Stub1 or Chic2 leads to reduced ubiquitination of Csf2rb. The effects of Stub1 and Chic2 on Csf2rb were greatest at reduced cytokine concentrations, suggesting that Stub1/Chic2-mediated regulation of Csf2rb is a mechanism of reducing cell surface accumulation when cytokine levels are low. Our study uncovers a mechanism of CSF2RB regulation through ubiquitination and lysosomal degradation and describes a role for CHIC2 in the regulation of a cytokine receptor.The IL-3, IL-5, and GM-CSF family of cytokines play an essential role in the growth, differentiation, and effector functions of multiple hematopoietic cell types. Receptors in this family are composed of cytokine-specific α chains and a common β chain (CSF2RB), responsible for the majority of downstream signaling. CSF2RB abundance and stability influence the magnitude of the cellular response to cytokine stimulation, but the exact mechanisms of regulation are not well understood. Here, we use genetic screens in multiple cellular contexts and cytokine conditions to identify STUB1, an E3 ubiquitin ligase, and CHIC2 as regulators of CSF2RB ubiquitination and protein stability. We demonstrate that Stub1 and Chic2 form a complex that binds Csf2rb and that genetic inactivation of either  Cytokine receptors convert extracellular signals into the activation of intracellular pathways that drive proliferation, differentiation, and effector functions of cells. The duration and magnitude of signaling are tightly regulated, and the modulation of cell surface receptor expression is a key mechanism to tune a cell’s response to extracellular signals. For example, CBL is a ubiquitin ligase that targets receptor tyrosine kinases for degradation following activation in order to terminate signaling, and mutations in the RING ubiquitin ligase domain lead to constitutive signaling that drives myeloid malignancies . Recent The IL-3, IL-5, and GM-CSF family of cytokines and respective cytokine receptors play a key role in the growth, differentiation, and effector functions of multiple hematopoietic cell types. Collectively, these cytokines provide a mechanism of communication between adaptive and innate immune cells, as they are primarily produced by activated T cells and stimulate myeloid cells at multiple stages of differentiation  . Similar results were found for endogenous CSF2RB protein levels (A and S1D). We also found that lysosomal acidification inhibition by Bafilomycin A1 led to increased CSF2RB reporter levels and endogenous protein levels in both cell lines, suggesting that lysosomal degradation also plays a role in CSF2RB regulation . In summary, our results indicate that in both human and murine cells, CSF2RB/Csf2rb is likely regulated by a noncullin ubiquitin ligase and lysosomal acidification.We expressed this human CSF2RB reporter in two cell lines: TF1 cells, a human, GM-CSF-dependent, myeloid cell line and 32D cells, a murine, IL-3-dependent, myeloid cell line. To test whether CSF2RB is regulated by the ubiquitin-proteasome system, we utilized small molecule inhibitors of E1 ubiquitin–activating enzymes (MLN7243), NEDD8-activating enzymes (MLN4924), and the proteasome (MG132). In both 32D and TF1 cells, E1 and proteasomal inhibition led to an increase in the CSF2RB GFP/mCherry ratio, while inhibition of neddylation, an essential modification for cullin-RING ubiquitin ligase activation, had minimal effect B and S1Cn levels A and S1DTo gain insight into the molecular machinery required for the ubiquitination and degradation of CSF2RB, we employed a CRISPR/Cas9 screen using the CSF2RB reporter and a previously described ubiquitin ligase–specific single-guide RNA (sgRNA) library  in two hSTUB1 was significantly enriched in the GFP/mCherry high population across multiple cell lines, regardless of cytokine conditions, indicating that STUB1 may destabilize CSF2RB. In TF1 cells cultured chronically in 5 ng/ml GM-CSF, sgRNAs targeting STUB1, UBE2N, UPS10, and UPS20 were significantly enriched in cells with stabilized CSF2RB (D). Surprisingly, across both cell lines and the varied cytokine conditions , STUB1 was the only consistently significant hit . Stub1 was again one of the most significantly enriched genes in this third cell line, and interestingly, no other known interactor of STUB1 was significantly enriched.While our ubiquitin ligase–specific CRISPR screens identified STUB1 as a hit across multiple cellular contexts, STUB1, like other ubiquitin ligases, is thought to work in concert with other proteins to interact with and ubiquitinate substrates . To idenFig. 1STUB1 identified was CHIC2 (inset of G), a poorly characterized protein with no known cellular function. Consistent with the codependency analysis, Chic2 behaved similarly to Stub1 in our genome-wide, reporter-based screen (F). This correlation and our genome-wide screen suggest that STUB1 and CHIC2 act in concert to regulate CSF2RB levels.To prioritize other potential hits from our genome-wide screen, we also employed a codependency analysis of the Cancer Dependency Map, a database of genome-wide CRISPR screens across 1054 cancer cell lines, which allows for the identification gene and protein level networks , 17, 18.inset of G, a poord screen F. This cStub1 and Chic2 with two independent sgRNAs each (A and B). Endogenous Csf2rb protein levels were increased with Stub1 and Chic2 KO, especially at lower concentrations of IL-3 (C and D). Quantification of Western blot replicates at 0.01 ng/ml IL-3 demonstrated a 2- to 3-fold increase in Csf2rb protein levels with Stub1 or Chic2 KO (A and S4B). To test whether this increase in endogenous Csf2rb protein expression was not due to an increase in mRNA transcription, we examined Csf2rb mRNA expression by quantitative RT-PCR. Expression of Csf2rb mRNA was not increased with Stub1 or Chic2 KO, consistent with posttranslational regulation of Csf2rb by Stub1 or Chic2 (C and S4D).Having identified a role for STUB1 and CHIC2 in regulating the CSF2RB reporter, we next sought to examine the effect of these proteins on endogenous CSF2RB. We generated 32D cells with CRISPR/Cas9 inactivation of NAs each , A and B of IL-3 , C and DChic2 KO A and S4Bor Chic2 C and S4DStub1 or Chic2 reduced the rate of Csf2rb steady-state destruction with cycloheximide treatment over an 8 h time course (E and F). Therefore, Stub1 and Chic2 directly regulate Csf2rb protein stability.We next sought to test whether this effect on Csf2rb was indeed on protein stability with a cycloheximide-chase experiment. KO of e course , E and FStub1 and Chic2 KO led to increased protein stability, we next sought to test whether Stub1 and Chic2 KO together leads to an additive effect on Csf2rb stability. We generated 32D Cas9 cells with Stub1 KO, Chic2 KO, and both Stub1/Chic2 double KO and measured Csf2rb protein levels by Western blot. Stub1 and Chic2 KO led to an equal increase in Csf2rb protein levels, and the Stub1/Chic2 double KO did not increase Csf2rb stability beyond the single KOs alone (G). Of note, Stub1 KO led to increased Chic2 protein levels, while Chic2 KO did not lead to increased Stub1 protein levels, suggesting that Chic2 may be consumed in Stub1’s mechanism of Csf2rb regulation. Taken together, this further supports the hypothesis that Stub1 and Chic2 likely regulate Csf2rb protein stability by the same mechanism and in the same pathway.Given that both Os alone G. Of notStub1 and Chic2 KO led to increased total protein levels of Csf2rb, we hypothesized that Stub1 and Chic2 KO specifically lead to cell surface accumulation of Csf2rb, where it is able to participate in active signaling. As measured by flow cytometry, both endogenous, cell surface Csf2rb and a Csf2rb reporter were both increased ∼2.5- to 3-fold with Stub1 and Chic2 KO in 0.01 ng/ml IL-3 . In the CHIC2 IP/MS, we likewise found that Stub1 was enriched, although not significantly, as well as Itm2b, Ifitm2, and Fcgr1 (B). Importantly, no interactions were identified between CHIC2 and heat shock proteins, suggesting that heat shock protein/STUB1 complexes are independent of the CHIC2-containing complexes. Furthermore, there were no common interactors identified between the STUB1 and CHIC2 IP/MS experiments, suggesting that the STUB1/CHIC2 complex may not have other components. In validation experiments, we found that V5-CHIC2 coimmunoprecipitated with endogenous Stub1 by IP-Western blot, demonstrating that STUB1 and CHIC2 do indeed interact (C). We also found that STUB1-V5 coimmunoprecipitated with endogenous Chic2 and that different concentrations of IL-3 did not alter this interaction  of overexpressed V5-tagged STUB1 or CHIC2 in 32D cells followed by mass spectrometry (MS). In the STUB1 IP/MS, we found that Chic2 and multiple known partners of STUB1, including heat shock proteins, were significantly enriched A. In thend Fcgr1 B. Importinteract C. We alseraction Figure 3Ivice versa, we preformed a STUB1 IP in Chic2 KO 32D cells and a CHIC2 IP in a Stub1 KO 32D cells, each followed by MS (D and E). Few STUB1 interactors were significantly lost  with Chic2 KO. These included Lmnb1 and Hspe1, but these proteins are thought to be located on the nuclear membrane and mitochondria, respectively, and not likely to play a role in cell surface regulation of Csf2rb  , the C-terminal amino acid sequences of CHIC1/CHIC2 are similar but distinct (G). Due to these differences, Ravalin et al., suggest that the CHIC1/CHIC2 C terminus may participate in a higher affinity interaction with STUB1 compared to HSP70 or HSP90 family members  domain of STUB1, a common structural motif known to mediate protein–protein