In response to emerging genetic variability, a new SARS-CoV-2 target (RNA-dependent RNA-polymerase) has been added to both tests: Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test. A rapid evaluation of both tests was performed in South Africa, using residual respiratory specimens. Residual respiratory specimens (n = 125) were used to evaluate the Xpert® Xpress CoV-2 plus test and included 50 genotyped specimens. The Xpert® Xpress CoV-2/Flu/RSV plus test was assessed using 45 genotyped SARS-CoV-2 specimens, 10 influenza A, 10 influenza B and 20 respiratory syncytial virus specimens. Results were compared to in-country standard-of-care tests. Genotyped specimens tested the performance of the test under pressure from circulating SARS-CoV-2 variants of concern. Reference material was included to assess the test limits and linearity. The Xpert® Xpress CoV-2 plus test performance compared to reference results across residual respiratory specimens was good (positive percentage agreement (PPA) = 95.2%, negative percentage agreement (NPA) = 95.0%) The Xpert® Xpress CoV-2/Flu/RSV plus test showed good performance across all residual respiratory specimens . All genotyped variants of concern were detected by both tests. The Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests can be used to diagnose SARS-CoV-2, and to diagnose and differentiate SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus, respectively. The NPA was lower than the recommended 99%, but was influenced by the low number of negative specimens tested. The variants of concern assessed did not affect test performance. It is recommended that sites perform their own assessments compared to in-country standard-of-care tests.The Xpert South Ance 2011 , as one ® Xpress SARS-CoV-2 test targets the SARS-CoV-2 specific nucleocapsid region two (N2) and pan sarbecovirus envelope (E) genes, and includes internal processing controls. This test has been widely evaluated [® Xpress CoV-2 plus , and this test has been successfully evaluated elsewhere [® Xpress SARS-CoV-2/Flu/RSV test allows the detection and differentiation of SARS-CoV-2, Flu A, Flu B and RSV from a single nasopharyngeal or anterior nasal swab. Cepheid has now included the RdRp target in this test: Xpert® Xpress CoV-2/Flu/RSV plus , influenza B (Flu B), respiratory syncytial virus (RSV)) and SARS-CoV-2, as disease symptoms are similar. Cepheidlsewhere ,30,31,32® Xpress CoV-2 plus and Xpert® Xpress CoV2/Flu/RSV plus tests in South Africa. Residual clinical specimens, including VOC across all local COVID-19 waves, and reference materials were used, and the performance of both tests is described.The design and performance of these updated assays (research use only (RUO) format) in the presence of NGTF and emerging VOC has been well-described previously . This st® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test (both Cepheid®) were rapidly evaluated using residual patient specimens. Specimen selection and workflow are shown in ® Xpress CoV-2 plus) and 1b (Xpert® Xpress CoV-2/Flu/RSV plus), respectively. The Xpert® Xpress CoV-2 plus test was compared to standard-of-care (SOC) results and the Xpert® Xpress SARS-CoV-2 test, while the Xpert® Xpress CoV-2/Flu/RSV plus test was compared only to SOC results. In addition to performance against the reference results, the potential impact of variants of concern (VOC) on the tests’ analytical accuracy was evaluated. Specimens were selected across the four major waves in South Africa (Wildtype (Wuhan with D614G): April–July 2020; Beta: November 2020–January 2021; Alpha/Delta: May–July 2021; Omicron: October 2021–April 2022). Limited reference material was also included in the study, and is described below.The XpertEthics approval for this evaluation was obtained from the University of the Witwatersrand Human Research Ethics Committee Medical  as an amendment to the approval to access residual clinical specimens for SARS-CoV-2 test testing: M1911201.® Xpress CoV-2 plus evaluation were supplied in a variety of media, including but not limited to universal transport medium (UTM), saline and phosphate-buffered saline (PBS). A limited number of SARS-CoV-2 negative specimens (n = 20) were included. Results were compared to two SOC comparators: cobas® SARS-CoV-2  and TaqMan™ TaqPath COVID-19 . A limited number of specimens (n = 35) were further compared to the Xpert® Xpress SARS-CoV-2 test currently in use in the NHLS in order to ensure non-inferiority of the Xpert® Xpress CoV-2 plus test, which is necessary for potential implementation into national programmes. Specimen wave was noted as an indicator of likely VOC, but the VOC of these specimens was not confirmed.Residual respiratory clinical specimens (n = 75), collected according to standard of care (SOC) protocols, were obtained from the National Health Laboratory Service (NHLS) and the National Institute of Communicable Diseases (NICD). Specimens were selected across local SARS-CoV-2 waves (as described above) and based on SOC SARS-CoV-2 results, and were stored at −80 °C until testing. The SARS-CoV-2 specimens used for the XpertThe potential impact of circulating SARS-CoV-2 VOC on test performance was assessed using an additional 50 genotyped residual patient specimens. These specimens were selected by COVID-19 wave and were previously genotyped by the AllPlex SARS-CoV-2 Variants I and II and NovaPlex SARS-CoV-2 Variants V tests  and/or TaqMan SARS-CoV-2 Mutation Panels  and/or MassARRAY SARS-CoV-2 Variant Panel v3 (RUO) , or by next generation sequencing performed at the NICD. In addition, AccuPlex SARS-CoV-2 variant reference material  was selected from the AccuPlex SARS-CoV-2 Variants Panel 1  and AccuPlex SARS-CoV-2 Variants Panel 2 (Delta) were used to assess the performance of the test on VOC material. Briefly, AccuPlex material (5000 copies per millilitre (cp/mL)) was diluted to 500 cp/mL with molecular grade water  and each was tested twice. Omicron reference material was not available at the time of this evaluation.® Xpress CoV-2 plus and Xpert® Xpress SARS-CoV-2 tests, with a single undiluted (5000 cp/mL) specimen also processed . Wildtype SARS-CoV-2 culture material dilutions , prepared and eluted according to previously described protocols [® Xpress CoV-2 plus test in triplicate to assess the precision of the test across the range.The reported limit of detection (LOD) of the test  and tesrotocols , were te® Xpress CoV-2-Flu-RSV plus evaluation were supplied in a variety of media (as described above), while the Flu and RSV specimens were collected in viral transport medium. In total, 45 SARS-CoV-2, 10 FluA, 10 FluB, 10 RSV A, 10 RSV B and 12 respiratory-virus negative specimens were tested. SOC testing was performed on multiple tests , and on the AllPlex™ SARS-CoV-2/FluA/FluB/RSV . The last test allows differentiation of both Flu A and B, and RSV A and B subtypes. Xpert® Xpress CoV-2/Flu/RSV plus results were compared to SOC results. In addition, the reported LOD of the test  = 0.007 TCID50/mL, Flu B(2019) = 12.9 CEID50/mL and RSV(2016) = 0.33 TCID50/mL [Residual respiratory clinical specimens (n = 97), collected and tested according to standard of care (SOC) protocols, were obtained from the NICD and the NHLS. Specimens were selected based on SOC results and were stored at −80 °C until testing. The SARS-CoV-2 specimens used for the XpertCID50/mL ) and tes® Xpress SARS-CoV-2, Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test testing were performed according to the manufacturer’s instructions for use (IFU) [® Xpress SARS-CoV-2, Xpert® Xpress CoV-2 plus or Xpert® Xpress CoV-2/Flu/RSV plus test definition file (ADF) respectively. Both 6-colour and 10-colour GeneXpert instruments were used for this study. The cartridges include a sample processing control (SPC) and a probe check control (PCC) to ensure the quality of the specimen  and the reagents (fluorescence check) respectively. Results are automatically generated by the GeneXpert™ DX System Version 6.2 software. While these tests report qualitative results, cycle threshold (Ct) values were available and were used for this analysis.Xpertse (IFU) ,35,36. B® Version 20.114  software packages, with raw data being captured in Microsoft® Excel . The positive percentage agreement (PPA), negative percentage agreement (NPA) [® Xpress SARS-CoV-2 (Xpert® Xpress CoV-2 plus only) were calculated; 95 percent confidence intervals (95% CI) were included. Errors and invalid specimens were noted, and these results were excluded from the accuracy analysis.Data were assessed using STATA SE 14.2 for Windows  and MedCalcnt (NPA) , agreement (NPA) ,39) and ® Xpress CoV-2 plus test. Seventy-one of these had cobas® SOC results, with a mean cobas® Ct of 27.9  for the E-gene and 27.1  for the ORF1a gene. The other 34 SARS-CoV-2 positive specimens had TaqPath SOC results, with mean TaqPath Ct of 23.1  for the ORF1ab gene, 23.4  for the N-gene and 23.7  for the S-gene. The specimen subset further tested on the Xpert® Xpress SARS-CoV-2 test (n = 30 positive specimens) had a SOC ORF1ab mean Ct of 26.9 . The SARS-CoV-2 VOC set (n = 50) had a mean SOC ORF1ab Ct of 24.0 . Complete data is available in A total of 105 SARS-CoV-2 positive specimens and 20 SARS-CoV-2 negative specimens were tested using the XpertAccuracy was determined using clinically relevant specimens (n = 157), including 125 residual clinical specimens, 12 viral culture specimens and 20 AccuPlex specimens . Only si® SARS-CoV-2 (SOC) negative specimen (specimen 52) was positive by both the Xpert® Xpress SARS-CoV-2 and Xpert® Xpress CoV-2 plus tests, but negative on Xpert® Xpress CoV-2 plus repeat testing. Five cobas® positive specimens  were negative by Xpert® Xpress CoV-2 plus, of which two were also negative by Xpert® Xpress SARS-CoV-2. The Xpert® Xpress CoV-2 plus test showed 100% agreement with the TaqPath test in 30 positive specimens tested.The test performance across all specimens (n = 157) when compared to SOC or expected reference material results gave PPA of 96.4%, NPA of 95.0% and overall agreement of 0.8416. Similarly, the test performance across only residual clinical specimens (n = 125) compared to SOC gave PPA of 95.2%, NPA of 95.0% and overall agreement of 0.8348. There were a total of six discordant SARS-CoV-2 specimens compared to the SOC test . One cob® Xpress SARS-CoV-2 test was not used as SOC for the residual specimens. However, this test is used within the national testing program and thus could be considered as a SOC test. When compared to the Xpert® Xpress SARS-CoV-2 test , PPA remained acceptable at 98.0%, with NPA decreasing to 87.5%. This was, however, influenced by the low number of negative specimens assessed (n = 7/55) and in reality reflects only one false negative specimen (specimen 119) with high Ct by the Xpert® Xpress SARS-CoV-2 test (E = 38.4 N2 = 41.4). Similarly, only one false positive (specimen 114), compared to Xpert® Xpress SARS-CoV-2, had high Ct by the Xpert® Xpress CoV-2 plus test  and by SOC testing. Agreement remained very good at 0.8363. Discordant specimens are shown in The Xpert® Xpress CoV-2 plus test across variants  than for the Xpert® Xpress SARS-CoV-2 test (mean Ct = 35.30). The Xpert® Xpress CoV-2 plus N2-gene (mean Ct = 37.06) and RdRp (mean Ct = 36.50) targets were detected at similar Ct to the Xpert® Xpress SARS-CoV-2 test N2-gene target (mean Ct = 37.55). The mean %CV across the tests  was consistently <5%  compared to the R2 values for the Xpert® Xpress SARS-CoV-2 test .AccuPlex reference material (Wildtype—Wuhan) was detected across all dilutions from 100 cp/mL to 1000 cp/mL by both the Xpert targets . Variabintly <5% . The R2 2 values a were sl® Xpress CoV-2/Flu/RSV plus evaluation were tested on a number of SOC tests  and SOC Ct were thus reported by gene target rather than by test. The SARS-CoV-2 N-gene target is common to the majority of tests , with 32 specimens reporting a mean Ct of 24.9 , which is reflective of the overall test Ct values. In addition, 23 results included the S-gene target (TaqPath or AllPlex) with a mean Ct of 23.7 , 20 results included the ORF1ab gene (cobas® or TaqPath) with a mean Ct of 24.5 , 15 results included the RdRp gene (AllPlex) with a mean Ct of 24.7  and 12 results included the E-gene (cobas® or Xpert® Xpress SARS-CoV-2) with a mean Ct of 25.0 . A limited