genomic epidemiology of global carbapenemase-producing