interactions, and the C terminus of proteins predicted an interaction between STUB1’s TPR domain and the last five amino acids on the C terminus of CHIC2 F 20). C. CF) (20distinct G. Due to members .H). Furthermore, we found that co-IP of endogenous Chic2 with STUB1-V5 was lost when we deleted the TPR domain of STUB1 (I). Taken together, our data indicate that CHIC2 and STUB1 interact and that both CHIC2’s C terminus and STUB1’s TPR domain are required for their interaction.Based on this modeling data, we hypothesized that the interaction between STUB1 and CHIC2 is dependent on STUB1’s TPR domain and CHIC2’s C terminus. We found that the co-IP of endogenous Stub1 with V5-CHIC2 was lost when the last five amino acids of CHIC2 (ΔIFRPD) were deleted H. Furtheof STUB1 I. Taken Given that STUB1 is known to function with multiple HSP family members and that HSP family members were not identified in our genetic screens or IP/MS analyses of CHIC2, we sought to directly test whether inhibition of HSP family members had any effect on CSF2RB protein stability. Since there is significant redundancy among HSP family members, we utilized small molecule inhibitors of HSPs, including Apoptazole, to inhibit HSP70 proteins and HSP990 to inhibit HSP90 proteins. Treatment of 32D cells with either inhibitor did not significantly increase Csf2rb protein levels, suggesting that HSP proteins likely do not play a role in the Stub1/Chic2 regulation of Csf2rb .Stub1 and Chic2 KO increased Csf2rb protein stability, we hypothesized that Stub1 and Chic2 interact directly with Csf2rb. We found that both STUB1-V5 and V5-CHIC2 coimmunoprecipitated with endogenous Csf2rb (A and B). To explore the components of the STUB1/CHIC2 complex required for the interaction with CSF2RB, we next tested the effect of deletion of the C terminus of CHIC2 and the deletion of the TPR domain of STUB1 on their interaction with Csf2rb. Loss of the TPR domain of STUB1, thereby preventing interaction with Chic2, blocked co-IP of endogenous Csf2rb (C). Deletion of the C terminus of CHIC2 (ΔIFRPD) did not affect the co-IP of Csf2rb with V5-CHIC2, while the interaction with Stub1 was again lost (D). This suggests that Chic2 binds to Csf2rb independent of Stub1, and we hypothesized that Chic2 may facilitate the interaction between Stub1 and Csf2rb. Chic2 KO reduced but did not block co-IP of Csf2rb with STUB1-V5 (E), suggesting that either Chic2 and Stub1 bind independently of each other to Csf2rb or there are yet other proteins in the complex not captured by our genetic and proteomic techniques.Because both s Csf2rb , A and Bs Csf2rb C. Deletiain lost D. This sSTUB1-V5 E, suggesF); however, this increased interaction was commensurate with the increased expression of Csf2rb in reduced IL-3 concentration, suggesting that the interaction between Stub1 and Csf2rb is more dependent on concentration of Csf2rb rather than inactivity. Indeed, treatment of 32D cells with Ruxolitinib to inhibit Jak2 phosphorylation of Csf2rb did not alter the interaction between STUB1-V5 and Csf2rb, suggesting that Stub1 likely binds to inactivated and activated Csf2rb equally (G).To understand the conditions required for the regulation and interaction of Stub1/Chic2 with Csf2rb, we tested the effects of cytokine concentration and receptor activation on the interaction between Csf2rb and Stub1/Chic2. We hypothesized that reduced cytokine concentration would enhance the interaction between Stub1 and Csf2rb. Endogenous Csf2rb co-IP with STUB1-V5 increased with reduced IL-3 concentration F; howeve equally G.Stub1 and Chic2 KO on Csf2rb protein stability, we hypothesized that Stub1 and Chic2 are involved in ubiquitination of Csf2rb. To test ubiquitination of endogenous Csf2rb, we employed Tandem Ubiquitin Binding Entity (TUBE) agarose beads, which selectively enrich ubiquitinated proteins. Western blot analysis of TUBE-enriched protein lysates showed that Stub1 or Chic2 KO led to reduced ubiquitination of Csf2rb (A). Therefore, Chic2 and Stub1 interact with Csf2rb, and while CHIC2 is not required for this interaction, both Chic2 and Stub1 KO reduce Csf2rb ubiquitination.Given the effects of f Csf2rb A. TherefStub1 or Chic2 KO 32D cells with a lysosomal acidification inhibitor, Bafilomycin A1. KO of Stub1 or Chic2 blocked the effect of Bafilomycin A1 on Csf2rb stability (B). Taken together, our data suggests that Stub1/Chic2 ubiquitinates Csf2rb, leading to lysosomal degradation.Given that lysosomal acidification inhibitors increased Csf2rb protein stability, we hypothesized that Stub1 and Chic2 may regulate Csf2rb through lysosomal degradation. To test this, we treated WT and tability B. Taken Stub1 and Chic2 KO lead to increased total and cell surface protein levels of Csf2rb at low cytokine concentrations, and these effects were limited only to the β chain and not the cytokine-specific α chain (Il3ra). Furthermore, we found that Stub1 interacts with Csf2rb at low cytokine concentrations and that Stub1 and Chic2 KO reduce ubiquitination of Csf2rb. Finally, Stub1 and Chic2 KO abrogated the effect of lysosomal acidification inhibitors on Csf2rb protein stability, suggesting that ubiquitination by Stub1/Chic2 likely leads to lysosomal degradation. These data are consistent with a model in which the STUB1/CHIC2 complex ubiquitinates CSF2RB leading to lysosomal degradation to prevent cell surface accumulation in the absence of cytokine.In this study, we used genetics screens, analysis of the Cancer Dependency Map, and IP/MS to discover a mechanism of CSF2RB regulation by a STUB1/CHIC2 complex. We found that STUB1 has been previously found to regulate diverse classes of receptors, both membrane bound and otherwise, including the glucocorticoid receptor, mineralocorticoid receptor, androgen receptor, estrogen receptor, epidermal growth factor receptor (EGFR and ERBB2), MET receptor, insulin receptor, toll-like receptor 4, and the IL-4 receptor , 30, 31.CHIC2-ETV6 fusions have been found in rare cases of AML , TF1 (ATCC CRL-2003), and THP1 (ATCC TIB-202) cells were maintained in in RPMI (Corning) with 10% fetal bovine serum (FBS) (Sigma) and 1x penicillin–streptomycin–glutamine supplement (Thermo Fisher Scientific). 32D medium was supplemented with 5 ng/ml recombinant murine IL-3 , and TF1 medium was supplemented with 5 ng/ml recombinant human GM-CSF . HEK293T cells (ATCC CRL-3216) for lentiviral production were maintained in Dulbecco's modified Eagle's medium (Corning) with 10% FBS. All cell lines were maintained at 37 °C and 5% CO2.STUB1 (sgStub1-1/2 and sgSTUB1-1/2) and CHIC2 (sgChic2-1/2 and sgCHIC2-1/2) or a nontargeting sgRNA (sgNT) were cloned into sgRNA expression vectors with fluorophores, U6.sgRNA.SFFV.TagBFP, TagRFP, or RFP657, as per the Zhang lab protocol  and CHIC2  complementary DNAs (cDNAs) were obtained from the Broad Institute’s Genomic Perturbation Platform in pDONR223. CSF2RB cDNA was obtained from Dharmacon Inc  in pDONR223. Murine Csf2rb cloned into the Artichoke backbone  was synthesized by Twist Biosciences. STUB1 and CHIC2 cDNAs were mutagenized with site-directed mutagenesis  in the pDONR223 plasmid. Oligonucleotides used for STUB1/CHIC2 cDNA mutagenesis are listed later. Lentiviral expression constructs for modified STUB1 and CHIC2 were generated via Gateway LR Clonase  reaction between the pDONR223 plasmid and lentiviral destination plasmid pRRL-SFFV-IRES-GFP or -dTomato . CSF2RB in pDONR223 was likewise cloned into a gateway reporter plasmid derived from the Artichoke plasmid . Cas9 was expressed using pXR101-Cas9  or pXPR311-Cas9  . All constructs were confirmed by Sanger sequencing (Eton Biosciences).Oligonucleotides (synthesized by Eton Bioscience) used for generating sgRNA constructs are listed in the table later. sgRNA targeting mouse and human protocol . sgRNA osgChic2-1 (Mouse): ATCTTCCTGTTAATGTGCGGsgChic2-2 (Mouse): GCACGTTAGGGTGCAGTATGsgStub1-1 (mouse): GGAGATGGAGAGTTATGATGsgStub1-2 (mouse): CGTGGGCCGCAAGTACCCGGsgNT (nontargeting): ACGGAGGCTAAGCGTCGCAAAdd V5 tag + stop codon to STUB1 (adds V5 tag = GKPIPNPLLGLDST and stop = TGA)STUB1_add_V5_STOP-F: gctgggcctggatagcacctgaTGCCCAACTTTCTTGTACAAAGDelete the TPR domain of STUB1 (deletes AA's 26-127)STUB1_add_V5_STOP-R: agcgggttcggaatcggtttgccGTAGTCCTCCACCCAGCCSTUB1_del_TPR-F: CGGCTGAACTTCGGGGACAdd stop codon to CHIC2 (adds stop codon = TGA)STUB1_del_TPR-R: GCTCGGGCTCTTCTCGGGCHIC2_add_STOP-F: tgaTGCCCAACTTTCTTGTACAdd N-terminal V5 tag to CHIC2 (adds V5 tag = GKPIPNPLLGLDST)CHIC2_add_STOP-R: ATCTGGTCGAAAAATCGGCHIC2_add_Nterm-V5-F: ctgctgggcctggatagcaccGCGGATTTCGACGAAATCDelete the last 5 aa's of CHIC2 (IFRPD) and add stop codon (deletes AA's 161-165)CHIC2_add_Nterm-V5-R: cgggttcggaatcggtttgccCATGCCAACTTTTTTGTACCHIC2_del_IFRPD-F: aTGCCCAACTTTCTTGTACCHIC2_del_IFRPD-R: caCGGTGTCTTTGGTAAAAATTC2 plate by transient transfection of ∼70% confluent HEK293T cells in 10 ml of media using 63 μl TransIT-LT1  in 600 μl Opti-MEM reduced serum medium  containing 10.5 μg lentiviral expression plasmid, 14 μg psPAX2 packaging plasmid , and 5.25 μg pCMV-VSV-G envelope plasmid . Lentiviral supernatant was harvested at 48 h post-transfection and passed through a 0.45 μm syringe filter .Lentivirus was produced in a 10 cm6 cells in 2 ml of appropriate culture media with 4 μg/ml polybrene  were infected in 6-well plates with 400 μl to 1 ml of lentiviral supernatant. Plates were centrifuged at 1050g at 37 °C in an Eppendorf 5910R centrifuge. Cells were then cultured overnight and washed 3x in PBS and then cultured as normal. Cells were transduced with pXR101-Cas9 (THP1) or pXPR311-Cas9 (TF1 and 32D) for stable expression of Cas9 