number of SARS-CoV-2 negative specimens (n = 12) were included, with the 40 respiratory specimens also listed as SARS-CoV-2 negative for the purposes of the study.The 45 SARS-CoV-2 specimens used for the Xpert® Xpress CoV-2/Flu/RSV test and the SOC Ct was thus reported as a single result.The respiratory specimens were all tested by AllPlex™ SARS-CoV-2/FluA/FluB/RSV and stratified by virus. The SOC mean Ct was 27.6  for the Flu A specimens (n = 10), 28.1  for the Flu B specimens (n = 10) and 25.5  for the RSV specimens (n = 10 RSV A and n = 10 RSV B). The RSV subtype is not differentiated on the XpertAccuracy was determined using residual clinical specimens (n = 97) and reference material. Only one error due to cartridge failure was observed  and was excluded from accuracy analysis. No difference in performance was observed between the 6-colour and 10-colour GeneXpert Dx platforms. Complete results are shown in ® SARS-CoV-2, both with high Xpert® Xpress CoV-2 plus Ct of 38.5 and 39.8, respectively. There were a further three RSV-positive specimens that were also positive for SARS-CoV-2 by the Xpert® Xpress CoV-2/Flu/RSV plus test . All had high Xpert® Xpress SARS-CoV-2 Ct values . Confirmatory SARS-CoV-2 testing by an alternate test was not possible due to limited specimen volume. No Flu A, Flu B or RSV specimens were misclassified amongst the residual patient specimens, with only one AccuPlex dilution (100 cp/mL) reporting a RSV negative result with a high Ct of 39.7. Two AccuPlex specimens were negative for Flu A2 , but positive for Flu A1, with overall positive Flu A results reported.When assessed across all disease targets , the tes® Xpress CoV-2/Flu/RSV plus test across SARS-CoV-2 VOC was possible as the 45 SARS-CoV-2 specimens were previously genotyped specimens. The test detected 100% of specimens  was detected by the Xpert targets . Variabi targets b in the ® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests using residual clinical specimens and compared to SOC results. There is currently limited published information on the new iterations of these tests [The evaluation of novel or redesigned diagnostics is important to ensure ongoing testing of expected quality, ultimately impacting patient care. This study describes a rapid evaluation of the Xpertse tests ,26, withse tests . While tse tests , the neg® Xpress CoV-2 plus test showed acceptable performance compared to SOC test results (cobas® SARS-CoV-2 and TaqMan® TaqPath COVID-19) for testing residual patient specimens. The PPA of the test (96.4%) is in line with that described for the Xpert® Xpress SARS-CoV-2 test in a recent meta-analysis (97.0%) [® SARS-CoV-2 mean Ct of 36.6 for the E-gene target and 34.9 for the ORF1a target), are believed to be near the LOD for the cobas® SARS-CoV-2, Xpert® Xpress SARS-CoV-2 and Xpert® Xpress CoV-2 plus tests, leading to increased variability in results. A brief report [® SARS-CoV-2 and the Xpert® Xpress SARS-CoV-2 tests at higher Ct values had similar results, with 1/35 specimens testing positive by cobas® SARS-CoV-2 and negative by Xpert® Xpress SARS-CoV-2. The results of the new test also concur with the original evaluations of the Xpert® Xpress SARS-CoV-2 test compared to seven SOC tests (99.5% PPA and 95.8% NPA) [® SARS-CoV-2 test (89% as opposed to 99%) [® Xpress CoV-2 plus test also showed acceptable PPA (98.0%) when compared to the Xpert® Xpress SARS-CoV-2 test. This is similar to the perfect agreement between the Xpert® Xpress SARS-CoV-2 and Xpert® Xpress CoV2 tests on 50 positive specimens, including VOC and NGTF specimens [® Xpress SARS-CoV-2 test was also very good [® Xpress CoV-2 plus and Xpert® Xpress SARS-CoV-2 tests were similar, with low SD (<1.2 Ct) and %CV (<3.2%) down to 100 cp/mL AccuPlex SARS-CoV-2. While larger studies with increased numbers of negative specimens are recommended, it is likely that the Xpert® Xpress CoV-2 plus test can be used equivalently to the Xpert® Xpress SARS-CoV-2 test. Few errors were noted for either test and no difference in results was observed between the 6-colour and 10-colour instruments. Additionally, the Xpert® Xpress CoV-2 plus test provides results more rapidly (~31 min) than the Xpert® Xpress SARS-CoV-2 test (~48 min).The Xpert (97.0%) , with ve (97.0%)  of 84.16 (97.0%)  was skewf report  describi.8% NPA) , althougpecimens . The decery good  at >80%.® Xpress SARS-CoV-2 test by Loeffelholz and colleagues [® Xpress SARS-CoV-2 RUO tests and included the RdRp target that was not included in the first Xpert® Xpress SARS-CoV-2 EUA test, noted that the presence of E, N2 and RdRp was consistent amongst concordant positive results, with discordant positive specimens usually showing only the E-gene or N2-gene targets. The sole specimen negative by cobas® SARS-CoV-2 and detected by Xpert® Xpress SARS-CoV-2  and Xpert® Xpress CoV-2 plus  showed all gene targets, including RdRp. Similarly, in our study, the E-gene target showed lower Ct values than the N2-gene and RdRp-gene targets, with the latter targets showing similar Ct throughout. Only one specimen (positive by cobas® SARS-CoV-2 and negative by Xpert® Xpress SARS-CoV-2) was Xpert® Xpress CoV-2 plus E-gene and N2-gene positive and RdRp-gene negative, so it is not possible to comment on the decreased sensitivity of RdRp observed in the earlier study [® Xpress SARS-CoV-2 N2-gene only positive specimens were also positive by digital drop PCR, indicating a low viral burden [® Ct values.It is of interest that the early evaluation of the Xpertlleagues , which wer study . The RdRer study . A recenl burden . However® Xpress CoV-2/Flu/RSV plus test showed comparable performance to the SOC tests, with an overall PPA of 100% and NPA of 98.3%. Individually, the Flu A, Flu B and RSV targets reported 100% agreement, while the SARS-CoV-2 target had 100% PPA and 90.4% NPA. This NPA is lower than the NPA reported in a recent multicentre evaluation of the original Xpert® Xpress SARS-CoV-2/Flu/RSV test (98.7%) against a range of tests [® Xpress SARS-CoV-2 (100%) and Xpert® Xpress Flu/RSV (>99.5%) tests in a separate study [® Xpress CoV-2/Flu/RSV plus test using the residual specimens from an evaluation of the Xpert® Xpress SARS-CoV-2/Flu/RSV [® Xpress CoV-2/Flu/RSV plus is acceptable and was skewed in the current study by the low number of negatives tested. The decreased NPA observed in this study is linked to five specimens with high Ct SARS-CoV-2-positive specimens by Xpert® Xpress CoV-2/Flu/RSV plus that were negative by SOC. A limitation of this study was the low specimen volume available, meaning that repeat testing was not possible, nor was the clinical profile available. While unlikely, given that each specimen was prepared and tested individually, cross-contamination cannot be ruled out. It is, however, possible that these specimens had low SARS-CoV-2 nucleic acid levels near the limit of the tests [® Xpress SARS-CoV-2/Flu/RSV test recorded comparable performance between this and the Xpert® Xpress SARS-CoV-2 test for the detection of SARS-CoV-2 [® Xpress CoV-2/Flu/RSV plus test produces results in 25–45 min (as expected), which is more rapid than the existing Xpert® Xpress SARS-CoV-2/Flu/RSV test (45–50 min) [The Xpertof tests , and agate study . An impote study  evaluate/Flu/RSV , with 10/Flu/RSV . The stu/Flu/RSV . The goohe tests ,45 and cRS-CoV-2 . The Xpe–50 min) , with re–50 min) . As SARS® Xpress SARS-CoV-2/Flu/RSV test, where a limited number of VOC were included [® Xpress CoV-2/Flu/RSV plus test, and by Burns et al. [® Xpress SARS-CoV-2 plus and the Xpert® Xpress CoV-2/Flu/RSV plus, with a focus on NGTF and VOC [It is concerning that the NPA of both tests under evaluation was lower than that observed in other studies. However, this is likely linked to study design, with a focus on SARS-CoV-2-positive specimens that negatively influenced the NPA of the tests, to the use of residual patient specimens only, and to the variability of molecular tests near the LOD ,45. Regaincluded , by Johnincluded  in theirs et al.  for both and VOC .® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests can be considered to replace existing Xpert® Xpress tests or other SOC tests in use. However, it is recommended that laboratories perform their own evaluations ahead of implementation and monitor test performance at scale, to ensure comparable performance compared to existing SOC and ongoing quality patient management.The Xpert


Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is responsible for the COVID-19 pandemic that has caused unprecedented loss of life and economic trouble all over the world, though the mechanism of its replication remains poorly understood. In this study, antibodies were generated and used to systematically determine the expression profile and subcellular distribution of 11 SARS-CoV-2 nonstructural replicase proteins  by Western blot and immunofluorescence assay. Nsp3, nsp5, and nsp8 were detected in perinuclear foci at different time points, with diffusion and stronger fluorescence observed over time. In particular, colocalization of nsp8 and nsp13 with different replicase proteins suggested viral protein-protein interaction, which may be key to understanding their functions and potential molecular mechanisms. Viral intermediate dsRNA was detected in perinuclear foci as early as 2-h postinfection, indicating the initiation of virus replication. With the passage of time, these perinuclear dsRNA foci became larger and brighter, and nearly all colocalized with N protein, consistent with viral growth over time. Thus, the development of these anti-nsp antibodies provides basic tools for the further study of replication and diagnosis of SARS-CoV-2.IMPORTANCE The intracellular localization of SARS-CoV-2 replicase nonstructural proteins (nsp) during infection has not been fully elucidated. In this study, we systematically analyzed the expression and subcellular localization of 11 distinct viral nsp and dsRNA over time in SARS-CoV-2-infected cells by using individual antibody against these replicase proteins. The data indicated that nsp gene expression is highly regulated in space and time, which could be useful to understand the function of viral replicases and future development of diagnostics and potential antiviral strategies against SARS-CoV-2. Coronavirinae of the family Coronaviridae is classified into four genera by the International Committee on Taxonomy of Viruses: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Coronaviruses (CoVs) are highly diverse RNA viruses with a broad host range; some are known to infect humans, mice, pigs, and cats among other mammalian species, causing mild to severe respiratory, intestinal and nervous system diseases –3. Curre–onavirus , 4.https://coronavirus.jhu.edu/map.html), there have been more than 430 million confirmed cases globally, leading to at least 5.9 million deaths as of February 2022. The ongoing challenge posed by variants of SARS-CoV-2 is unprecedented, especially the “Omicron” strain, which is more contagious. In many countries, “Omicron” has become the dominant strain in circulation, including in those with high COVID-19 vaccination rates, which increases the risk of continual pandemic waves  in Wuhan, Hubei Province in China . The mosic waves .SARS-CoV-2 is a positive-sense, single-stranded RNA virus with an ~30 kb genome that contains 12 putative open reading frames (ORFs) encoding at least 29 proteins. About two thirds of the genome consists of a large replicase (Rep) gene (ORF1a and ORF1b), followed by structural and accessory genes , 6. ORF1The SARS-CoV-2 replicase polyproteins are regulated by proteases encoded by two viral proteins, the 3-chymotrypsin-like protease (3CLpro or nsp5) and papain-like proteinase (PLP) within nsp3 . The SARIn addition, the SARS-CoV-2 structural gene encodes four structural proteins, including spike glycoprotein (S protein), membrane protein (M protein), envelope protein (E protein), and nuclear protein (N protein) , 6, in aA number of studies on the SARS-CoV-2 replicase proteins have been published, from potential inhibitors, drug screens, structure, and function –32. The –NC_045512.2) was used to construct expression plasmids of each individual replicase protein encoded by ORF1a and ORF1b , with 10 unimmunized mice used to collect negative-control sera. A small