and selected with blasticidin   for 7 days starting the day after transduction. Cells transduced with the fluorescent reporter plasmid were selected with 2 μg/ml puromycin  for 3 days starting the day after transduction. Cells transduced with lentiviral constructs containing fluorescent proteins (sgRNA expression vectors and pRRL-SFFV expression vectors) were sorted using a Sony SH800S or MA900 cell sorter.For lentiviral transduction, 1-2 × 10Relevant cells were treated with 1 μM MLN7243 , 5 μM MLN4924 , 10 μM MG132 , 10 μM Chloroquine , 100 nM Bafilomycin A1 , 1 μM Apoptozole , 100 nM HSP990 , or dimethyl sulfoxide  for the reported amount of time.4 events. FlowJo software  was used to analyze the data. The data were gated on the mCherry+ cell population to ensure all cells analyzed contained the reporter, and the mean GFP and mCherry signals (mean fluorescence intensity [MFI]) was measured. The reported GFP/mCherry ratio was calculated as the (MFI of GFP)/(MFI of mCherry) and then normalized to the relevant condition.Cells were cultured in relevant conditions in 96-well flat bottom plates. Cells were mixed by pipetting, and a CytoFLEX S flow cytometer (Beckman) was used to directly measure the GFP and mCherry signal from 105 cells were sorted from the top 5% GFP/mCherry high population and the bottom 5% GFP/mCherry low population. Sorted cells were pelleted and frozen at −20 °C prior to genomic DNA (gDNA) isolation.TF1 Cas9 or THP1 Cas9 cells were infected with the custom, ubiquitin ligase–specific sgRNA library cloned into the lentiGuide-puro plasmid  . Cells w6 cells were sorted from the top 10% GFP/mCherry high population and the bottom 10% GFP/mCherry low population in quadruplicate. Sorted cells were pelleted and frozen at −20 °C prior to gDNA isolation.For the whole genome screen, 32D Cas9 cells were infected with the Brie whole genome, murine sgRNA library (Addgene #73632). Infected cells were cultured in 0.1 ng/ml IL-3 RPMI + 10% FBS for 6 days and passaged to maintain library representation at 500x. On the sixth day, cells were washed and cultured in 0.01 ng/ml IL-3 RPMI + 10% FBS overnight prior to sorting. After the appropriate treatments, cells were harvested and put on ice for sorting. Cells were sorted using a Sony SH800S or SH900 cell sorter. Approximately 2 × 102, 3 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10 mM Tris pH 7.5, 0.2 mg/ml Proteinase K) with incubation for 10 min at 65 °C and 15 min at 95 °C. sgRNA sequences were amplified and barcoded from the gDNA and next generation sequencing library was prepared by a two-step PCR. The sgRNA sequence was amplified in a first PCR reaction with eight staggered forward primers. Twenty microliters of direct lysed cells was mixed with 0.04 U Titanium Taq (Takara Bio 639210), 0.5x Titanium Taq buffer, 800 μM dNTP mix, 200 nM SBS3-Stagger-pXPR003 forward primer, 200 nM SBS12-pXPR003 reverse primer in a 50 μl reaction , 2 min at 72 °C). Two microliters of the first PCR reaction was used as the template for 15 cycles of the second PCR, where Illumina adapters and barcodes were added . An equal amount of all samples was pooled and subjected to preparative agarose electrophoresis followed by gel purification using a QIAquick gel extraction kit . Eluted DNA was further purified by NaOAc and isopropanol precipitation. Amplified sgRNAs were then sequenced using an Illumina NextSeq.gDNA from sorted cells was isolated using a direct lysis buffer . R statistical software  was used to calculate the Pearson correlation coefficient and p-value for correlations between the CERES scores for STUB1 and the CERES scores for every other gene.DepMap Public 21Q4 data containing the CERES scores for all genes across 1054 cancer cell lines was downloaded from the DepMap portal , and the ΔΔCt method was used to calculate the relative expression.Total RNA was isolated from samples with a RNeasy mini kit . RNA concentration was measured using a NanoDrop 8000 (ThermoScientific), and SuperScript IV Vilo master mix  was used to generate cDNA from 1 μg of RNA. Multiplexed real-time quantitative PCR (RT-qPCR) was conducted with 250 ng of cDNA, TaqMan gene expression master mix , and TaqMan primers from ThermoFisher Scientific, noted later. Four technical replicates and three biological replicates were analyzed for each condition in 20 μl reactions in a 384-well plate. RT-qPCR was executed with an Applied Biosciences QuantStudio 6. C5 cells per sgRNA per well, 2 × 105 total cells per well) in a 96-well plate. A Tecan D300e digital dispenser was used to dispense the appropriate amount of 5 μg/ml recombinant mouse IL-3 in dH2O + 0.1% Triton X-100 as per the manufacturer’s instructions. Each condition was done in triplicate. On the second day (day 1) and every following 2 to 3 days, cells were passaged 1:10 days into fresh media, and the remaining percentage of BFP+ and RFP+ cells was measured by flow cytometry on a CytoFLEX S flow cytometer (Beckman).32D Cas9 cells expressing relevant sgRNA with BFP or RFP were mixed at 1:1 ratio  was again used to analyze the data and calculate the median fluorescence intensity of the PE signal.Approximately 2 × 10https://imagej.nih.gov/ij/).Cells were lysed in a NP40-based lysis buffer ) for 15 min on ice. Protein lysates were precleared by centrifuging at 14,800 RPM for 10 min at 4 °C. Protein lysates were harvested, and protein concentration was measured and normalized using the Pierce bicinchoninic acid (BCA) protein assay kit . Samples were prepared in NuPAGE LDS sample buffer  and NuPAGE sample reducing agent  and then boiled at 70 °C for 10 min. Samples were then resolved by SDS-PAGE using NuPAGE 4% to 12% Bis-Tris protein gels , and transferred by electrophoresis at 90 to 100V for 2 h on to 0.45 μm nitrocellulose membranes . Membranes were blocked in Odyssey blocking buffer  for 1 h at room temperature (RT). Membranes were incubated in primary antibodies, detailed below, overnight at 4 °C in Odyssey blocking buffer. Membranes were washed in 1x Tris-buffered saline with Tween-20  3x for 5 min at RT and then incubated in secondary antibodies, detailed later, for 1 h at RT in Odyssey blocking buffer. Membranes were again washed 3x for 5 min at RT in 1x Tris-buffered saline with Tween-20 and then visualized using a Li-Cor Odyssey CLx. Western blots were quantified at relevant exposures using ImageJ  overnight at 4 °C. For TUBE immunoprecipitation, 2 mg of total protein were incubated with 20 μl of 50% control  or TUBE1 agarose beads . Following incubation, beads were separated by magnet and washed 1× with lysis buffer and 2× with wash buffer . Samples were eluted from magnetic beads by boiling in 1x LDS NuPAGE sample buffer with 1x NuPAGE reducing agent at 70 °C for 10 min. Samples were analyzed by immunoblot as described before.For IP/MS experiments, IPs were performed as before, and following washing steps with lysis/wash buffer, samples were prepared for MS. Immunoprecipitation eluates were reduced with 10 mM tris(2-carboxyethyl)phosphine for 30 min at RT and then alkylated with 15 mM iodoacetamide for 45 min at RT in the dark. Alkylation was quenched by the addition of 10 mM DTT. Proteins were isolated by methanol-chloroform precipitation. The protein pellets were dried and then resuspended in 50 μL 200 mM EPPS pH 8.0. The resuspended protein samples were digested with 2 μg LysC overnight at RT followed by the addition of 0.5 μg Trypsin for 6 h at 37 °C. Protein digests were dried, resuspended in 100 μL 1% formic acid, and desalted using 10-layer C18 Stage-tips before being analyzed by LC-MS.Data were collected using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a FAIMS Pro Interface and coupled with a UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a 75 μm inner diameter microcapillary column (Thermo Fisher Scientific). Peptides were separated using a 180 min gradient of 10% to 25% acetonitrile in 1.0% formic acid with a flow rate of 350 nl/min.m/z 380 to 1200, resolution 60,000, AGC target 3 × 106, auto maximum injection time, dynamic exclusion of 30 s, and charge states of 2 to 6. Top N 40 data-dependent MS2 spectra were acquired with a scan range starting at m/z 110, resolution 15,000, isolation window of 1.6 m/z, normalized collision energy set at 30%, AGC target 1 x 105 and the automatic maximum injection time.Each analysis used a TopN data-dependent method. The FAIMS Pro Interface compensation voltages were set to -50 and -70. The data were acquired using a mass range of Proteome Discoverer 2.4 (Thermo Fisher Scientific) was used for .RAW file processing and controlling peptide and protein level false discovery rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against a Uniprot human database (December 2019) with both the forward and reverse sequences as well as known contaminants such as human keratins. Database search criteria were as follows: tryptic with two missed cleavages, a precursor mass tolerance of 20 ppm, fragment ion mass tolerance of 0.6 Da, static alkylation of cysteine (57.02146 Da), and variable oxidation of methionine (15.99491 Da). Peptides were quantified using the MS1 Area, and peptide abundance values were summed to yield the protein abundance values.t test as implemented in the limma package within the R framework , and relevant statistical analyses are described in the corresponding figure legends. All relevant data are included within this article and supporting information. Plasmids will be made available upon reasonable requests.This article has the following The authors declare that they have no conflicts of interest with the contents of this article.


A novel molecularassembly of a cobalt–sulfate coordinationpolymer and melamine is synthesized under acidic conditions. Bar-shapedpink monocrystals as long as 1 mm are found to align along magneticfield lines in the proximity of a strong magnet. Magnetometry showsno hysteresis at temperatures down to 2 K but instead magnetic anisotropyand antiferromagnetic coupling. X-ray diffraction on a single crystalreveals that the cobalt–sulfate chains are along the shortestlattice vector or the crystal’s long axis. The crystal alignmentalong the magnetic flux can be attributed to single-ion anisotropythat results in longitudinal antiferromagnetic coupling along thechain. Both structurally and magnetically isotropic crystals of metal–organichybrid materials can be highly useful as elemental components in magneto-optics. Coordination polymersas molecular magnets can exhibit various magnetic properties due todirectional magnetic coupling among magnetic metals through low-dimensionallyarranged molecular units. They are vital for understanding of thefundamentals of magnetic coupling and the magneto–structurecorrelation. In particular, those with higher critical temperaturescould be useful in high-density magnetic information storage and quantumcomputation.10 while the use of melamine is not very common in coordinationchemistry because of the low solubility in organic solvents.11 Synthesis parameters are optimized to yieldmillimeter-long single crystals. X-ray diffraction reveals layersof cobalt–sulfate chains packed with stacks of melamine byhydrogen bonding. Crystals align along the magnetic flux at room temperaturelike compass needles, making this polymer a good candidate for a molecularmagnet. Magnetometry reveals no hysteresis but instead antiferromagneticcoupling at low temperatures and magnetic anisotropy. The weak butdirectional response of bar-shaped crystals to the external magneticfield can be attributed to the single-ion anisotropy that resultsin longitudinal antiferromagnetic coupling along the chain.In this contribution, a novel molecular frameworkof a cobalt–sulfatecoordination polymer and melamine is synthesized. Melamine is a richsource of hydrogen bonds. Its molecular symmetry and skeleton rigiditysee 1A areus4·7H2O and an acetic acid solutionof melamine, followed by heating the mixture in a sealed glass vial.Unlessotherwise noted, all crystals (referredto as CoS-M) have been synthesized by mixing an aqueous solution ofCoSO4·7H2O and melamine at 70 °C,which ensures the solubility of melamine in acetic acid. In each case,1 mL of the aqueous solution of CoSO4·7H2O was placed in a 10 mL glass vial and then 3 mL of the acetic acidsolution of melamine was added. The concentrations of CoSO4·7H2O in water tested are ρCo =0.025 and 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, and 0.8 mol/L, and the concentrationsof melamine in acetic acid are ρMelamine = 0.025,0.05, 0.1, and 0.2 mol/L. Purple and pink denote the best and marginalcrystal quality, respectively, and white denotes no crystal formation.At ρCo = 0.8 mol/L, no crystals were formed. Thelargest crystals, approximately 500 μm long, as in Co, ρMelamine) = . When the sample is under the same conditions but pressurizedin an autoclave (1.38 bar), the crystal length reaches around 1000μm (see the Supporting Information (SI) for more details on the hydrothermal synthesis).The table in 12 At the lowconcentrations, the mobility is high but nucleation does not occur.13Thehigher concentrations hinder the formation of the crystals,possibly due to the increased ionic strength of both cations Co(II)and anions SI-1). As a result, no crystalsare formed. This indicates that melamine plays a vital role in theformation of the crystal, ensuring its presence in the solid structure.At 90 °C, marginal quality crystals grow only at (1:1) and (1:3).A high temperature promotes the diffusion of reactants and consequentlythe nucleation. On the other hand, it hinders relaxation steps requiredfor crystal formation.15 Therefore, temperatures higherthan 70 °C are not optimal for the growth of CoS-M crystals.The best-quality crystals grow at 70°C and (1:3). At lowertemperatures, melamine does not dissolve, as concluded in the solubilitytests (see 4·7H2O) does not fullydissolve.As for the influence of the volume ratio, (water/acetic acid) =(1:3) is optimal. In the solvent with excess water (or acetic acid),melamine .10 The crystal is composedof cobalt–sulfate chains along the a-axisin which adjacent cobalt cations are bridged by two HSO4–1 anionsthrough Co–O bonds. Two H2O molecules are also coordinatedto the cobalt by their oxygen through Co–O bonds (2.08 Å).Each cobalt ion lies in an octahedral coordination geometry. The cobalt–sulfatechains lying in the (001) plane establish N–H···Obonds with melamine molecules, which are arranged in a zigzag fashionapproximately within the (1 1 2) plane or facing the [2 2 1] direction.Importantly, the direction of the Co–S chains (axis a) was found to be along the long axis of the crystal.Single-crystal X-ray diffractionreveals that CoS-M is a coordination polymer that consists of cobaltcations, sulfate anions, water and melamine, as shown in The crystal’s orientations are susceptibleto an external magnetic field. When exposed to strong magnetic fieldsof a neodymium magnet, the long axis of the crystal becomes alignedalong the field direction, as shown in 7. The ligand field strength determines the splittingbetween the t2g and eg orbitals in an octahedralgeometry. In a weak ligand field, the configuration is t2g5 e2, resulting in a high spin state with total spin S = 3/2. The ground-state term of this quartet state is 4T1g. In a strong ligand field, the configuration is t2g6 e1, i.e., a low-spin state with total spin S = 1/2.The ground-state term of this singlet state is 2Eg.16The electronic configuration of divalent cobaltion, Co(II), is[Ar] 3dh is hard to realize, and inmost cases the octahedron is axiallydistorted to a tetragonal Dh4 geometry.As shown in 2g level,namely, the dxz and dyz orbitals move to a lower energy and the dxy orbital moves to a higher energy. The eg levelsplits into low-energy 4A2g and 4Eg, respectively, and are split by energy Δ.The 4A2g state further splits into two Kramersdoublets, ms = ±1/2and ±3/2, as a result of spin–orbit coupling, with thezero-field splitting (ZFS) energy denoted by D in ms = ±1/2 for D > 0 and ms = ±3/2for D < 0.A perfect octahedral coordinationgeometry OSI for the χT plot and the AC susceptibility.C/(T – θ)where C is the Curie constant and θ the Weisstemperature. Using the inverse Curie–Weiss relation, the curveof best fit (the cyan curve in C = 3.45 cm3 mol–1 K) and θ = −9.53 K. The Curie constant is in agreementwith the values for high-spin Co(II) in an octahedral environment(C = 2.8–3.4 cm3 mol–1 K) reported in literature.27 The corresponding effective magnetic moment is μeff = (8C)1/2 = 5.25. The negative Weisstemperature suggests an antiferromagnetic coupling, likely betweenthe nearest neighbor spins of the CoS chains  D in 4 into ac∥) and that in the perpendicular direction(χ⊥) are derived as41N is the Avogadro constant, k isthe the Boltzmann constant, and T istemperature.The magnetic susceptibility in the parallel direction(χD = 5.2 ± 0.49 meV (= 42.1 ± 3.95 cm–1), and gave. = g∥ = g⊥ = 2.399 ± 0.006. The best fit for χ′ (cyan curvein D = 0.97 ±0.76 meV (= 7.8 ± 6.12 cm–1) and gave. = g∥ = g⊥ = 2.405 ± 0.007.The zero-field splitting energy evaluated from χ′ isapproximately five-times smaller than that evaluated from χ.As the Hamiltonian in The averaged magnetic susceptibility is given asD reported forcobalt complexes are in the range from −38 to +73 cm–1 when the coordination numbers are 5 and 4 and from +25 to +83 cm–1 when the coordination number is 6.43D = 0.97 ± 0.76 meV (= 7.8 ± 6.12 cm–1) obtained here from the χ′ is very small,approximately the thermal energy at 1 K. This means that in the temperaturerange of 100–300 K the zero-field splitting contributes toonly a minor or no deviation from the S = 3/2 ground state.Thezero-field splitting energies B/Co) ofthe Brillouin function for S = 1/2, supporting the S = 3/2 ground state at 2 K. The magnetization curve deviatessignificantly from the Brillouin function for paramagnetism and exhibitsa sigmoidal shape. See the SI for the magnetizationisotherms of roughly aligned crystals measured in fields up to 9 Tboth parallel and normal to the dominant crystal orientation.B at 7 T and seems to coincide withthe saturation level (3.0 μB/Co) for S = 3/2 in higherfields. The Zeeman energy in weakmagnetic fields is proportional to ±(1/2)gμBB for mS = ±1/2 and ±(3/2)gμBB for mS = ±3/2.The magnetic moment reaches avalue of 2.17 μD = 0.97 ± 0.76 meV may occur in a magneticfield of ≥17 T, which is much higher than the measured magneticfield range. Additionally, this small zero-field splitting is visibleonly at temperatures below 1 K. Furthermore, the magnetization curveat 2 K is smaller than the Brillouin function for S = 1/2 in fields below 4.5 T. Hence, the sigmoidal magnetizationcurve can be attributed to the antiferromagnetic coupling of 3/2 spinsin the Co–S chain.The level crossing of the groundand first excited level separatedby As shown in xy, xy orbital lies off the ligand.In 3 hybridorbitals of sulfur in HSO4 have no favored exchange andshould accommodate antiferromagnetically coupled spins in order torealize the antiferromagetic coupling of the adjacent cobalt ions.Hence, the antiferromagnetic coupling between cobalt atoms can beattributed to more elaborate interactions through super-exchange viaHSO4, whose sign depends largely on the bonding environmentbut has been reported to be antiferromagnetic in many metal sulfatesand related materials.Now, we discuss mechanisms forthe antiferromagnetic coupling.As shown in Finally, the origin of the longitudinalspin orientation couldbe the single-ion anisotropy, anisotropic diamagnetism due to themolecular orientation, or the demagnetizing field due to the crystal’sanisotropic shape. All melamine molecules face the [2 2 1] direction.Their aromatic rings exhibit anisotropic diamagnetism, which is thegreatest when the rings face the field direction. The resulting demagnetizingfield should favor the [2 2 1] direction along the field, but thecrystals are aligned so as to make the [1 0 0] direction parallelto the field direction. In turn, the demagnetizing field for a typicalcrystal shape, e.g., 625 × 50 × 50 μm as in A cobalt–sulfate coordination polymerand melamine are assembledto form millimeter-long magnetic monocrystals. X-ray diffraction ona single crystal reveals that the cobalt–sulfate chains arealong the crystal’s long axis. Magnetometry evidences the longitudinalantiferromagnetic coupling of cobalt spins. As a result of the single-ionmagnetic anisotropy, these magnetic crystals are aligned along magneticfield lines, which can be highly useful in magneto-optics.