amount of tail blood was collected for ELISA determination, and then the antisera were pooled from all mice in each group. Antiserum titers for all individual mice injected with viral nsp were greater than 1:10,000 (data not shown). Serum specificity was tested by IFA in BHK-21 cells transfected with Myc-labeled recombinant nsp proteins (pRK5-SARS-CoV-2-nsp). At 24-h posttransfection , all eigBy WB analysis, the anti-nsp1 serum detected a discrete band of 14 kDa, while the anti-nsp2, anti-nsp5, and anti-nsp8 sera detected discrete bands at 75, 33, and 22 kDa, respectively . No staiTo characterize the expression of SARS-CoV-2 replicase proteins during infection of cultured A549-ACE2 cells, we used the polyclonal antisera generated above to detect at 24-h postinfection (hpi). The anti-nsp1 serum detected a single protein band with an observed mass of 14 kDa, consistent with the predicted nsp1 protein cleaved at 180G↓A181 . The antThe CoV N protein encapsidates viral genome RNA, playing a vital role in the viral life cycle, and is abundantly expressed during infection . EarlierOur previous study indicated that nsp3 of swine acute diarrhea syndrome (SADS)-CoV, an alphacoronavirus, was detected in perinuclear foci at 4 hpi . Stertz SARS-CoV-2 is an RNA virus that replicates its genome, translates proteins, and assembles progeny viral particles in the cytoplasm. It depends on the infrastructure of the host cell and metabolism for nearly all stages of its replication cycle. Release of the viral genome into the cytoplasm of host cells begins a complex program of viral gene expression, which is highly regulated in space and time , 39. HowTo determine the intracellular localization and timing of expression of the viral replicase proteins in greater detail, a confocal time course study of nsp3, nsp5, and nsp8 was performed. These three proteins were chosen because the observed staining intensity with the prepared antibodies was higher than that of the others tested . A549-ACIt has been shown that point mutations in nsp7 and nsp8 of SARS-CoV delayed viral growth, identifying those residues as critical for viral replication . Discretin situ (CoVs produce double-stranded RNA (dsRNA) intermediates during the process of replication , and thein situ , 43. Sucin situ . To detein situ , similarin situ . Given tin situ , B.Analysis of viral protein-protein interaction is an important step to understand the function of viral proteins and their potential molecular mechanisms. Nsp8 has been found to be associated with RdRp and its function is defined as a primer for RNA synthesis , 44. NspMoreover, we analyzed the colocalization between different SARS-CoV-2 proteins using rabbit anti-nsp8 antibody and home-made mouse immune sera against the other replicase proteins. Dual-labeling experiments were carried out in A549-ACE2 cells at 6 hpi . We founThe nsp13 helicase has been identified as an ideal target for antiviral drug development because of its conserved sequence and its indispensability in all CoVs , 49. We Several studies have found that the nsp7 and nsp8 (nsp7-nsp8) supercomplex are essential cofactors for nsp12 polymerase, which revealed the structural basis for RNA replication . In addiIn summary, we systematically analyzed the expression profile and localization of nsp1, nsp2, nsp3, nsp5, nsp7, nsp8, nsp9, and nsp14 proteins and dynamic changes of nsp3, nsp5, nsp8, and nsp13 proteins over time. Our data demonstrate that viral nsp gene expression is highly regulated in space and time, and these details may be helpful for understanding the function of viral replicases and future development of diagnostics, potential antiviral strategies, and nsp-antigen-based vaccines against SARS-CoV-2.l-glutamine (Gibco). The A549-ACE2 clonal cell lines were generated by lentiviral transduction of the human ACE2 gene. All cells were grown at 37°C with 5% CO2. The SARS-CoV-2 nCoV-SH01 strain (GenBank accession no. MT121215) clone Sdel was propagated in Vero E6 cells in the absence of trypsin, and the resulting infectious virus stock used in this study had a 21-nt deletion in the spike gene  supplemented with 10% fetal bovine serum  and 1% (wt/vol) antibiotics . A549 cells (ATCC #CCL-185) were cultured in DMEM supplemented with 10% fetal bovine serum , 1% penicillin and streptomycin (HyClone), 1x nonessential amino acids (HyClone), 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid , and 2 mM ike gene . All infNC_045512.2), which is made up of nsp1, nsp2, nsp3, nsp5, nsp7, nsp8, nsp9, nsp10, nsp13, nsp14, and nsp15, gene fragment were cloned into the prokaryotic expression vector pET-30a with an N/C-terminal His-tag, yielding plasmids pET-30a-SARS-CoV-2-nsps. These plasmids were transformed into Escherichia coli BL21(DE3) cells, and His-tagged proteins were purified using Ni-NTA His-Tagged (TransGen Biotech) according to the manufacturer’s protocol. Similarly, replicase gene fragments were also cloned into the eukaryotic expression vector pRK5 with a C-terminal Myc tag (pRK5-SARS-CoV-2-nsps) for detection by antisera. Similarly, replicase gene fragments were also cloned into the eukaryotic expression vector pRK5 with an N/C-terminal Myc or Flag tag (pRK5-SARS-CoV-2-nsps) for detection by antisera.To express the SARS-CoV-2 replicase proteins . After 3 weeks, the mice were immunized with the same dose, and blood samples were collected about 2 weeks after the second immunization.The recombinant eukaryotic plasmids were transfected into BHK-21 cells grown in 24-well plates to 70% to 80% confluence using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After 24 h, the medium was removed and wells were washed twice with phosphate-buffered saline (PBS), then fixed with 4% paraformaldehyde (PFA) for about 15 min at room temperature (RT). After washing three times with PBS for 3 min, cells were permeabilized in 0.5% Triton X-100 for 10 min at RT and washed with PBS repeatedly, then incubated with the primary antibody (mouse serums) diluted in 3% bovine serum albumin (BSA)/PBS for 1 h at 37°C. After washing with PBS, the samples were incubated with secondary antibody diluted in PBS for 1 h at 37°C, washed with PBS, and stained with 4’,6-diamidino-2-phenylindole (DAPI) for about 5 min at RT. For the staining of virus-infected cells, A549-ACE2 cells grown on 12-mm coverslips in a 24-well plate were infected with SARS-CoV-2 at an MOI of 2 as described previously . ImmunofThe following home-made mouse antibodies (and dilutions) were used: anti-nsp1 (1:200); anti-nsp2 (1:200); anti-nsp3 (1:200); anti-nsp5 (1:200); anti-nsp7 (1:200); anti-nsp8 (1:200); anti-nsp9 (1:200); anti-nsp14 (1:200); anti-nucleocapsid (N) protein .The following commercial antibodies were used in the study: mouse anti-dsRNA monoclonal antibody J2 ; rabbit anti-SARS nsp8 ; rabbit anti-SARS nsp13 ; rabbit anti-SARS N protein ; goat anti-mouse antibody conjugated with Alexa Fluor 488 ; goat anti-mouse antibody conjugated with Alexa Fluor 555 ; goat anti-rabbit antibody conjugated with Alexa Fluor 488 ; and goat anti-rabbit antibody conjugated with Alexa Fluor 555 .Confocal fluorescent images were obtained by confocal laser scanning microscopy  using an inverted oil immersion lens with a 63x objective. DAPI was excited with a 405-nm diode laser, Alexa Fluor 488 with a 488-nm argon laser, and Alexa Fluor 555 with a 561-nm DPSS laser. For fluorescence detection, respective emission filters were used, and images were analyzed by Leica LAS X system software. All immunofluorescence assays were performed at least three times.3VO4, 10 mM NaF, 25 mM Tris-HCl, 200 mM NaCl, 25 mM β-glycerophosphate, 1% NP40 and protease cocktail ) at 4°C for 15 min, centrifuged 12,000 × g for 15 min at 4°C, and collected for SDS-PAGE or WB analysis. For co-IP, whole-cell lysates (WCLs) were preadsorbed onto Dynabeads protein G (Thermo Fisher Scientific) and incubated with anti-Flag antibodies in order to precipitate Flag-tagged proteins for 12 h at 4°C on a rotator. After washing with PBST three times, the resins were eluted with elution buffer  for WB. Samples were transferred onto a polyvinylidene difluoride (PVDF) membrane that was subsequently blocked with Tris-buffered saline and Tween 20 (TBST) containing 5% nonfat powdered milk (Sangon Biotech) for about 2 h at RT. After washing with TBST three times, the primary antibody (mouse serums) was added and incubated overnight at 4°C. After washing with TBST, the horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Fisher Scientific) was incubated with the membrane for 1 h. Finally, the samples of proteins were detected using an ECL kit (FDbio science).Transfected cells were lysed in lysis buffer  containing protease inhibitors (Sigma-Aldrich # S8830), and proteins were separated via 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk (BD Difco #232100) in TBST for 1 h at RT, and probed with the primary antibodies at 4°C overnight. After washing with TBST, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. Then blots were washed again with TBST, and developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher #34577).The following home-made antibodies (and dilutions) were used: anti-nsp1 (1:500); anti-nsp2 (1:500); anti-nsp3 (1:500); anti-nsp5 (1:500); anti-nsp8 (1:500); anti-nsp9 (1:500). The following commercial antibodies were used: rabbit anti-β-actin ; mouse anti-Flag M2 antibody ; mouse anti-Myc antibody ; goat anti-mouse conjugated with HRP ; and goat anti-rabbit conjugated with HRP .P value ≤ 0.05 used as the threshold for statistical significance.Confocal images for analysis of intracellular localization of SARS-CoV-2 replicase nonstructural proteins were processed using ImageJ or Fiji software. Statistical analysis was performed using GraphPad Prism 8.4.3 software. One-way analysis of variance (ANOVA) was used for multiple comparisons between the protein of interest and other different nonstructural proteins, with a


These findings taken together unveil molecular basis of immune recognition in IECs in response to SARS-CoV-2, and provide insights that intestinal immune cross-talk with other immune cells triggers amplified immunity and probably contributes to immunopathogenesis of COVID-19.SARS-CoV-2 causes a spectrum of clinical symptoms from respiratory damage to gastrointestinal disorders. Intestinal infection of SARS-CoV-2 triggers immune response. However, the cellular mechanism that how SARS-CoV-2 initiates and induces intestinal immunity is not understood. Here, we exploited SARS-CoV-2-GFP/ΔN trVLP pseudo-virus system and demonstrated that RIG-I and DHX15 are required for sensing SARS-CoV-2 and inducing cellular immune response through MAVS signaling in intestinal epithelial cells (IECs) upon SARS-CoV-2 infection. NLRP6 also engages in the regulation of SARS-CoV-2 immunity by producing IL-18. Furthermore, primary cellular immune response provoked by SARS-CoV-2 in IECs further cascades activation of MAIT cells and produces cytotoxic cytokines including IFN-γ, granzyme B  The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused over 500 million infections and more than 6 million deaths worldwide . InfectiEx vivo studies of SARS-CoV-2 with human colon organoids or intestinal enteroids demonstrate that SARS-CoV-2 infection initiates antiviral innate immune response by producing type I interferon (IFN) and type III IFN  intermediates during genome replication and virion propagation . As in oIn viral infections, the crucial role of inflammasome in control of immune response and viral infections has been elucidated. NLRP3 inflammasome activation in infected macrophages/monocyte has been reported to contribute to SARS-CoV-2 related inflammation . A promiNlrp6 activation signals proinflammatory cytokine IL-18 production . In SARSHere, we exploited SARS-CoV-2-GFP/ΔN trVLP system, as previously described , to infeIntestinal epithelial cells (IECs) are highly permissive for SARS-CoV-2 infection due to high expression of entry receptor ACE2 and TMPRSS serine protease which facilitates virus entry into host cells . Recentlvia lentiviral transduction into Caco-2-ACE2 cell  intermediates. Numerous viral RNA sensors have been reported to sense viral RNAs and initiate cellular immune response, of which RIG-I, MDA5, DHX-15, LGP-2 were demonstrated responsible for recognizing several enteric viruses, such