Garcinia mangostana, “Queen of Fruit” of Thailand, is known as a rich source of xanthones with potent cytotoxic properties against various cancer cells. Study on xanthones is provoking not only due to the structural diversity but also a wide variety of pharmacological activities. Hence the aim of the current study is to determine the effects of metabolites from G. mangostana root on cell proliferation and migration of hepatocellular carcinoma cells. Twenty-two metabolites, including two new benzophenones and one new biphenyl, were isolated and characterized. Five xanthones with a prenyl moiety showed significant cytotoxicity against both HCC cells tested; however, only dulxanthone D displayed the most promising activity on the migration of Huh7 HCC cells, comparable to sorafenib, a standard drug. Moreover, the compound dose-dependently induced apoptosis in Huh7 cells via mitochondrial pathway. Accordingly, dulxanthone D held a great potential for development as a novel migration inhibitor for effective HCC treatment.Liver cancer refers primarily to hepatocellular carcinoma (HCC) accounting for over 90% of cases and is the highest incidence in men in Thailand. Over the past decades, the incidence of HCC dramatically increased with a strong rise of mortality rates.  Interestingly, carcinogenic infection from human papillomavirus (HPV), hepatitis B and C virus could lead approximately 25–40% to HCC even though the mechanism underlying progression still remains unclear. According to Thailand-based Cancer Registry reported, in 2015, liver cancer accounted for 15,912 patients out of 20,617 new cancer cases to deaths. Moreover, it occurs more often in men than women. Despite metastatic liver cancer being the most frequently seen type, for instance, spreading from breasts or colons, hepatocellular carcinoma (HCC) is also the most common primary cancer account for 80% of all primary liver cancers3. This is due to the typically late diagnosis with a median survival following diagnosis of approximately 6–20 months4. Currently, general drugs approved by the Food and Drug Administration (FDA) for liver cancer are extensively depends on patient’s symptoms. For examples, sorafenib tosylate, a kinase inhibitor drug is used with the HCC that cannot be removed by surgery6, and Bevacizumab, an angiogenesis inhibitor is given to patients who have not received systemic therapy or in metastatic stage7.Hepatocellular carcinoma (HCC) is number one deadliest disease in Thailand from the report of Ministry of Public Health. The risk factors for cancer are various, it could be from environment, life routine and human genetic8. For explanation, metastasis occurs when the primary cancer travels from its original place through the bloodstream and lymph node, then forms a secondary or metastatic tumor in the nearest organs. More importantly, HCC has a high recurrent rate of intrahepatic metastatic spread as well as extrahepatic metastasis to the colon10. Thus, this could estimate lessen of survival time in patient with around 11–13 months in nearly last stage. Nowadays, there are many treatments use to prevent cancer, such as immunotherapy, radiation therapy, or targeted therapy11. However, among various treatments, chemotherapy is the most often use to treat cancers. In addition, more than 50% of chemotherapy drugs are produced from natural compounds. This is due to a fewer toxicity and side-effects against normal cells12.In addition, liver cancer has been grouping into five stages to prevent medical intervention harms in patients with cirrhosis. Liver cancer metastasis appears in fourth of five stages classify as advanced stage, indicating that the cancer could spread from origin to other organsGarcinia mangostana L.), a large perennial plant with lushes of leathery thick leaves, grown well and widely in tropical weather countries specially in Southeast Asia. It was known as ‘Queen of fruits’ due to the unique sweet-but-sour taste and the round, dark purplish appearance with thick green sepals arranged like a crown13. Additionally, not only a flavor that make mangosteen becomes well known but also because of its variety in medicinal applications. In many countries including Thailand, mangosteen has been used as a traditional medicine for recent past. Various parts from the mangosteen plant can be used in numerous ways. For instance, extracts of ripening fruits were used as diarrhea treatment whereas stem barks were used as wound infection treatment14. Nowadays, there are prevalent studies about metabolite compositions from mangosteen. The most common classes found are xanthones with α- and β-mangostin as major components. These compounds have been reported with many beneficial bioactivities such as anti-oxidation, anti-inflammatory, antimicrobial and anticancer15. Mangosteen also contains other class compositions, for examples, flavonoids, anthocyanins and benzophenones16.Mangosteen , and one new found in naturally occurring, 4,5-dimethoxy-3-ol (20), along with eighteen known compounds: isojacareubin (1), α-mangostin (2), β-mangostin (3), γ-mangostin (4), mangostanaxanthone IV (5), dulxanthone D (6), toxyloxanthone B (7), 1,7-dihydroxy-3-methoxy-2-prenylxanthone (8), euxanthone (9), norathyriol (10), 8-deoxygartanin (11), maclurin (14), 2,3′,4,6-tetrahydroxybenzophenone (15), mangaphenone (16), (3-hydroxy-4-methoxyphenyl)methanone (17), garciosine A (19), 3-hydroxy-4-geranyl-5-methoxybiphenyl (21) and epicatechin (22) as shown in Fig. 12, 13 and 18 were elucidated by analysis of spectroscopic data, while the known metabolites were identified by comparison of their NMR data with those reported in the literature36.Chemical investigation of EtOAc crude extract of mangosteen roots afforded two new benzophenones and one new biphenyl, namely mangostanones I–III (12) was obtained as a dark-yellow solid. HRESIMS analysis showed a sodium adduct ion peak at m/z 313.0690 [M+Na]+ . The 1H and 13C NMR spectra of compound 12  and 6.15 ], a 1,3,5-trisubstituted benzene ring [δH 6.60  and 6.51 ], and two methoxy groups at δH 3.88  and 3.61 . The 13C NMR spectrum assigned with the help of the HSQC data exhibited 15 signals due to two methyls, four sp2 methines, and six non-protonated carbons representing one carbonyl (δC 202.4) and five oxygenated carbons. The aromatic protons at δH 6.60 and 6.13 were correlated with the carbonyl group at δC 202.4 in the HMBC spectrum  to C-4 (δC 166.2), H-3 to C-1, C-5, H-5 to C-1, C-3, and 6-OMe (δH 3.59) to C-6 (δC 162.5) confirmed the location of two methoxy units at C-3 and C-5 on ring A. From the above data, compound 12 was proposed structurally as shown.Mangostanone I 2 was obt13) was obtained as a brown-yellow solid with the molecular formula of C15H14O6 determined by HRESIMS at m/z 299.0537 [M+Na]+ . Indeed, NMR spectroscopic data of 13 were similar to those of mangostanone I (12) , 7.09 , and 6.85 ], while 12 had a 1,3,5-trisubstituted benzene on ring B. In addition, compound 12 had two methoxy groups on ring A, while compound 13 showed one methoxy at C-4. The location of the methoxy group at C-4 was indicated by the HMBC correlations of 4-OMe (δH 3.58) to C-4 (δC 161.4).Mangostanone II 3 was obt18) was obtained as a white powder. The molecular formula was C14H14O4 as determined by HRESIMS at m/z 269.0799 [M+Na]+ . The IR spectrum of 18 showed absorption bands at 3465 cm−1 for free hydroxyl group, 3001 cm−1 for aromatic C–H stretch, and 1596 and 1576 cm-1 for C=C stretch. The UV spectrum showed absorption bands at λmax at 221 and 267 nm which are typical of a hydroxygenated benzene derivative33. The 1H and 13C NMR spectroscopic data  and 6.63 . The remaining resonances at δH 7.42  and 6.87  suggested a 1,4-disubstituted benzene unit on ring B. The correlation of OH-3  to C-2 (δC 106.7), C-3 (δC 149.5), and C-4 (δC 134.9) in the HMBC spectrum  and OCH3-5 to C-5.Mangostanone III 8 was obt1–6 and 11 were considerable active on both cell lines tested with the IC50 value less than 40 μM, the compounds, except for β-(3) and γ-mangostin (4), were thus subjected to further anti-migration assay. This is because α-mangostin (2) with highly isolated amounts was thought to be a representative molecule of the compound series, as well as the effect of compound 3 on HCC cells has been reported37.To investigate anticancer properties of these compounds, preliminary screening of all isolated compounds was observed by their cytotoxic ability towards two hepatocellular carcinomas HepG2 and Huh7) using MTT method. Sorafenib and doxorubicin were used as positive drug controls. The results , (IC50*2), and 3 μM following the IC50 value of sorafenib described in previous study. Sorafenib is a standard drug approved to treat many cancer types such as hepatocellular carcinoma, renal cell carcinoma, and thyroid cancer38. Because of this, 3 μM were used to compare between tested compounds and standard drug in the same concentration. As a result, compound 6 showed fascinating results in inhibited cells migration. There were no increased of tissue repair percentage in all conditions, including at the same dose of sorafenib (3 μM) since 24 h ). Untreated cells were used as control. The Annexin V/phosphatidylserine (PS) double stain was performed to study apoptotic cells and determined by Muse® Cell Analyzer. The results indicated that compound 6 was able to induce Huh7 apoptosis in dose-dependent manner, and it could be seen clearly when treated the cells with a 24 μM concentration , as well as one new naturally occurring, 4,5-dimethoxy-3-ol (20). The structures of the new compounds were characterized by analysis of the spectroscopic data, particularly 1D and 2D NMR and MS data. Inspired by previous studies, the cytotoxic abilities of all isolated compounds towards two hepatocellular carcinomas (HepG2 and Huh7) were evaluated using MTT assay. Sorafenib and doxorubicin were used as positive drug controls. The present study revealed that compounds 1˗6 and 11 were significantly active against both investigated cell lines with the IC50 values less than 40 μM. As expected, only xanthone displayed cytotoxic effect on the cancer cells tested, whereas most benzophenones and biphenyls (12–21) were inactive and/or less active. Additionally, results  mutation and represented the advance stage of HCC. We may have missed the data about the effect of compounds on anticancer activity in HepG2 cell line, which occurred TP53 wildtype and might represent the intermediate state of cancer40. To assess the cell migration inhibitory activity of the selected xanthones 1, 2, 5, 6 and 11 on HCC cells, monolayer Huh7 cells were treated with the compounds at various concentrations under and above the concentration providing 50% proliferative inhibition, compared to sorafenib (3 μM). As demonstrated in Fig. 6) after 24 h treatment in all conditions, including at 3 μM, the same dose of sorafenib, by the gap of injured cells being no different from the 0 h treatment. Furthermore, it was found that treatment of the compound at higher concentration or longer than 24 h did not cause seriously cell damage.Cell metastasis is one of cancer cells process occurring when primary tumor spread through lymphatic and circulatory system to the nearest organs, as well as is reported to be responsible for over 90% of cancer deaths. Cell migration (motility) is a critical cause of tissue invasion supporting primary tumors to disseminate and metastasize41. The ERK pathway represents an attractive target for anticancer drug development. In the current study, since dulxanthone D 6 could suppress proliferation and migration of HCC cells, the effect on phophor-ERK1/2 expression was determined, compared to that of the level of ERK1/2, and 6 exerted the reduction in phophor-ERK1/2 expression as expected. This explained dulxanthone D 6 might target the components of the ERK pathway.The extracellular signal-regulated kinase (ERK) signaling pathway is dominantly associated in diverse cellular processes including proliferation, survival, differentiation and motility. In addition to often up-regulation in human cancers, its multiple roles in a complex malignant lead to expect the specific blockade of the pathway results in both anti-proliferative and anti-metastatic effects in cancer cells42. Normally, there are two main pathways of apoptosis including intrinsic and extrinsic pathway. In this study, assessment of apoptosis was performed to analyze whether dulxanthone D 6 was able to induce apoptosis in Huh7 cell line. The results revealed 6 was able to induce Huh7 apoptosis in dose-dependent manner, clearly seen at a 24 μM concentration by 29% apoptotic rate , displaying the most promising antimigration, dose-dependently induced apoptosis in Huh7 cells. Its apoptotic activation was proved to be intrinsic pathway, by involving in the decrease in Bcl-2 and Bcl-XL and the increase in Bax expression level. These findings make dulxanthone D 6 an interesting lead in the development of new anti-HCC cancer agent. Therefore, the anti-metastatic potency of 6 and its underlying mechanism in details is under further study.Phytochemical investigation of root extract of mangosteen led to the isolation and identification of twenty-two metabolites including three new compounds. Among of them, prenylated xanthones displayed the most potent cytotoxicity against HCC cells, HepG2 and Huh7. Dulxanthone D (Garcinia mangostana) roots were collected in May 2019 from Nakhon Sri Thammarat province, Thailand, with permission of Mrs. Narumon Choodej, the farm owner. The plant used in this study is not wild. Identification was performed by a staff of Royal Forest Department, Nakhon Si Thammarat Province. A voucher specimen was assigned with the code CUCHEM2019-003 and is deposited at Department of Chemistry, Faculty of Science, Chulalongkorn University. All methods were carried out in accordance with relevant guidelines in the method section.The mangosteen . The extract was chromatographed on SiO2 column using EtOAc:n-hexane (2:8) to give ten fractions (A-J). Fraction H (2.9 g) was separated by Sephadex LH-20 (MeOH) to gain three subfractions (H1-H3). Subfraction H1 was meticulously separated with SiO2 column using a mixture of acetone:n-hexane (3:7) to afford compounds 1 and 10 (3.8 and 31.7 mg respectively). Subfraction H3 was separated SiO2 column with a 4:6 ratio of acetone:n-hexane to yield compound 12 (5.7 mg), together with eight fractions (H3.1-H3.8). Later, subfraction H3.7 was then re-chromatographed with ODS (C-18) column using a mixture of H2O:MeOH (2:8) and provided compounds 13 (32.9 mg) and 14 (10.9 mg). Subfraction H2 was treated similarly to subfraction H1 to obtain compound 16 (13.3 mg) with another subfraction H2.1. The subfraction H2.1 was then subjected to ODS (C-18) column with H2O:MeOH (2:8) as eluent, gave out compound 15 (98.7 mg). Subsequently, Fraction E (2.6 g) was separated by Sephadex LH-20 column with MeOH and give six subfractions (E1-E6) which E3 was crystalized and yielded compound 3 (281 mg). Fraction E5 was further purified by reversed-phase ODS (C-18) column using H2O:MeCN (2:8) mixture to obtain compound 2 (301.6 mg). Following by fraction C (346.9 mg), was subjected to SiO2 column utilizing acetone:n-hexane (1:9) to obtain six subfractions (C1-C6). Subfraction C4 then was subjected to SiO2 column using CH2Cl2:n-hexane gradient from 6:4 to 9:1 and gave compounds 5 and 20 with weight 5.8 and 120.2 mg, respectively. Fraction B was separated by SiO2 column using EtOAc:n-hexane (1:9) mixture to give nine subfractions (B1-B9). Subfraction B4 was picked to separate with Sephadex LH-20 using MeOH and gave out compound 21 (58.8 mg). For fraction D, the chromatography was performed using SiO2 column with CH2Cl2:n-hexane from 7:3 to 9:1 gradient elution to yield fifteen subfractions (D1-D15). Subfractions D4 was subjected to Sephadex LH-20 column (MeOH), followed by SiO2 column with EtOAc:n-hexane (2:8) mixture to yield compound 9 (5.8 mg). Subfractions D6 was separated by SiO2 column with acetone:n-hexane (2:8) mixture to give nine fractions (D6a-D6i). D6d was further purified by to Sephadex LH-20 eluting by MeOH, followed by SiO2 column with a gradient of EtOAc:n-hexane (1:9 to 2:8) to yield compound 11 (7.8 mg). Subfraction D14 was separated by ODS column with a 2:8 mixture of H2O:MeOH (2:8) to give compound 8 (1.3 mg). Furthermore, fraction G was subjected with acetone:n-hexane (2:8) elution on SiO2 column to obtain ten subfractions (G1-G10). Combined G6 and G7 fraction was then purified by ODS (C-18) column with H2O:MeOH (1:9) to yield compound 17 (30.4 mg). Fraction J was eluted over Sephadex LH-20 with MeOH to obtain seven subfractions (J1–J7) and one pure compound . Subfraction J3 was further chromatographed with SiO2 column utilizing acetone:n-hexane (4:6), and, lastly, ODS (C-18) column chromatography of subfraction J3e afforded compound 4 (3.6 mg). Fraction F was subjected to SiO2 column utilizing EtOAc:CH2Cl2 gradient from 0:10 to 1:9 to obtain ten subfractions (F1-F10). Compound 19 (60 mg) was obtained by separating subfraction F7 with SiO2 column using acetone:n-hexane (3:7) together with impure compound 18. The impure compound was further purified by ODS (C-18) column utilizing H2O:MeCN (2:8) to yield pure compound 18 (31.4 mg). Subfraction F10 was isolated by Sephadex LH-20 using MeOH to afford compound 7 (1.9 mg) and another five subfractions (F10a–F10e). Fraction F10b was subjected to ODS (C-18) column eluted with H2O:MeOH (3:7) to provide compound 6 (3.9 mg).The powdered mangosteen roots (2.4 kg) were extracted 3 times with MeOH for 3 days (5 L each per time) at room temperature. After concentrated under reduced pressure, the latter was partitioned between H1H, 13C, and 2D NMR spectroscopic data were obtained from Bruker AV400 spectrometer . Deuterated CDCl3 and acetone-d6 were used as NMR solvent. HRESIMS data was obtained with a Bruker micrOTOF. FT-IR data was obtained from Perkin-Elmer Model 1760X spectrophotometer.2 humidified incubator.Human Hepatocellular Carcinoma (HCC) cell lines, HepG2 (JCRB1054) and Huh7 (JCRB0403) were purchased from Japanese Collection of Research Bioresources (JCRB), National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan. Both were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a 5% CO4 cells/well. Then, the cells were incubated at 37 °C with 5% CO2 for 24 h. After removed the medium supernatant, the cells were treated for 48 h with serial dilution of the tested compounds along with sorafenib and doxorubicin as standard drug control in the presence of serum-free DMEM media. MTT solution (0.5 mg/ml DMEM) was added to each well and then incubated for 4 h in a humidified atmosphere. After the incubation period, the supernatant was removed and DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm using microplate reader. The results were calculated by using GraphPad Prism™ (version 8.0) and presented as the percentage of cells viability and half-maximal inhibitory concentration (IC50).Cytotoxicity activity was performed using MTT assay. Briefly, HCC cells were seeded in 96-well plate with a density of 1 × 105 cells/well. The wound was created by scratching with micropipette tip (1 mm diameter). The medium was removed and replaced with serum-free DMEM containing compound 1, 2, 3, 4, 7, 8, and 21 . Sorafenib (3 μM) was used as a positive control and cells treated only with serum-free medium were used as standard control. The cells were observed and photographed using microscope at 0, 24, and 48 h. The wound healing progression was calculated and presented as percentage of tissue repair (%Tissue repair).0 h: length of gap between scratched cells (μm) at 0 h.Wound healing assay was performed to identify cell migration. Huh7 monolayer cells were prepared in 12-well plate with a density of 5.5 × 10n h: length of gap between scratched cells (μm) at n h.Wound space5 cells/well and incubated for 24 h with 5% CO2 at 37 °C. Cells were treated with compound 6  for 24 h. Then, cells were detached with trypsin and collected to wash with PBS and resuspended in 1 ml of DMEM. Then, Annexin V-7AAD reagent  was added in the cell’s suspension with 1:1 ratio and stained for 30 min at room temperature in the dark. Live, dead, and apoptosis cell populations were determined using Muse® Cell Analyzer. The results were analyzed and calculated as apoptosis profile.Apoptosis was determined by measuring external phosphatidylserine, which located on the outer of cell membrane. Huh7 cells were seeded in 24-well plate with a density of 2.5 × 106 at IC50 concentration and double in value for 24 h with density of 2.5 × 105 cells/well in 24 wells-plate, cells were harvested at time-depended and total protein were collected using ice cold lysis buffer. Protein samples then calculated and loaded in SDS-PAGE to separate using electrophoresis. Further, protein was transferred from gel to polyvinylidene fluoride (PVDF) transmembrane and blocked with 5% skimmed milk. The primary antibodies then added and incubated for 24 h in 4 °C. Membrane was washed with PBST afterward, then secondary antibodies were added and incubated with in optimal times. Membrane was washed again with PBST and performed color developer to visualize in the dark room. The results were photographed and analyze using Image J program.After treated Huh7 with compound p < 0.05 was considered as statistically significance.Data are presented as mean ± SD values. GraphPad prism™ ver. 8 program with one-way analysis of variation was used for testing the significance, Supplementary Information.


We performed QTLs analysis for GL using a recombinant inbred lines (RILs) population derived from the cross between japonica variety GY8 (short grains) and LX1 (long grains) in four environments.Grain length (GL) that is directly associated with appearance quality is a key target of selection in rice breeding. Although abundant quantitative trait locus (QTL) associated with GL have been identified, it was still relatively weak to identify QTL for GL from qGL5.3, qGL6.1 and qGL11 were detected to control GL by individual environmental analyses and multi-environment joint analysis. Of these, a major-effect and stable QTL qGL6.1 was identified to be a novel QTL, and its LX1 allele had a positive effect on GL. For fine-mapping qGL6.1, a BC1F2 population consisting of 2,487 individuals was developed from a backcross between GY8 and R176, one line with long grain. Eight key informative recombinants were identified by nine kompetitive allele specific PCR (KASP) markers. By analyzing key recombinants, the qGL6.1 locus was narrowed down to a 40.41 kb genomic interval on chromosome 6. One candidate gene LOC_Os06g43304.1 encoding cytochrome P450 (CYP71D55) was finally selected based on the difference in the transcriptional expression and variations in its upstream and downstream region.A total of 197 RILs were genotyped with 285 polymorphic SNP markers. Three QTLs qGL5.3, qGL6.1 and qGL11 were identified to control grain length in rice. One novel QTL qGL6.1 was fine mapped within 40.41 kb region, and LOC_Os06g43304.1 encoding cytochrome P450 (CYP71D55) may be its candidate gene. We propose that the further cloning of the qGL6.1 will facilitate improving appearance quality in japonica varieties.Three QTLs The online version contains supplementary material available at 10.1186/s12284-022-00606-z. Oryza sativa L.) breeding.  