as rotavirus, reovirus, EMCV. To identify the specific sensors that involve in recognizing SARS-CoV-2 RNA and contribute to immunity induction in intestinal epithelial cells, we established RNA sensors RIG-I, MDA5, DHX15, LGP-2, TLR3 deficient models with CRISPR-Cas9 knockout respectively in basis of Caco-2-ACE2-N cell line. In addition, the key adapter MAVS responsible for IFNs induction was also depleted efficiently for use  and cytokine production was measured by ELISA. As shown in Recently, NLRP6 was demonstrated to mediate cellular immunity in intestinal epithelia cells in response to enteric RNA virus infection, by interacting with DHX15. In addition, activation of NLRP6 inflammasome accounts for IL18 maturation and production. To determine whether NLRP6 engages in the regulation of SARS-CoV-2 immunity in IECs, we established NLRP6 knockout cell line in basis of Caco-2-ACE2-N IECs and evaluated the cytokine production with SARS-CoV-2 GFP/ΔN-trVLP infection. We confirmed constitutive expression of NLRP6 in Caco-2-ACE2-N IECs, and found SARS-CoV-2 infection. Incompetent SARS-CoV-2 virus were incapable of increasing NLRP6 expression  in Transwells. Transcriptional change of immunity related genes was profiled with RT2 Profiler PCR Array Gene Expression Analysis. We found that CoV-2 epithelia-immunocytes coculture induced a profound inflammatory response in immunocytes, distinct from the immunological profile of Caco-2-ACE2-N cells infected with SARS-CoV-2  . GastroiManipulating infectious pathogenic virus is strictly restricted in BSL-3 laboratories, which hinders broad experimental research. Reverse genetics have profoundly advanced experimental viral study. Recently, several reverse genetics systems have been successfully created to produce fully infectious recombinant SARS-CoV-2 virus and replicons, providing powerful tools for broader study on SARS-CoV-2 beyond clinical isolates . In thisIn this study, we found that unlike lung epithelial cells, intestinal epithelial cell line Caco-2 presented relatively moderate immune response upon SARS-CoV-2 infection. Specifically, canonical anti-virus IFN, IFN-β, was not significantly induced, whereas IFNλ was dominant in anti-virus cellular immune response, which is coordinated with the findings in colon enteroid studies in response to SARS-CoV-2. The moderate anti-viral IFN production may partially explain why human colon epithelial cell line Caco-2 can support extensive viral replication and produce large amounts of viral particles in infection. ISG gene activation indicated inflammatory immune response. Of the induced proinflammatory cytokines, IL-18 was substantially increased. TNF-α, CXCL1, IP-10 were also mounted in the context of SARS-CoV-2 infection at variant timepoints. Thus, SARS-CoV-2 infection induces moderate but considerable anti-virus and inflammatory immune response in intestinal epithelial cells. The recently epidemic Omicron variant displayed less pathogenic in clinical. Research study reported that unlike previous SARS-COV-2 variant, Omicron isolates present less replication and lower viral load in Caco-2 cell when infected, and are highly sensitive to interferon treatment . This suSARS-CoV-2 is a positive-sense RNA virus belonging to the β-coronavirus genus. The innate immune system defense against RNA virus infection by developing multiple RNA recognizing mechanisms to induce numerous host defense molecules, including type I and type III interferons and proinflammatory cytokines and chemokines. The RLRs are commonly identified sensors that recognize viral RNA in the cytosol and are essential for triggering the innate immune response to RNA viruses in most cell types . The RLRWe also demonstrated that RIG-I and DHX15 were required for sensing SARS-CoV-2 and inducing cellular immune response through MAVS signaling in intestinal epithelial cells upon SARS-CoV-2 infection. Of note, RIG-I was mainly responsible for antiviral immunity and inflammatory cytokine production, while DHX15 also participated in regulation of IFNs but was specifically responsible for IL-18 production. The overlapping but distinctive contribution to SARS-CoV-2 immunity suggested interaction between DHX15 and RIG-I signaling. DHX15 appeared to interact with both K63- and K48-poly-Ub . UnanchoFurthermore, we found that NLRP6 engaged in the regulation of SARS-CoV-2 immunity in IECs. Depletion of NLRP6 diminished the immune response and cytokine production, especially IL-18. It has been reported that DHX15–NLRP6 sense RNA and subsequently regulate IFNs and IL-18 secretion in the control of intestinal viral infections . HoweverInfection of SARS-CoV-2 potentially provokes excessive production of proinflammatory cytokines and even systemic hyperinflammation, resulting in multiorgan failure to lethal damage. A few research studies have emphasized that immune dysregulation contributes to SARS-CoV-2 immunopathogenesis, dissecting the molecular mechanism that profound alteration in myeloid cells, T cells drives hyperinflammation and severe clinical manifestations . In our In summary, this work unveils the molecular basis of immune recognition of the SARS-CoV-2 virus, depicts mechanism of cellular response in IECs, and provides insights that intestinal immune cross-talk with other immune cells triggers amplified immunity Figure 7Human colon carcinoma cell line T84 (ATCC CCL-248) and human intestinal epithelial cells (IECs) line HT-29 (ATCC HTB-38) were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human colorectal adenocarcinoma Caco-2 (ATCC HTB-37) and Vero E6 (ATCC CRL 1586) were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.Mouse primary intestinal epithelial cells (IECs) were isolated from 6-week-old C57BL/6 mouse intestines. Briefly, intestines were cut into pieces and digested with 0.72mg/mL Dispase and 2.5mg/mL Collagenase IV for 30min. Cell suspension was collected and separated by Percoll gradient . Purified IECs were cultured in DMEM, supplemented with 10% FBS, 4mM glutamine, 20mM HEPES, and 1% penicillin/streptomycin. After culture, the purity of IECs was confirmed by flow cytometry with PE-anti-E-Cadherin antibody and FITC-anti-Cytokeratin 18 antibody.Caco-2 cells stably expressing ACE2 and nucleocapsid were generated by transduction with lentivirus containing ACE2/IRES-puro or nucleocapsid/IRES-G418 vector. Stably transduced cells were maintained in corresponding culture medium with 1μg/ml puromycin, and/or 1 mg/ml G418.For CRISPR-Cas9-mediated gene disruption, Caco-2-ACE2-N cells were transduced with Cas9 and sgRNA LentiVector and selected with antibiotics for at least 10 days. cells were subcloned and then lysed for western blot to identify knockout clones.To determine permissiveness of intestinal cell lines to SARS-CoV-2 viral entry, pseudo-typed virus VSV-SARS-CoV-2 with luciferase was used for infection assay. Cells were seeded in 96 well plates. VSV-SARS-CoV-2 was added at the indicated MOI. At 24 hpi, cells were harvested for luciferase assays following the Luciferase Assay System protocol . Luminescence was detected by Perkinelmer EnSight Microplate Reader.5 Caco-ACE2-N cells were seeded in 12-well plates overnight. Harvested culture supernatants from infected Caco-ACE2-N cells were 10-fold serially diluted with fresh medium. Seeded cells were inoculated with diluted supernatants respectively. After 1h infection, 1 mL of 0.6% microcrystalline cellulose  was added. The plates were incubated for another 3 days in incubator. On day 4, MCC was removed. 4% formalin was used to fix the cells. Plaques were quantified and recorded as PFU/mL.Viral titers in cell culture supernatant were determined by a modified plaque-forming assay with Caco-ACE2-N cells. Briefly, 5x10Total cellular RNA was isolated using Qiagen RNeasy Mini Kit according to the manufacturer’s instructions. Reverse transcription was performed using iScript™ cDNA Synthesis Kit (Bio-Rad). The primers for viral RNA were as follows: Vprimer1-CGAAAGGTAAGATGGAGAGCC and Vprimer2- TGTTGACGTGCCTCTGATAAG. Real-time qPCR was performed in 96-well plates in triplicates using SYBR Green Supermix (Bio-Rad) and StepOne Plus system (Applied Biosystems). For other immune genes detection, Taqman probes, GAPDH (Hs99999905_m1), ISG15 (Hs01921425_s1), OAS1 (Hs00973637_m1), IFITM3 (Hs03057129_s1), IFNB1 (Hs01077958_s1), IFNL1 (Hs00601677_g1), and IFNL2 (Hs00820125_g1), etc. were used for quantification of mRNA expression.The RT2 first strand kit (Qiagen) was used for the synthesis of the cDNA strand using 500ng of total RNA from samples extracted using the RNeasy kit (Qiagen). The customized RT2 Profiler PCR array kit was used for profiling immunological genes expression according to the manufacturer’s instructions. The average of the threshold cycle (CT) values from mock controls were used to normalize gene expression. Fold changes of mRNA expression between the noninfected and the infected conditions were analyzed using the ΔΔCT values.The supernatant from different groups were collected and cytokines were measured by human IFN-λ2, IFN-λ3, IL-18, CXCL1 and TNF-α ELISA kit (Biolegend) following manufacturer’s instructions.6 PBMCs were cocultured with 2×105 SARS-CoV-2 GFP/ΔN trVLP-infected (MOI=0.5) or mock Caco-2-ACE2-N cells in a 96-well U-bottomed plate and incubated for 16 h with the addition of brefeldin A at 10 h. After 16h coculture, cells were harvested and fixed with Fixation Buffer (Biolegend) for FACS staining.Human PBMC were purchased from SAILY Bio. 1x10For Transwell coculture, Caco-2-ACE2-N cells were inoculated and infected with mock control or pseuo-SARS-CoV-2. After 48h inoculation, supernatants were replaced with fresh medium suitable for PBMC. Then CoV-2-infected or mock Caco-2-ACE2-N cells were replated onto Transwell inserts. PBMCs were placed into the bottom chamber for 16h of coculture. After that, PBMC were harvested for immune genes profiling with customized RT2 Profiler PCR Array Gene Expression Kit (Qiagen). Cocultures were performed with at least three biological replicates per condition. For depletion assay, 20μg/mL αIL-18 neutralizing antibody was added at the start of the coculture. For conditional medium assay, conditional medium of Caco-2-ACE2-N cells with 60h CoV-2 infection was added into PBMC with 1:1 ratio of PBMC culture medium. After 16h culturing, PBMC were harvested and MAIT cells were analyzed by flow cytometry. For rIL18 addition, 10ng/mL rIL18 was added into PBMC culture for 16hs.Flow cytometry was exploited for analysis of cytokine expression after coculture. Surface staining was performed as follows. MAIT cells were defined by anti-CD3-FITC (BioLegend), anti-CD161-APC/Cy7 (BD Pharmingen) and anti-Vα7.2-APC (BD Pharmingen). NK cells were identified by anti-CD56-BV421 (BioLegend). After surface staining, cells were aliquoted into 3 parts and incubated with anti-IFNγ-PE (clone B27), anti-TNFα-PE (clone MAb11), or anti-GzB-PE (clone GB11) for intracellular staining at 4°C for 30min. Dead cells were excluded with 7-AAD. Cells were acquired on BD Lyric analyzer (BD Biosciences) and analyzed with FlowJo software.Cells were washed twice with PBS on ice and lysed with lysis buffer  containing protease inhibitor Cocktail (ThermoFisher Scientific) for immunoblot analysis. Protein samples were dissolved in SDS sample buffer. After electrophoresis, separated proteins were transferred onto PVDF membrane. The membrane was then blocked with 5% nonfat milk. After incubation with specific primary antibody, HRP-conjugated secondary antibody was applied. The membranes were scanned by an enhanced chemiluminescence system (ThermoFisher Scientific).All data were presented as means ± SEM as indicated and analyzed by GraphPad PRISM software. Unpaired t test, Student’s paired t test, one-way ANOVA with multiple comparisons were used. *p < 0.05; **p < 0.01; ***p < 0.001.The original contributions presented in the study are included in the article/YL designed the experiments and wrote the manuscript. LZ, YZ and RW mainly designed and conducted the experiment. LZ and YZ contributed to equal work. RW conducted flow cytometry, qPCR and cell culture. XL completed molecular construction and provided plasmids. JZ performed western blot and ELISA, MT helped to revise the manuscript. All authors contributed to the article and approved the submitted version.


TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.A limited number of studies are devoted to regulating  TRIP6, 7q22.1) gene encodes for highly conserved 50 kDa protein (476 amino acid residues). TRIP6 is structurally organized into an N-terminal proline-rich domain, Crm-1-dependent nuclear export signal (NES), and three tandemly arrayed LIM domains  followed by PDZ-binding motif TTDC  at its C terminus , pGL4.24[luc2P/minP], and pNL1.1TK[Nluc/TK] vectors were purchased from Promega . 5′ flanking sequence of the TRIP6 was taken from Ensembl genome browser (Homo sapiens GRCh38.p12) for TRIP6-201 transcript (ENST00000200457.9). Sequence −936/+111 (where +1 means TRIP6 transcription start) was amplified from MCF-7/PacR genomic DNA by PCR and cloned into pGL3-Promoter vector via KpnI and NcoI sites. The inserted sequence was subcloned by PCR into pGL4.10[luc2] and pGL4.24 vectors . The construction of plasmids, primers and PCR conditions are summarized in pGL3-Promoter, pGL4.10 vector  and SLC12A9-TRIP6 intergenic region (sequence −375 to −1) (] vector . In fact5 to −1) .TRIP6 proximal promoter (sequence −157 to −12 relative to the TRIP6 transcription start) but not the TRIP6 distal promoter (sequence −936 to −157) is sufficient to drive TRIP6 expression in both MCF-7 cells and taxane-resistant MCF-7 sublines (We assessed firefly (Fluc2P) and deep-sea shrimp Nanoluc (Nluc) luciferase activities in MCF-7 cells and taxane-resistant MCF-7 sublines co-transfected with a series of 5′ and 3′ truncated constructs and the normalization pNL1.1.TK[Nluc/TK] vector. Firstly, the experiments showed that the sublines . Secondlcis-acting regulatory elements in the active human TRIP6 proximal promoter, we scanned the −200 to −1 sequence with Jaspar 2022 transcription factor binding profiles (≥93% relative profile score threshold) (TRIP6 transcription in MCF-7 cells.To identify reshold)  55]. We. Wecis-areshold) A. Remarkreshold) B, indicap = 0.036) Fluc2P/Nluc activity in MCF-7 cells but not in PacR and 0035R cells . Yet, the other element(s), probably the GC box [Furthermore, the −117/−72 construct exhibited a two-fold increase in Fluc2P/Nluc activity compared to the −72 /−12 construct in all tested cells B. The ree GC box  or M-CATe GC box , increasTRIP6 transcription in MCF-7 cells and taxane-resistant MCF-7 sublines. The predicted E box, GC box, CT box, and M-CAT might contribute to the TRIP6 promoter activity; however, there would still be other unidentified motifs. In addition, the AP1 site likely enhances TRIP6 transcription only in MCF-7 cells, as it does not appear to modulate the response in PacR and 0035R cells.Collectively, CRE unambiguously promotes cis [TRIP6 dependence on the CRE motif   with CpG methylation level, indicating that DNA methylation might regulate TRIP6 expression also in breast tumors.Furthermore, we assessed A study) B. TRIP6 TRIP6 differential methylation between MCF-7 cells and taxane-resistant MCF-7 sublines , we employed 16 probes that targeted to defined gene regions TSS200 , TSS1500 , 1st Exon, gene body (region between ATG and stop codon), and 5′ and 3′ untranslated regions (UTRs). It is worth noting that the distal promoter region substantially overlaps with the last exon of the SLC12A9 gene. Although the whole TRIP6 gene sequence analysis showed higher methylation in PacR cells , in fact, methylation of the distal TRIP6 promoter region (TSS1500) significantly changed. We found no differential methylation between MCF-7 cells and taxane-resistant MCF-7 sublines in the TRIP6 proximal promoter (TSS200), gene body, and 1st exon, in line with bisulfite sequencing data  and protein expression only in a small number of samples (N = 20) due to limitations in sample size. Whereas all breast tumor tissues expressed TRIP6 mRNA, we detected TRIP6 protein expression by immunoblotting in 17 of the 20 examined samples  in breast cancer tissues. (We assessed  samples A. TRIP6 tissues. B.TRIP6 mRNA expression levels between adjuvant (N = 82) and neoadjuvant (N = 13) cohorts (p = 0.86). Furthermore, we found no statistically significant correlation between TRIP6 mRNA level and DFS or OS, independent of the type of therapy. High TRIP6 mRNA expression was observed in premenopausal (p = 0.033) and progesterone receptor positive (p = 0.020) breast cancer in the adjuvant cohort of breast cancer patients but not in the neoadjuvant cohort  (We found no difference in ctively) .TRIP6 mRNA expression in human organs except for skeletal muscle, brain, and leukocytes [TRIP6 expression in various neoplasms might indicate disrupted gene regulatory mechanisms during cancerogenesis [TRIP6 overexpression in paclitaxel-resistant MCF-7/PacR subline [TRIP6 expression in MCF-7 cells as well in paclitaxel-resistant cells have not been described in detail.An early study demonstrated a ubiquitous 1.8-Kb ukocytes . Recentlukocytes . Additioogenesis ,61. Furt subline , yet theTRIP6 copy number gain and the activity of the cyclic-AMP response element in the hypomethylated TRIP6 proximal promoter contribute to the high TRIP6 protein level in parental MCF-7 cells. Although the AP-1 motif seems more important in parental MCF-7 cells, copy number gain but not altered regulation of the TRIP6 promoter instead contribute to TRIP6 overexpression in both taxane-resistant MCF-7 sublines  gene copy number and mRNA level increased in parallel with the ABCB1  gene copy number and mRNA level. In agreement, FISH analyses unambiguously validate TRIP6/ABCB1 co-amplification in taxane-resistant MCF-7 sublines  and mitoxantrone-resistant (EPG85-257RNOV) human gastric carcinoma cells, the former cells having also upregulated ABCB1 [Strikingly, the sublines . Despitecompound . To dateed ABCB1 .ABCB1 amplified region, referred to as ABCB1 amplicon, is often documented in drug-resistant sublines [ABCB1 amplicon core as a 1 Mb region commonly detected in ABCB1 overexpressing cells [ABCB1 amplicon was bordered by semaphorin 3D  and cyclin-dependent kinase 6  [TRIP6  might indicate the extraordinary size of the ABCB1 amplicon in MCF-7 sublines.The sublines ,65,66,67The most famous breakage–fusion–break (BFB) mechanism of amplicon formation leverages specific sequences referred to as fragile sites . What meABCB1/TRIP6 co-amplification, we noticed the loss of der(18)t in both taxane-resistant MCF-7 sublines. The impact of this aberration in the context of taxane resistance is unknown.Beyond TRIP6 expression could theoretically be caused by different transcriptional regulations at the TRIP6 promoter level, we analyzed the responsiveness of the TRIP6 promoter by dual-luciferase assay. The TRIP6 proximal promoter region (−157 to −45) controlled luciferase expression in MCF-7 cells  relative to gene transcription start [TRIP6 remains to be determined in further studies.Since the enhanced -7 cells . The mos-7 cells . Disruptgulation . A genompromoter , but itson start  and methon start . As testTRIP6 mRNA expression. Recently, we revealed no clinicopathological association of the TRIP6 mRNA expression level in ovarian cancer [TRIP6 expression in sensitive and taxane-resistant MCF-7 breast cancer cell lines, we evaluated TRIP6 mRNA expression against clinical data of breast cancer patients who had undergone taxane-containing regimens. So far, Zhao et al. have analyzed TRIP6 protein expression in breast cancer from the Chinese cohort [Finally, we evaluated the clinical data of breast cancer patients with n cancer . To highe cohort . UnfortuTRIP6 expression in MCF-7 cells. Furthermore, increased TRIP6 copy number contributes to high TRIP6 expression in MCF-7 cells in vitro. Co-amplification of TRIP6 with ABCB1 underlies TRIP6 upregulation in two taxane-resistant MCF-7 sublines. Cytogenetic analyses showed that amplicon arose from intact chromosome 7. In addition, we observed a loss of derivative chromosome der(18)t in both sublines, with an unknown relation to taxane resistance. Moreover, the present study has not found direct prognostic or predictive relevance of TRIP6 for better tailoring breast cancer management at the clinics. Instead, the analysis of breast tumor of a neoadjuvant cohort revealed TRIP6 mRNA expression level associations with positive progesterone receptor expression status and premenopausal status.This study presents compelling evidence that the cyclic AMP response element (CRE) located within the stable hypomethylated proximal promoter controls TRIP6 proximal promoter might act as another important regulatory site in regulation of TRIP6 expression. The relevance of our functionally valid observation for clinical course of breast and other cancer(s), including eventual utility of TRIP6 as a target for new therapy design, shall be evaluated by follow-up studies.Collectively, we propose that


Mitochondrial disruption leads to the release of cytochrome c to activate caspase-9 and the downstream caspase cascade for the execution of apoptosis. However, cell death can proceed efficiently in the absence of caspase-9 following mitochondrial disruption, suggesting the existence of caspase-9-independent cell death mechanisms. Through a genome-wide siRNA library screening, we identified a network of genes that mediate caspase-9-independent cell death, through ROS production and Alox5-dependent membrane lipid peroxidation. Erk1-dependent phosphorylation of Alox5 is critical for targeting Alox5 to the nuclear membrane to mediate lipid peroxidation, resulting in nuclear translocation of cytolytic molecules to induce DNA damage and cell death. Consistently, double knockouts of caspase-9 and Alox5 in mice, but not deletion of either gene alone, led to significant T cell expansion with inhibited cell death, indicating that caspase-9- and Alox5-dependent pathways function in parallel to regulate T cell death in vivo. This unbiased whole-genome screening reveals an Erk1-Alox5-mediated pathway that promotes membrane lipid peroxidation and nuclear translocation of cytolytic molecules, leading to the execution of cell death in parallel to the caspase-9 signaling cascade. During intrinsic apoptosis, mitochondrial outer membrane permeabilization leads to the release of cytochrome c into the cytosol to form apoptosome with Apaf-1 and caspase-9, resulting in the activation of caspase-9 ,3,4. CasAlthough the caspase-9-dependent pathway can rapidly trigger apoptosis after mitochondrial disruption, it appears that interruption of caspase-9-dependent apoptosis may shift cells to engage other cell death mechanisms. Caspase-9-knockout mice display perinatal lethality with prominent defects in brain development that may be associated with defective cell death in neuronal progenitor cells ,24. Howe−/− mice [−/−). However, caspase-9−/− T cells efficiently underwent cell death induced by different stimuli. Moreover, we did not detect T cell accumulation in T/casp9−/− mice, suggesting that the existence of other cell death mechanisms independent of caspase-9 in T cells. To define the caspase-9-independent cell death pathway downstream of mitochondrion disruption, we performed a genome-wide siRNA library screening for genes involved in cell death using caspase-9-deficent Jurkat T cells. Our data suggest that mitochondrial disruption leads to Erk1-dependent phosphorylation and nuclear membrane translocation of Alox5, resulting in membrane lipid peroxidation, nuclear entry of nucleases and cytolytic molecules to induce cell death.Caspase-8-independent cell death mechanisms have been intensively studied. Rip1 and Rip3 have been shown to mediate necroptosis when caspase-8 activation is inhibited ,39,40,41−/− mice ,24. We tflox mice [flox/flox mice with T cell-specific knockout of caspase-9 (T/casp9−/−). Alox5−/− mice (The Jackson Laboratory) were crossed with T/casp9−/− mice to obtain T/casp9−/−Alox5−/− mice. Experiments were performed according to federal and institutional guidelines and with the approval of the Institutional Animal Care and Use Committee of Baylor College of Medicine (AN-2099) and the Houston Methodist Research Institute (IS00006582).Lck-cre mice (The Jackson Laboratory) were bred with caspase-9lox mice  to obtaiJMR cells , a caspaCells were cultured in 96-well tissue culture plates in the presence or absence of indicated treatments for different time. The cells were stained with APC-annexin V  and 5 μg/mL propidium iodide (PI) (ThermoFisher), followed by flow cytometry analyses. Percentage of cell death induced by treatments with different apoptosis stimuli was calculated from live cells of untreated and treated groups as follows: (untreated-treated)/treated × 100%. In some experiments, 10 μM necrostatin-1, necroX-5  or carbobenzoxy-valyl-alanyl-aspartyl-O-methyl-fluoromethylketone  was included in the culture. Cells were also stained with APC-conjugated Annexin V  in some assays. Mouse T cells were stimulated cultured with 5 μg/mL Concanavalin A (con A) ConA and 100 U/mL IL-2 for 48 h and used for different assays.S271A-GFP or GFP alone were transfected into JMR cells by electroporation using the Neon transfection system (Life Technologies). After 24 h culture, the cells were treated with 6 μM ABT-263 or solvent control. The loss of live GFP+ cells after ABT-263 treatment was determined 24 h later.To determine the effects of phosphorylation of Alox5, substitution of Serine 271 to Alanine (S271A) in Alox5 was performed using the site-directed mutagenesis kit . Plasmids expressing Alox5-GFP , Alox5To measure ROS, JMR cells were cultured in the presence or absence of ABT-263 for 12 h. The cells were then incubated with 5 μM Mito-SOX (Life Technologies) at 37 °C for 30 min. In some experiments, cells were also stained with APC-annexin V, followed by flow cytometry. To determine lipid peroxidation, JMR cells with or without ABT-263 treatment as above were incubated with 5 μM BODIPY 581/591 C11 (Life Technologies) at 37 °C for 1 h and analyzed by flow cytometry.S271A-GFP expression plasmids were transfected into JMR cells by electroporation using the Neon transfection system (Life Technologies) and cultured for 24 h. The cells were then treated with ABT-263 or solvent control for 8 h, followed by cytospin onto glass slides and DAPI staining. The staining was examined using a Nikon Eclipse 80i fluorescence microscope.Cells treated with cell death stimuli for 12 h were added to glass slides by cytospin and incubated with monoclonal mouse anti-TIA-1 , monoclonal mouse anti-AIF  or polyclonal rabbit anti-EndoG . The cells were then incubated with Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit IgG (Life Technologies). The nuclei were counterstained with DAPI (Thermo Scientific). Cells treated with cell death stimuli for 24 h were used for TUNEL staining using the Click-iT TUNEL assay kit (Life Technologies). The nuclei were counterstained with DAPI. Alox5-GFP or Alox5S217 . The blots were also probed with anti-GFP. Total lysates were used for Western blot by probing with anti-FLAG . JMR cells transfected with siRNA targeting specific genes or non-targeting siRNA control were lysed for Western blot by probing with specific antibodies. The following antibodies were used for Western blot: rabbit polyclonal or monoclonal antibodies to Alox5, ERK1, p38 MAPK, p44/42 MAPK (ERK1/2), phosphor-p44/p42 MAPK (p-ERK1/ERK2), RAD51, SAPK/JNK, SESN2 , Alox5AP, DHODH, HADHA, MGST1, TIA-1 (Abcam), EndoG (ProSci), OXR1  and PDP1 (Sigma); and mouse monoclonal antibodies to AIFM1, API5, PNKP (Santa Cruz Biotechnology), caspase-3, caspase-6 caspase-7, caspse-9, phosphor-p38 MAPK, phosphor-SPAK/JNK , FLAG (Sigma) and GFP (Origene). The blots were also probed with mouse monoclonal anti-α tubulin (Santa Cruz Biotechnology) to ensure equal loading.pCMV6-Alox5-GFP (Origene) was transfected into 293T cells with vectors expressing FLAG-tagged MAPKs. The cells were lysed 24 h later and immunoprecipitated with anti-GFP (Origene), followed by Western blot with anti-phospho-Alox5p values were determined by a two-tailed Student’s t-test using the GraphPad Prism software.Data are presented as mean ± SD, and flox mice [−/−). As expected, caspase-9 deletion led to virtually complete suppression of activation of effector caspases, including caspases-3, -6 and -7, in T cells treated with etoposide  in an Alox5-GFP fusion construct. Interestingly, transfection with Alox5S271A-GFP significantly suppressed ABT-263-induced cell death in JMR cells, whereas wild-type Alox5-GFP had no such effect (It has been shown that phosphorylation of Alox5 at Serine 271 affects nuclear membrane localization of Alox5 ,66. Wheth effect b. This sS271A-GFP did not show nuclear membrane translocation after treatment with ABT-263 (MAP kinases play an important role in the regulation of cell survival and cell death ,68. Our  ABT-263 a, indica ABT-263 b. Togeth−/− mice with Alox5−/− mice. We did not observe T cell accumulation in T/casp9−/− or Alox5−/− mice compared to wild-type controls (−/−Alox5−/− mice displayed significant increases in the percentages and total numbers of CD4+ and CD8+ T cells (−/− and Alox5−/− T cells treated with etoposide or staurosporine showed similar levels of cell death (−/−Alox5−/− T cells displayed impaired cell death (−/−Alox5−/− T cells was significantly reduced compared to that in wild-type, caspase-9−/− and Alox5−/− T cells (−/−Alox5−/−, but not caspase-9−/− or Alox5−/− T cells (−/− T cells, but not in Alox5−/− or caspase-9−/−Alox5−/− T cells (−/− and caspase-9−/−Alox5−/− T cells, only caspase-9−/−Alox5−/− T cells showed significant reduction in nuclear DNA damage by TUNEL staining (In order to determine whether Alox5 is also important for the induction of caspase-9-independent cell death in vivo, we crossed T/casp9controls a. In con T cells a, while  T cells . This inll death b. Howevell death b. Simila T cells b. TUNEL  T cells c. Treatm T cells d. While staining c. In casCaspase-9-independent cell death involved nuclear DNA damages as indicated by TUNEL staining c. SeveraThe protective functions of DNA repair enzymes suggest that DNA damage is important for the execution of caspase-9-independent cell death. In caspase-dependent apoptosis, nuclear translocation of DDF40/CAD after cleavage of DDF45/ICAD leads to DNA fragmentation and apoptosis ,8,9. Lip−/− or Alox5−/− mice (−/−Alox5−/− mice did not display EndoG nuclear translocation after ABT-263 treatment (−/− and Alox5−/− T cells, but not in caspase-9−/−Alox5−/− T cells (−/−Alox5−/− T cells (We next examined whether double knockouts of caspase-9 and Alox5 affect nuclear import of cytolytic molecules during cell death. We observed nuclear translocation of EndoG after ABT-263 treatment in T cells from wild-type, T/caspase-9−/− mice a. In conreatment a. Endogereatment b. After  T cells b. Nuclea T cells c. TogethThrough a genome-wide siRNA screening using a caspase-9-deficient Jurkat T cell line, we identified an Erk1-Alox5-mediated cell death pathway in parallel to caspase-9 signaling cascade. Several mitochondrial proteins, including DHODH, HADHA and PDP1, were found to promote ROS production after treatment with a Bcl-2/Bcl-xL inhibitor, ABT-263. Mitochondrial disruption led to ERK1-dependent phosphorylation of Alox5 at Serine 271 and promoted translocation of Alox5 to nuclear membranes. An Alox5 mutant with Serine 271 substitution dominantly interfered with caspase-9-independent cell death. Activation of Alox5 was critical for inducing nuclear translocation of nucleases and cytolytic molecules and the induction of DNA damage. While knockout of caspase-9 in T cells did not cause T cell expansion in mice, double knockouts of caspase-9 and Alox5 in T cells led to severe defects in cell death with significant T cell accumulation. This study suggests that mitochondrial disruption induces the activation of Alox5-dependent membrane lipid peroxidation and translocation of nucleases to the nucleus, resulting in caspase-9-independent cell death. Although the features of caspase-9-independent cell death were grossly similar to the classic caspase-dependent apoptosis with nuclear translocation of DNases and DNA damages, it employed distinct cell death machinery involving oxidative stress, MAPK activation and lipid peroxidation to promote nuclear entry of cell death molecules.Our genome-wide screening suggests that several sets of genes with opposite functions in regulating oxidative stress and DNA fragmentation also counteract each other in mediating caspase-9-independent cell death. Notably, genes promoting ROS production and oxidative stress, including PDP1, DHODH, HADHA, Alox5 and Alox5AP, were involved in enhancing caspase-9-independent cell death. In contrast, genes with anti-oxidative functions, including MGST1, SESN2 and OXR1, showed significant protection of cell survival. In the downstream, genes involved in DNA fragmentation, such as EndoG and TIA-1, promoted cell death, while DNA repair enzymes, RAD51 and PNKP, inhibited cell death. These data support an important role for oxidative stress and nuclear DNA fragmentation in the induction and execution of caspase-9-independent cell death.Activation of Erk1 leads to its translocation to the nucleus where it phosphorylates transcription factors and cell cycle regulators to promote cell survival and proliferation . A deathROS production and lipid peroxidation have been implicated in apoptosis, autophagic cell death and ferroptosis . Alox5-d−/− or Alox5−/− mice suggests that caspase-9- and Alox5-dependent cell death mechanisms function in parallel of each other. T cell accumulation in T/caspase-9−/−Alox5−/− mice indicates that Alox5-dependent cell death is indeed critical for carrying out intrinsic cell death when caspase-9-dependent pathway is absent. Caspase-9-independent intrinsic cell death is mediated by distinct signaling molecules without the involvement of caspase-9-dependent caspase cascade. Although the early signaling events are different from caspase-dependent apoptosis, the late stages, including nuclear translocation of DNase and DNA damages, are similar. This suggests that cells undergoing such caspase-9-independent intrinsic cell death are efficiently cleared by phagocytosis without causing inflammation.A lack of T cell accumulation in T/caspase-9The Erk1-Alox5-dependent peroxidation of nuclear membrane is a potentially important signaling pathway for the execution of cell death shared by apoptosis and non-apoptotic forms of cell death. Its relationship to apoptosis, necrosis and ferroptosis remains to be determined. This caspase-9-independent cell death mechanism may play an important role in the regulation of diverse biological and pathological processes . Erk-dep