is the main factor that determines appearance in rice, and affect milling, cooking and eating quality, and is therefore an important agronomic traits in rice . In addition to dense and erect panicles and more grains per panicle, one of the most significant morphological features of LX1 is their long grains, which were higher than those of other dense and erect panicle varieties. The objective of present study was (1) to detect the QTLs for GL by using a recombinant inbred lines (RILs) population derived from a cross between Gangyuan8 (GY8) and LX1 in four environments, (2) to localize the major-effect QTL qGL6.1 to a narrow genomic region by using the BC1F2 population, and (3) to identify the candidate gene of qGL6.1. Our results will provide important information to unearth the QTL for rice GL and help to understand how to achieve high yield and long grains in the LX1.In our previous studies, to improve the GL of erect panicle varieties, the P < 0.01), indicating that the GL was consistent across environments  > 2.5) as well as QTL × environment interaction effects (LOD(AbyE) > 2.5), but these additive contribution rates were higher than those between the additive effect and environment interaction, indicating that these additive effects were the main contributors to phenotypic variation. The qGL6.1 had significant additive effect (LOD(A) > 2.5) rather than additive-environment interaction (LOD(AbyE) < 2.5), which were considered to be expressed independently. These three QTL qGL5.3, qGL6.1 and qGL11 were detected by the joint analysis with phenotypic contribution of 2.86%, 17.51% and 5.45%, respectively.A total of 15 QTLs that controlled GL was detected based on joint analysis in multiple environments and showed in the physical map Fig. . Three sis Table . By usinqGL6.1 for GL was detected in an approximate 97.46-kb interval on chromosomes 6  to backcrossed with recipient parent GY8, as they carried different allele in qGL6.1 locus but had 68% similar in genetic background by 8 K-SNP chip analysis. A total of 2, 487 BC1F2 plants were detected by nine KASP markers across the qGL6.1 target region to identify eight key informative recombinants  and SNP7  , this region contains four annotated genes, LOC_Os06g43290.1, LOC_Os06g43304.1, LOC_Os06g43320.1 and LOC_Os06g43330.1. The LOC_Os06g43290.1 and LOC_Os06g43330.1 encodes putative Ty1-copia subclass retrotransposon protein, and LOC_Os06g43304.1 and LOC_Os06g43320.1 encodes cytochrome P450, respectively.By individual environmental analyses and multi-environment joint analysis, the major-effect QTL s 6 Fig. A. To obtnts Fig. B. Among rains 7.6 mm to baLOC_Os06g43304.1 differentially expressed between GY8 and R176 among these four annotated genes , SDT (LOC_Os06g44034) and qGL-6 (LOC_Os06g48950) between the GY8 and LX1  between GY8 and LX1, they not expected to affect the protein , which cause reduction or loss of function of GL6, thus affecting rice grain length  and SNP7 , covering a 40.41-kb region in the Nipponbare genome.For fine mapping of qGL6.1 interval, only the LOC_Os06g43304.1 encoding a cytochrome P450 (CYP71D55) was significantly differentially expressed in the inflorescence meristem at the stage of primary and secondary rachis branch formation between GY8 and R176, which was most likely caused by the variations in its upstream and downstream region. As we known, the cytochrome P450 monooxygenases catalyze numerous monooxygenation/hydroxylation reactions, play an important role in different biochemical pathways, plant metabolism, cell proliferation, and expansion  was finally selected based on the difference in the transcriptional expression. The cloning and genetic mechanism study of the qGL6.1 will facilitate improving appearance quality in japonica, especially in erect panicle varieties.In this study, using the RIL population, three QTL japonica variety LX1 and short grains japonica variety GY8. The mapping population consisted of 197 F6:9 and F6:10 RILs developed from a cross between GY8 × LX1.The parental lines in this study were the long grains 2, and the seedlings were transplanted at a spacing of 30 cm between rows and 13 cm between plants. Protection lines were set up everywhere, and field management followed local standards of production for rice.All 197 RILs and parents were grown at Donggang , Shenyang  and Panjin  in Liaoning Province in 2018 and 2019 cropping seasons  for evaluation of GL. Each RIL and parents were sown in a seedling nursery on April and with one seedling being transplanted per hill on May 24 ~ 30. A completely random block design was repeated three times in each environment. Each plot area was 2.5 mAt maturity, the panicles of 10 plants of each line, and their parents were harvested from each plot. The distribution of primary branch number per panicle was firstly measured for each plot. Ten panicles with the largest distribution proportion of primary branch number in each plot were selected to collect the fully filled grains. After the grains were dried for 72 h at 50℃, the lengths of twenty randomly chosen grains from each plot were estimated as the lengthwise distance between opposite tips using a vernier caliper, and the values were averaged and used as the measurements for the line. In 2018DG, the GL of only 142 RILs were measured because of serious lodging of some lines, while that of all RILs were measured in the other three environments.2 = σg2/(σg2 + σge2 /e + σε2/re), where σg2 is (MSf − MSfe)/re, σge2 is (MSfe − MSe)/r and σε2 is MSe; σg2 = genetic variance, σge2 = genotype × environment interaction variance, σε2 = error variance, MSf = mean square of genotypes, MSfe = mean square of genotype × environment interaction, MSe = mean square of error, r = number of replications, and e = number of environments.The phenotypic values of RILs in each environment were analyzed. SPSS 19.0 was used for correlation analysis, and AOV function in QTL IciMapping (version 4.2) was used for variance analysis, with default parameters. The information in the ANOVA table was used to calculate broad sense heritability (H2) of each trait: Hhttp://www.R-project.org/). Genotype, environment, genotype × environment interaction, and nested repeats in the environment are all considered random factors.The best linear unbiased prediction (BLUP) of each parameter is calculated in the mixed linear model by using the lmer function in package lme4 of R software . The Synergy H1 fullfunction microplate reader  was used to read the fluorescence signal. Then the linkage relationship between markers and the qGL6.1 locus was analysed for fine-mapping.After phenotypic and genotypic data analysis, one line with long grains, carrying the LX1 allele in major-effect QTL for GL but having similar genetic background to GY8, was selected in RIL population. For QTL fine-mapping, this line was backcrossed with GY8 to generate the BC−△△CT  according to the manufacturer’s instructions. The first strand of cDNAs were synthesized from DNaseI-treated total RNA using a Primer Script RT reagent Kit with gDNA Eraser . Reverse-transcribed RNA was used as PCR template for gene-specific primers , exon and intron region, as well as downstream region 1000 bp) of related genes were amplified from genomic DNA using KOD plus Neo FX . The primer used to sequence these locus was listed in Additional file 000 bp ofAdditional file 1. Table S1 Correlation of grain length in different environments. Table S2 Grain length of rice recombinant inbred lines in different environments. Table S3 The Kompetitive Allele Specific PCR markers for fine mapping of qGL6.1. Table S4 The Kompetitive Allele Specific PCR markers for seven QTLs in this study. Table S5 The gene-specific primers for qPCR. Table S6 The gene-specific primers for DNA sequencing.Additional file 2. Fig. S1 The genotypic data at qGL11, qGL5.3, qGL5.1, qGL5.2, qGL6.2, qGL7.2 and qGL10 locus of some progenies derived from the heterozygous recombinants R5.Additional file 3. Fig. S2 Variations in the upstream, intron and downstream region of GW6a (A), SDT (B), qGL-6 (C), TGW6 (D) and GL6 (E) between Gangyuan8 (GY8) and Liaoxing1 (LX1).Additional file 4. Fig. S3 The expression level of GW6a, SDT, qGL-6, TGW6 and GL6 by qPCR analysis.


Background: The purpose of this study was to identify genetic variations associated with the metabolism of dabigatran in healthy Chinese subjects, with particular focus given to pharmacokinetics (PK) and pharmacodynamics (PD).Methods: Healthy Chinese adults aged 18–65 years with unknown genotypes from a bioequivalence trial were included according to the protocol registered at ClinicalTrial.org (NCT03161496). All subjects received a single dose (150 mg) of dabigatran etexilate. PK  and PD  parameters were evaluated. Whole-exome sequencing and genome-wide association analyses were performed. Additionally, candidate gene association analyses related to dabigatran were conducted.Results: A total of 118 healthy Chinese subjects were enrolled in this study. According to the p-value suggestive threshold (1.0 × 10−4), the following three SNPs were found to be associated with the AUC0–t of total dabigatran: SLC4A4 SNP rs138389345 (p = 5.99 × 10−5), FRAS1 SNP rs6835769 (p = 6.88 × 10−5), and SULT1A1 SNP rs9282862 (p = 7.44 × 10−5). Furthermore, these SNPs were also found to have significant influences on the AUC0–t of free dabigatran, maximum plasma concentration, and anti-FIIa activity (p < 0.05). Moreover, we identified 30 new potential SNPs of 13 reported candidate genes  that were associated with drug metabolism.Conclusion: Genetic variations were indeed found to impact dabigatran metabolism in a population of healthy Chinese subjects. Further research is needed to explore the more detailed functions of these SNPs. Additionally, our results should be verified in studies that use larger sample sizes and investigate other ethnicities. Oral direct thrombin inhibitor (DTI), a new type of direct oral anticoagulants (DOACs), has been increasingly used in recent years for the prevention of stroke and systemic embolism in cases of non-valvular atrial fibrillation (NVAF) and are currently recommended as the first line of medication-based treatment for this condition, according to American CHEST Guidelines . DabigatAlthough the above characteristics of dabigatran are either equivalent or superior to Vitamin k Antagonists , more atABCB1 gene and generally not metabolized by cytochrome oxidase P450 (CYP450), except at supra-therapeutic concentrations  and CES1 . Only two studies , which has been verified in a recent Asian study , in healthy subjects under fasting and fed conditions  with 240 ml of warm water after either being fed a standard high-fat meal  trial conducted in China, which was mainly performed to assess the BE of domestic generic dabigatran etexilate capsules, with reference to the original product  and the Third Hospital of Changsha . The Serial blood samples used for PK parameter analysis were collected from the BE trial. Venous blood samples (4 ml) were collected before dosing and then at 17–18 time points after drug administration . We also2 tubes and centrifuged for 10 min at 4°C and 1700 g within 1 hour of sampling. Plasma samples were transferred to cryovials and stored at −80°C until PK analysis. For PD analysis, blood samples were collected in sodium citrate (3.2% v/v) tubes and centrifuged for 15 min at room temperature and 2500 g within 1 hour of sampling. Plasma samples were transferred to cryovials that were stored at −80°C, and were subjected to PD analysis within 6 months of sampling. For genetic polymorphism testing, blood samples were collected in EDTA-K2 tubes, transferred to cryovials, and stored at below −60°C until genotype testing.For PK analysis, blood samples were collected in EDTA-Kmax) and the time to Cmax (Tmax) were recorded from the observed data. Values for the terminal half-life (t1/2) and area under the curve (AUC) for plasma concentration at time zero versus the last measurable time point (AUC0-t) were calculated. AUC0-t, Cmax, Tmax, and t1/2 reflected the PK parameters of total dabigatran, while AUC0-t(free), Cmax(free), Tmax(free), and t1/2(free) represented that of free dabigatran. No significant matrix effect was observed. All samples were analyzed within their established storage stability periods.Plasma concentrations of total and free (nonconjugated) dabigatran from BE trial center samples were determined by a home-validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which has been previously published . PK para® CS-2100i fully automated multiparameter hemostasis analyzer  at Peking University First Hospital. PT and APTT were measured by validated Coagulation Method Assay Kits . During the 2018 International Council for Standardization in Hematology (ICSH) (p < 0.001) was demonstrated in our previous study of Chinese patients . Specific calibrators and controls were used for dabigatran . The results of the anti-FIIa assay were reported in dabigatran units (with limits of detection for dabigatran of 0–550 ng/ml). The two QC sample concentrations used were 24 and 399 ng/ml. The inter- and