K14-Cre;c-Ang1 and Col2.3-Cre;c-Ang1 transgenic mice that systemically and locally overexpress cartilage oligomeric matrix protein-Ang1, respectively. We also investigated the impacts of Tie2 inhibitor and AMD3100 on hematopoietic development. Transgenic mice exhibited excessive angiogenic phenotypes, but K14-Cre;c-Ang1 mice showed more severe defects in growth and life span with higher presence of Ang1 compared with Col2.3-Cre;c-Ang1 mice. Dissimilar to K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not show impaired BM retention or senescence of HSCs, erythropoietic defect, or disruption of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis. However, these mice exhibited a defect in platelet production depending on the expression of Tie2 and globin transcription factor 1 (GATA-1), but not GATA-2, in megakaryocyte progenitor (MP) cells. Treatment with Tie2 inhibitor recovered GATA-1 expression in MP cells and platelet production without changes in circulating RBC in transgenic mice. Consecutive AMD3100 administration not only induced irrecoverable senescence of HSCs but also suppressed formation of RBC, but not platelets, via correlated decreases in number of erythroblasts and their GATA-1 expression in B6 mice. Our results indicate that genetic overexpression of Ang1 impairs hematopoietic development depending on its level, in which megakaryopoiesis is preferentially impaired via activation of Ang1/Tie2 signaling, whereas erythropoietic defect is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.While supplemental angiopoietin-1 (Ang1) improves hematopoiesis, excessive Ang1 induces bone marrow (BM) impairment, hematopoietic stem cell (HSC) senescence, and erythropoietic defect. Here, we examined how excessive Ang1 disturbs hematopoiesis and explored whether hematopoietic defects were related to its level using  Genetic overexpression of COMP-Ang1 impairs BM microenvironment, induces HSC senescence, and leads to anemic and thrombocytopenic disorders depending on its systemic and local overexpression through a dysregulated activation of the Ang1/Tie2 signaling and/or SDF-1/CXCR4 retention axis. The underlying mechanisms through which transgenic overexpression of COMP-Ang1 regulates megakaryopoietic and erythropoietic developments were investigated. This study demonstrates that genetic overexpression of COMP-Ang1 preferentially impairs megakaryopoiesis via activation of Ang1/Tie2 signaling. The current findings also highlight that erythropoietic defect by excessive COMP-Ang1 is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.1 Although the exact role of Ang1 on hematopoiesis is not yet well understood, numerous studies have indicated that the Ang1/Tie2 signaling axis plays essential roles in the long-term adhesion of hematopoietic stem cells (HSCs) to bone marrow (BM) niches, maintenance of HSCs in the BM, and hematopoietic development.2-6 Studies have also demonstrated the crucial roles of the Ang1/Tie2 signaling axis in regulating proliferation, differentiation, and maturation of megakaryocytes8 and erythroblasts.10 These studies suggest a relationship between angiogenesis and hematopoietic development, in which the Ang1/Tie2 axis acts as a key signaling regulator.Angiopoietin-1 (Ang1) controls vascular development and maintenance through its interaction with its specific receptor, Tie2.11 While supplemental Ang1 shows various impacts on endothelial cell survival, vascular anti-inflammation, and neovascularization,12-14 studies have also supported its beneficial role in hematopoiesis. Administration of Ang1 via plasmid injection facilitated hematopoietic recovery in radiated mice by increasing Tie2 and Notch expression.15 Genetic overexpression of Ang1 in type I collagen (Col1a1)-expressing osteoblasts increased long bone mass and mineral density.16 These reports not only indicate an interaction among angiogenesis, hematopoiesis, and osteogenesis but also imply the induction of BM microenvironmental rebuilding and hematopoietic recovery in response to the supplementation with Ang1.Total body irradiation, chemotherapy, and/or conditioned BM transplantation often impair the BM microenvironments that are critical for BM retention and functional maintenance of HSCs and hematopoietic progenitor cells (HPCs).17 Long-term administration of COMP-Ang1 by adenoviral vector expression exerted greater therapeutic benefits than supplementation with the recombinant human (rh) protein.18 We previously generated transgenic mice that overexpress COMP-Ang1 in Keratin 14 (K14)-expressing cells and investigated whether genetic supplementation with COMP-Ang1 also positively affected bone growth and function of HSCs. The transgenic mice exhibited severe and excessive angiogenesis in the body together with an impaired BM microenvironment and HSC senescence.19 The mice also exhibited a defect in erythroblastic maturation, increased production of inflammatory cytokines, dysregulated expression of globin transcription factor 1 (GATA-1), and imbalanced retention of the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis in the BM.20 Our previous findings indicated that persistent and prolonged supplementation with Ang1 by transgenic overexpression did not induce a benefit on the BM microenvironment and hematopoiesis, rather than it dysregulates hematopoietic development via an imbalanced activation or disruption of the Ang1/Tie2 signaling axis, SDF-1/CXCR4 retention axis, GATA-1 expression, or all of these mechanisms.Cartilage oligomeric matrix protein (COMP)-Ang1 is a chimera of Ang1 that has higher solubility and stability with greater angiogenic potential than native Ang1.Col1a1-expressing cells and compared the results with hematopoietic events derived from mice that express Ang1 in K14-expressing cells  production, and this defect is closely linked to HSC senescence and disruption of the SDF-1/CXCR4 retention axis.Here, we investigated the underlying mechanisms through which transgenic overexpression of COMP-Ang1 regulates megakaryopoietic and erythropoietic developments. We explored whether erythropoiesis and megakaryopoiesis are differently affected in relation to the amount of COMP-Ang1 generated. We generated mice that express local COMP-Ang1 in ng cells . To clarK14 (2.2 kb)-Cre;inducible-COMP-Ang1-transgenic  and Col1a1 (2.3 kb)-Cre;inducible-COMP-Ang1-transgenic  mice by crossing the K14-Cre22 and the Col2.3-Cre mice with inducible-COMP-Ang1-transgenic mice,23 respectively  analysis following the methods described previously22 and used in all experiments regardless of gender. All animals were cared for in the guidelines of the Animal Care Committee of Jeonbuk National University.This study was carried out in strict accordance with the recommendations in the Guide for Animal Care and Use of Jeonbuk National University. Before experiments, all procedures were approved by the University Committee on Ethics in the Care and Use of Laboratory Animals according to the ARRIVE guidelines. We generated ectively . Here, wrhCOMP-Ang1 or in combination with Tie2 kinase inhibitor (250 nM). After incubation, these cells were processed for flow cytometric determination of GATA-1 expression in MP cells. Parts of transgenic mice and their littermate controls were peritoneally injected with Tie2 kinase inhibitor (10 mg/kg), and levels of circulating platelets and GATA-1 expression in BM-conserved MP cells were determined. Alternatively, B6 mice (4-weeks-old) received subcutaneous injection of AMD3100  diluted in sterile endotoxin-free phosphate buffered saline (PBS) for 1 day or 21 consecutive days. Cells from the BM, spleen, and peripheral blood (PB) were collected 12 h after the last administration of AMD3100, and the role of SDF-1/CXCR4 axis on hematopoietic development was further investigated.Megakaryocytic progenitor (MP) cells were isolated from C57BL/6 (B6) mice  and incubated with various concentrations (0–1000 ng/mL) of -Sca-1+c-Kit+ (LSK) cells and CD150+CD48-LSK cells were phenotypically identified using following antibodies: lineage markers PE-Cy7-conjuaged anti-CD3 (cat.#552774), anti-B220 , anti-CD11b (cat.#552850), anti-Gr-1 (cat.#552958), or anti-TER-119 (cat.#557853) (All of these markers were from BD Biosciences); FITC-conjugated anti-Sca-1  or PE-conjugated anti-Sca-1 ; APC-conjugated anti-c-Kit ; perCP/Cy5.5-conjuated anti-CD150 ; and APC-Cy7conjugated anti-CD48 . Senescence-associated-β-galactosidase  activity in LSK, CD150+CD48-LSK, or Lin-Sca-1+CD29+CD105+ cells that had been already incubated with the cell surface markers were analyzed using C12FDG . In addition, HPCs including granulocyte-monocyte progenitors (GMPs), common myeloid progenitors (CMPs), and megakaryocyte-erythroid progenitor (MEPs) were defined using PE-conjugated anti-FcR (BD Biosciences), perCP/Cy5.5-conjuated anti-CD34 (BioLegend), or PE-conjugated anti-IL-7R (BD Biosciences) as a basis for LSK cell markers. Erythroblasts at four stages  were discriminatively gated with PE- or FITC-conjugated anti-CD71 (BD Biosciences) and PE-Cy7-conjugated anti-TER-119 (BD Biosciences) as described previously.20 MP cells were analyzed with PE-Cy7-conjugated anti-lineage markers (BD Biosciences), APC-Cy7-conjugated anti-Sca-1 , Texas Red-conjugated anti-c-Kit , PE-conjugated anti-FcR , APC-conjugated anti-CD9 , and PerCP-eFluor-conjugated anti-CD41 . In this study, MP cells were defined as Lineage−, IL7Rα−, Sca-1−, c-Kit+, CD9+, FcRlow, and CD41+ cells. Levels of GATA-1 (cat.#939101), GATA-2 (cat.#614003), and CXCR4 (cat.