intra-day precision (RSD%) were measured as < 6.76% and 7.53%, whereas inter- and intra-day accuracy were determined to be 97.71% and 86.69%. Relative changes in PT and APTT were also used for PD assessments, which involved taking the ratio after administration to before administration .The prothrombin time (PT), activated partial thromboplastin time (APTT), and levels of anti-FIIa activity were assessed using the Sysmexy (ICSH) , anti-FIpatients . Anti-FI® Spectrophotometer ; 3) DNA concentration was measured using a Qubit® DNA Assay Kit in a Qubit® 3.0 Flurometer .For DNA preparation, the quality of isolated genomic DNA was assessed by using the following three methods in combination: 1) DNA degradation and contamination were monitored on 0.8% agarose gels; 2) DNA purity was checked using the NanoPhotometerXT Human All Exon V6 Kit  according to the manufacturer’s standard protocol. Briefly, qualified genomic DNA was fragmented to an average size of 200bp. The ends of the DNA fragments were subjected to end repair processes, followed by A-tailing and adapter ligation. DNA fragments with adapters ligated on both ends were selectively enriched by polymerase chain reaction (PCR). After PCR, library hybridization and exome capture were performed using biotin labeled probes and magnetic bead selection. Captured libraries were enriched and tagged by PCR to prepare for sequencing. The final libraries were quantified using a KAPA Library Quantification Kit  and an Agilent 2100 Bioanalyzer. Paired-end sequencing (2 × 150 bp) was performed on an Illumina NovaSeq 6000 sequencer .For library preparation and sequencing, the whole exome library was prepared using the Agilent SureSelectp-value was >10–6. Based on the high-quality data obtained after quality control, PCA conducted in PLINK 1.09 was used for stratified population assessment to remove outlier samples. Finally, 75,348 SNPs were tested for association with PK and PD parameters of dabigatran.Variant filtering and prediction were performed on 347,010 single-nucleotide polymorphisms (SNPs) detected in exons, with 200 bp extensions on either end. SNPs were retained for association analysis when the missing rate was <10%, minor allele frequency was >5%, and Hardy-Weinberg equilibrium p-value significance threshold was set at the Bonferroni limit of 6.6 × 10–7 in the discovery phase–SNPs reaching this threshold were taken forward for replication. The significance level for successful replication was set at p = 0.05/75,348 of SNPs for which replication was attempted. Following the genome-wide analyses of the merged and imputed discovery and replication phase data, follow-up analyses were performed by adding each top hit SNP as an additional covariate in GWA. This procedure was performed in a sequential manner until no significant genome-wide signals remained. Regional plots were generated with LocusZoom  to correct for residual population stratification  and the Mann-Whitney or nonparametric Kruskal–Wallis H test to analyze the data. Age, sex, food, and HDL were adjusted for by multivariate linear regression. Two-sided p-values < 0.05 were considered statistically significant.To extensively investigate the influences of previously reported candidate genes on PK and PD parameters of dabigatran in our study population, we also identified related genes and made detailed analyses of these genes. According to previous pharmacogenomic studies , respectively. Basic participant characteristics are presented in 0–t: 1247.43 ± 442.08 ng h/mL, Cmax: 139.99 ± 55.61 ng/ml, Tmax: 3.61 ± 2.09 h, and t1/2: 8.78 ± 1.33 h; for free dabigatran, AUC0–t: 1110.42 ± 404.57 ng h/mL, Cmax: 125.22 ± 48.26 ng/ml, Tmax: 3.65 ± 2.10 h, and t1/2: 8.57 ± 1.57 h.A total of 118 healthy Chinese subjects were enrolled, of which mean age and BMI were 24 years (range: 18–41 years) and 22.2 kg/mp = 6.6 × 10–7), no SNP was identified as significant. Considering the sample size of this exploratory experiment was small (n = 118), the p-value was adjusted to 1.0 × 10−4 for screening suggestive genetic variations on the main metabolic parameter of AUC0–t. Three SNPs, SLC4A4 SNP rs138389345 (p = 5.99 × 10−5), FRAS1 SNP rs6835769 (p = 6.88 × 10−5), and SULT1A1 SNP rs9282862 (p = 7.44 × 10−5), exceeded our suggestive threshold for association with AUC0–t , while the minor allele of FRAS1 SNP rs6835769 was associated with a higher AUC0–t. The trends of influence on AUC0–t(free) for these suggestive SNPs could be also shown significantly (p < 1.0 × 10−3), with regional association plots presented in max, Cmax(free), and IIa2h were also similar (p < 0.05). However, these SNPs had no significant effect on the Tmax or t1/2 of total or free dabigatran. As for the other PD parameters, the minor allele of SLC4A4 SNP rs138389345 corresponded to a significantly lower APTT2h, and the minor allele of SULT1A1 SNP rs9282862 had a significantly lower ∆PT2h (p < 0.05). However, FRAS1 SNP rs6835769 was not associated with any index of APTT or PT.According to the genome-wide significance threshold  were associated with Cmax, six  with Tmax, and two (CES1 rs56278207 and CYP1A2 rs4646427) with t1/2. For the PD parameters, only two SNPs (CYP2B6 rs2279342 and rs34433978) might influence IIa2h, where the latter has also been associated with APTT2h, PT2h, and ∆PT2h. SLCO1B1 SNP rs11045748 might influence APTT2h, ∆APTT2h, and ∆PT2h. CYP2C19 SNPs rs12769205, rs3758580, and rs4244285 might affect both APTT2h and PT2h. The remaining two (ABCG2 rs2231138 and SLC22A1 rs622591) and one (CYP1A2 rs4646427) SNPs were associated with APTT2h and ∆APTT2h, respectively. At last, CYP3A5 SNPs rs15524 and rs4646453 had effects on ∆PT2h.For correlation analyses of candidate genes, the following 18 previously reported genes were finally included in this study: SLC4A4 rs138389345, FRAS1 rs6835769, and SULT1A1 rs9282862) that were associated with the metabolism of dabigatran. AUC0–t, AUC0–t(free), Cmax, Cmax(free), and IIa2h values in the minor alleles of SLC4A4 rs138389345 and SULT1A1 rs9282862 were significantly lower compared to in their wild type counterparts. Conversely, these values were significantly higher in FRAS1 SNP rs6835769. Additionally, SLC4A4 rs138389345 and SULT1A1 rs9282862 were found to have significant influences on APTT and PT, respectively. Moreover, through correlation analyses of reported candidate genes, we found inconsistencies in the results for three previously reported SNPs  regarding their associations with AUC0–t, Cmax, and PD indexes. Furthermore, we identified 30 new potential SNPs of 13 reported candidate genes in our study. ABCB1, ABCC2, ABCG2, CYP2B6, CYP1A2, CES1, SLCO1B1, UGT1A1, UGT1A9, and UGT2B7 might influence PK parameters, while ABCG2, CYP1A2, CYP2B6, CYP2C19, CYP3A5, SLCO1B1, and SLC22A1 might influence PD characteristics. Since our study was based on an original BE trial in healthy subjects, the accuracy of the PK/PD results was high and no known diseases or other factors affected drug metabolism. These same advantages of the previous study, which followed a similar experimental design , variable peak concentration, steady state concentration attainment within two to 3 days, bi-exponential distribution phase, volume of distribution (50–70 L), 35% bind rate to plasma proteins, hepatic conjugation with glucuronide, and ability for renal elimination (80%) . As a BCSLC4A4, located in chromosome 4q13.3, encodes an anion exchange protein named electrogenic sodium bicarbonate cotransporter 1 (NBCe1) (SLC4A4 SNP rs138389345 is a variant of SLC4A4 in which a single G base is deleted (G > -). From the HPA RNA-seq normal tissue data, the SLC4A4 gene is most expressed in the kidney. NBCe1 plays a major role in renal bicarbonate absorption via the proximal tubules and maintaining normal blood pH  . Additionally, this SNP resulted in increased values for AUC0-t(free), Cmax, Cmax(free), IIa2h, and APTT2h.Drug transporters, including members of the ATP-binding cassette (ABC) and solute carrier (SLC) superfamilies, are recognized as important determinants for governing the therapeutic response of many drugs . SLC4A4, (NBCe1) . NBCe1 i (NBCe1) . SLC4A4 blood pH . AbnormaH values , it is rFRAS1 gene, also known as KIAA1500, is located in chromosome 4q21.21, encodes the extracellular matrix (ECM) proteins, and regulates epidermal-basement membrane adhesion and organogenesis during development (FRAS1 SNP rs6835769 involves the mutation of a C base to a T base (C > T) within the gene, which can lead to the A817V mutation at the protein level. FRAS1 mutations have been confirmed to cause an extremely rare autosomal recessive genetic disorder named Fraser Syndrome (FRASRS), and seem to be responsible for non-syndromic unilateral renal agenesis  and 667A > G . Previous reports have confirmed that these alleles have significantly different frequencies depending on race , as seen in data from the Gambian Genome Variation Project (GGVP). SLC4A4 SNP rs138389345 showed a significantly decreased AUC0-t , with similar trends observed in AUC0-t(free), Cmax, Cmax(free), IIa2h, PT2h, and ∆PT2h. Although this linkage disequilibrium needs to be verified in Asian populations, and UGTs were the main phase II enzymes involved, SULT1A1 variation may have the potential to be a new biomarker for antithrombotic therapy–it would certainly be worthy to conduct more detailed studies to investigate this possibility.Drugs usually undergo two phases of metabolism in the body. The main purpose of phase II metabolic reactions is to combine reactions, increase drug polarity, and facilitate drug exclusion from the body. The most common phase II biotransformation enzymes include sulfotransferases (SULTs), N-acetyltransferases (NATs), and methyltransferases (MTs). SULTs utilizes 3′-phospho-5′-adenylyl sulfate (PAPS) as a sulfonate donor to catalyze the sulfate conjugation of a wide variety of acceptor molecules that bear a hydroxyl or an amine group . Sulfonalatelets . The two on race . SULT1A1efficacy , and havefficacy . Althougefficacy  has inveabolites . For theabolites . Severalactivity . SNP rs9ABCB1, ABCC2, ABCG2, CYP2B6, CYP1A2, CYP2C19, CYP3A5, CES1, SLCO1B1, SLC22A1, UGT1A1, UGT1A9, and UGT2B7) were found to have significant effects on drug metabolism. The most commonly reported SNPs, including ABCB1 rs1045642 and CES1 rs2244613, had no influence on any PK or PD parameters in our study, which was a finding consistent with Ji Q’s research , CYP, and SLC gene families and dabigatran metabolism in humans  had positive change. ABCG2 and CYP3A5 variants on bleeding and thromboembolic events in Finnish patients who were given dabigatran, where the results were all negative. In our study, the positive SNPs which had the most effects on PK or PD parameters included SLCO1B1 rs11045748 , SLCO1B1 rs2291075 & rs4149056 (AUC0-t and Cmax), UGT1A9 rs7586110 , UGT1A1 rs4148323 & rs4148327, ABCC2 rs2273697 & rs4148395 (AUC0-t and Cmax), CYP2B6 rs56156262 (AUC0-t and Cmax), and CYP2B6 rs34433978 . The reported positive SNPs of CYP2D6, SLC22A1, CYP3A5, and ABCG2 were not consistent with previous studies  should be performed to document their detailed pathways in drug metabolism.There were a few key limitations to our study worth discussing. First, as the sample size was limited, no SNP was found to meet the genome-wide significance threshold. Therefore, future multi-center studies with larger sample sizes should be conducted. Second, not all potential SNPs could be detected by our genotyping method, such as the reported SNPs of  UGT2B15 . Supplem UGT2B15 . Our finSLC4A4, FRAS1, and SULT1A1 may be novel biomarkers and potential targets for anticoagulation therapy involving dabigatran. Furthermore, newly identified SNPs of 13 reported candidate genes  may also affect dabigatran drug metabolism. Further studies are required to explore the more detailed functions of these SNPs. Additionally, the results of our study should be verified in a larger study among patients and to investigate their relationships with the clinical outcomes of antithrombotic therapy.Our study found that genetic variations do impact the metabolism of dabigatran in healthy Chinese populations. The suggestive genes of