#85578) in BM- and spleen-conserved MP cells were determined with PE-conjugated antibodies  after fixation and permeabilization. Numbers of Ki-67-positive BM-derived MP cells were measured using FITC-conjugated anti-Ki-67 antibody . BM-derived hematopoietic stem progenitor cells (HSPCs) expressing Tie2 were measured using flow cytometer  after staining with PE-conjugated antibody . To evaluate the impacts of COMP-Ang1 overexpression in Col2.3-Cre;c-Ang1 mice on BM-mesenchymal stem cells (MSCs), populations of Lin-Sca-1+CD29+CD105+ cells were phenotypically identified as MSCs using the same PE-Cy7-conjugated lineages as used for identification of hematopoietic cells. Unless specified otherwise, experiments using transgenic mice and their littermate controls were performed at 3 weeks of age.Cells were isolated from BM, spleen, and PB of transgenic mice, littermate controls, or B6 mice. After removal of RBC and washing with PBS, frequencies of cells were analyzed using a flow cytometer  installed at the Center for University-Wide Research Facilities of Jeonbuk National University. Cell populations were sequentially gated using FlowJo software program . In brief, populations of Lin5 cells) were also collected from PBS (vehicle)- or AMD3100 (5 mg/kg)-injected mice (CD45.2). These cells were transplanted by tail vein injection together with BM cells (4 × 105 cells) of competitor mice (CD45.1) into recipients (CD45.1/2) that were lethally irradiated with 10 Gy as described above. Donor cell-derived repopulating capacity was assessed using PB of the recipients after 5 months of transplantation. In addition, levels of p15, p16, p19, and p21 mRNAs in BM cells of vehicle- and consecutive AMD3100 (5 mg/kg)-treated mice were determined by real time PCR analysis. In this assay, cDNA was synthesized using 1 µg of BM cell-derived total RNA for each sample and AmpiGeneTM cDNA Synthesis kit (Enzo Life Sciences). Power SYBR® Green PCR Master Mix (Life Technologies) was used to detect accumulated PCR products during cycling using ABI StepOnePlus sequence detection system (Applied Biosystems). Oligonucleotide primers specific to p15, p16, p19, p21, and GAPDH were designed as follows; ccctgccacccttaccaga (forward) and cagatacctcgcaatgtcacg (reverse) for p15, gtcgcaggttcttggtcact (forward) and tctgcaccgtagttgagcag (reverse) for p16, gccggcaaatgatcatagag (forward) and cagcaagagctggatcagaa (reverse) for p19, tgtccgtcagaacccatc (forward) and aaagtcgaagttccatcgcc (reverse) for p21, and gacggccgcatcttcttgt (forward) and cacaccgaccttcaccatttt (reverse) for GAPDH.BM, spleen, and PB cells were collected from B6 mice administered with AMD3100 (0–5 mg/kg) for 1 day or consecutive 21 days. The frequencies of BM- and PB-conserved cells were analyzed using a flow cytometer . The frequencies of erythroblasts at 4 stages and MP cells and GATA-1 expression in these cells were determined as described above. BM cells  staining, circulating blood cell counting, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) see the t-test (n ≥ 6) or by a non-parametric test . One-way ANOVA followed by the Scheffe’s multiple range test was used for multiple comparisons among more than two groups. The Kolmogorov–Smirnov test was used to test the normality of data sets. A value of P < 0.05 was considered statistically significant.All data are expressed as the mean ± SD and analyzed using SPSS program (ver. 12.0). Differences between two groups were analyzed by unpaired Student’s K14-Cre;c-Ang1 mice,20 the Col2.3-Cre;c-Ang1 mice exhibited more branched and enlarged vessels around the hind limbs -positive CD150+CD48-LSK cells in Col2.3-Cre;c-Ang1 mice were comparable to those in littermate controls  and SDF-1 in BM supernatants , the Col2.3-Cre;c-Ang1 mice showed a similar number of RBC to that in littermate controls . Numbers of circulating platelets . The Col2.3-Cre;c-Ang1 mice also exhibited significantly fewer numbers of BM MP cells  in GATA-1 expression in BM-conserved MP cells compared with their littermate controls, respectively  in GATA-1 expression even in spleen-conserved MP cells compared with their littermate controls  isolated from transgenic mice and their littermate controls into conditioned recipient mice  of rhCOMP-Ang1, and GATA-1 expression in these cells was reduced in a dose-independent manner (rhCOMP-Ang1 (500 ng/mL)-exposed MP cells was almost completely recovered by treatment with the Tie2 kinase inhibitor (250 nM)  into transgenic mice and their littermate controls. GATA-1 expression in BM MP cells of the K14-Cre;c-Ang1 mice was recovered up to the level similar to that of untreated littermate control group  . Tie2 inol group . Circulanhibitor . In addipression  and circpression  similar 26 rapidly increases numbers of circulating LSK and CD150+CD48-LSK cells, consecutive administration of AMD3100 leads to senescence of BM HSCs via disruption of the SDF-1/CXCR4 retention axis.27 Here, we explored the regulatory roles of the SDF-1/CXCR4 axis on BM retention and senescence of HSCs using B6 mice that were subcutaneously injected with AMD3100 (5 mg/kg) for 1 day or 21 consecutive days. The single injection, but not consecutive treatment, with AMD3100 facilitated mobilization of BM-conserved HSCs into PB in the mice  (+CD48−LSK cells positive for C12FDG in a dose-dependent manner (P < 0.05), p16 (P < 0.001), p19 (P < 0.001), and p21 (P < 0.01) in BM cells compared with levels in PBS control (P < 0.01) fewer numbers of donor cells compared with the mice with PBS control-derived cells  . Howevert manner . B6 mice control . To veri control . The reced cells . These rP < 0.05) reduced peripheral RBC level in the mice (P < 0.05) reduced in B6 mice injected consecutively with 5 mg/kg AMD3100 compared with numbers in the PBS control  did not alter circulating numbers of platelets  and WBC the mice . B6 micethe mice  and GATAthe mice  in these control . A decre control . These r28-30 Single AMD treatment rapidly promotes HSPC mobilization into PB, but its long-term administration may induce hematopoietic damage via impaired retention of the SDF-1/CXCR4 axis.31-33 Here, we demonstrate that different from K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not exhibit impaired BM retention and senescence of HSCs and anemic disorder, and these differences were correlated with the SDF-1/CXCR4 retention axis. Our results also indicate that disruption of the SDF-1/CXCR4 retention axis mediates HSC senescence and erythropoietic impairment, whereas it does not directly disturb megakaryopoiesis and platelet production. Although the CXCR4 antagonist AMD3100 had no a direct relationship with the transgenic Ang1 overexpression models, our data may support crucial roles of SDF-1/CXCR4 axis on hematopoietic development.The SDF-1/CXCR4 axis plays important roles in maintaining the HSC pool and in regulating HSC mobilization and homing.8 Our experiments in transgenic mice indicate that genetic overexpression of COMP-Ang1 impairs megakaryocytic development and platelet production regardless of the promoter. In vitro treatment of rhCOMP-Ang1 in BM cells including Tie2-positive MP cells decreased GATA1-positive MP cells (%). Although this decrease was not correlated with the concentration of rhCOMP-Ang1, several experiments using Tie2 inhibitor indicated important role of Ang1/Tie2 signaling axis on the regulation of GATA1 expression in MP cells. Consequently, our results support that megakaryotic defects in the transgenic mice are closely associated with the Ang1/Tie2 signaling axis, as well as the nature of MP cells to highly express Tie2. In addition, in vitro and in vivo experiments using rhCOMP-Ang1, the Tie2 inhibitor, or both suggest that activation of the Ang1/Tie2 signaling axis results in defects in circulating platelet formation, rather than RBC production.Activation of the Ang1/Tie2 signaling axis is required for megakaryocyte maturation and platelet formation.34-37 In addition to the impacts of GATA-1 on erythrocytic maturation and RBC production,38-40 considerable findings support that the lack of GATA-2 causes a defective hematopoiesis, whereas its overexpression stimulates megakaryocyte differentiation and impairs erythroid differentiation.41 To further understand the cellular mechanisms by which transgenic overexpression of COMP-Ang1 induces hematopoietic defects, specifically thrombocytopenic and anemic disorders, we determined the numbers and expression of transcription factors GATA-1, GATA-2, or both in MP cells and erythroblasts in transgenic mice, irradiated recipients, or AMD3100-injected B6 mice. Our results suggest that numbers of MP cells and erythroblasts are closely associated with their expression of GATA-1, but not GATA-2. Our data indicate an association of GATA-1 with excessive Ang1-mediated impairments in platelets and RBC production. As shown by the unchanged numbers of MP cells positive for Ki-67 or Annexin V/PI, our findings also indicate that impaired platelet formation in transgenic mice is not directly associated with an alteration of MP cell death and proliferation.Transcription factors GATA-1 and GATA-2 play central roles in the development and maturation of megakaryocytes and erythrocytes into platelets and RBC.42 Based on the effects of Tie2 inhibitor on GATA-1 expression and production of platelets in transgenic mice, we postulated that activation of the Ang1/Tie2 signaling axis may directly cause thrombocytopenic damage rather than anemia via dysregulated GATA-1 expression in MP cells. In contrast, our results indicate that anemic disorder induced in transgenic mice is related to impaired retention of the SDF-1/CXCR4 axis along with decreased GATA-1 expression in erythroblasts at late stages. The current findings along with previous reports20 also suggest that the impaired SDF-1/CXCR4 retention axis in transgenic mice is correlated with the amount of COMP-Ang1 generated. It is important to consider that HSC senescence is not directly associated with megakaryocyte maturation and platelet formation, but it rather mediates erythropoietic disorder.43 In addition, inflammatory responses repress GATA-1 expression and erythropoietic development.36 Taken together, the current findings with previous reports20 indicate that in addition to the dysregulated expression of SDF-1 and GATA-1, the increases in inflammatory cytokines, BM niche injury, and HSC senescence are orchestrated with defected erythrocyte maturation and RBC production under systemic overexpression of COMP-Ang1.While GATA-1 expression appeared to be tightly correlated with the levels of MP cells and erythroblasts in transgenic mice, our results also strongly suggest the existence of different mechanisms by which GATA-1 expression in these cells is affected in relation to the amount of Ang1. One report showed that megakaryopoiesis is more sensitively affected by dysregulated GATA-1 function than erythropoiesis.44 Administration of rhCOMP-Ang1 via a local delivery or adenoviral transfection system exhibits beneficial effects on vasculogenesis, angiogenesis, and regenerative medicines. It has been also considered that a prolonged and persisted supplementation with COMP-Ang1 exert further great potency in its use for medicinal and pharmacological application. We could not exclude the fact that the transgenic mice used in this study are artificial models and thus can evoke a limitation in a clinical application of the current findings. However, our results provide evidence that an appropriate activation of the Ang1/Tie2 signaling and/or a suitable level of Ang1 via a local supplementation of rhCOMP-Ang1, but not its genetic overexpression, are required for the maintenance of HSC functions as well as for production of megakaryocytes and erythroid progenitor-derived cells.In summary, numerous studies have shown therapeutic potential of COMP-Ang1, an engineered angiopoietin-1 variant, at both vascular and systemic levels.This study demonstrates that long-term supplementation with COMP-Ang1 by its transgenic overexpression induced impaired BM retention and senescence of HSCs and led to defects in erythropoiesis, megakaryopoiesis, or both depending on its systemic and local expression . The maisxac080_suppl_Supplementary_MaterialClick here for additional data file.