Journal: Science Advances

Article Title: Steroid nuclear receptor coactivator 2 controls immune tolerance by promoting induced T reg differentiation via up-regulating Nr4a2

doi: 10.1126/sciadv.abn7662

Figure Lengend Snippet: ( A ) Representative flow cytometric analysis of Foxp3 − cells in sorted naive CD4 + cells from indicated mice before adoptive transfer into Rag1 −/− mice. ( B ) Representative flow cytometric analysis (left panels) and percentage (right panel) of Foxp3 + CD4 + T regs in spleen and mLN of Rag1 −/− mice 3 weeks after adoptive transfer of 0.4 × 10 6 naive CD4 + cells ( n ≥ 3 per genotype). ( C ) Representative flow cytometric analysis (left panels) and percentage (right panel) of Foxp3 + CD4 + T regs in colon, mLN, iLN, and spleen of Rag1 −/− mice transferred with 3 × 10 6 naive OT-II/SRC2 fl/fl or OT-II/SRC2 fl/fl /CD4 Cre CD4 + cells and subsequently treated with OVA (20 mg/ml) for 5 days ( n = 3 per genotype). ( D ) Mean clinical EAE scores of indicated mice at different days after EAE induction with MOG 35–55 . ( E ) Representative flow cytometric analysis (left panels) and the percentage and Foxp3 MFI (right panels) of Foxp3 + CD4 + T regs recovered from the CNS of EAE-induced mice ( n = 5 per genotype). ( F ) Number of Foxp3 + CD4 + T regs recovered from the CNS of EAE-induced mice ( n = 3 per genotype). ( G ) Representative flow cytometric analysis (left panels) and the percentage (right panel) of interferon-γ–positive (IFNγ + ) and IL-17A + cells among CD4 + T cells recovered from the CNS of EAE-induced mice ( n ≥ 7 per genotype). Boxed area: Cell population of interest. Data are from three experiments (D, presented as means ± SEM; B, C, E, and G, right panels, presented as means ± SD) or are from one representative of three independent experiments (A; B, C, E, and G, left panels). * P < 0.05, ** P < 0.01, and *** P < 0.001 (two-tailed Student’s t test).

Article Snippet: Antibodies against SRC2 (dilution ratio, 1:2000; A300-346A, Bethyl), rabbit IgG (P120-101, Bethyl), NFAT1 (dilution ratio, 1:1000; 5861S, Cell Signaling Technology), Nr4a2 (dilution ratio, 1:150; sc-376984, Santa Cruz Biotechnology), and β-actin (dilution ratio, 1:1000; SC-8422, Santa Cruz Biotechnology) were used for immunoblot analysis.

Techniques: Adoptive Transfer Assay, Two Tailed Test

Journal: Science Advances

Article Title: Steroid nuclear receptor coactivator 2 controls immune tolerance by promoting induced T reg differentiation via up-regulating Nr4a2

doi: 10.1126/sciadv.abn7662

Figure Lengend Snippet: ( A ) Volcano plot comparison of gene expression between Foxp3 YFP-Cre and SRC2 fl/fl /Foxp3 YFP-Cre CD4 + cells ( n = 3 per genotype). Differentially up-regulated genes (red) and down-regulated genes (blue) with a cutoff at P < 0.05 and fold change (FC) > 1.4 are shown. ( B ) Heatmap of Ncoa2 and other genes known to regulate T reg differentiation. ( C to E ) qPCR analysis of Foxp3, Ncoa2, Foxo1, Myb, Irf4, Nr4a2, and Stat5a (control) mRNA in CD4 + cells 36 hours after T reg polarization (C and E) and naive CD4 + cells (D) derived from indicated mice ( n ≥ 4 per genotype per group). ( F and G ) Representative flow cytometric analysis of protein levels (left panels) and relative MFI (right panel) for Nr4a2 in indicated genotypes of naive CD4 + cells versus CD4 + cells 24 hours after T reg polarization [ n = 4 per genotype for (F) and n = 5 per genotype for (G)]. ( H ) Representative flow cytometric analysis of protein levels (left panels) and relative MFI (right panel) for Nr4a2 in SRC2 fl/fl /CD4 Cre CD4 + T cells transduced with retrovirus expressing ± Nr4a2 and differentiated under T reg polarization conditions for 24 or 48 hours ( n = 4 per genotype). ( I and J ) Representative flow cytometric analysis (top panels) and the percentage (bottom panels) of Foxp3 + CD4 + T regs among indicated genotypes of NGFR + CD4 + cells transduced with retrovirus expressing ± Nr4a2 and polarized for 48 hours under T reg conditions ( n ≥ 4 per genotype per group). EV, empty vector; NGFR, marker of transduction; boxed region, cell population of interest. Data are from three experiments (A to H, right panels; I and J, bottom panels; presented as means ± SD) or are from one representative of three independent experiments (F to H, left panels; I and J, top panels). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0005 (two-tailed Student’s t test).

Article Snippet: Antibodies against SRC2 (dilution ratio, 1:2000; A300-346A, Bethyl), rabbit IgG (P120-101, Bethyl), NFAT1 (dilution ratio, 1:1000; 5861S, Cell Signaling Technology), Nr4a2 (dilution ratio, 1:150; sc-376984, Santa Cruz Biotechnology), and β-actin (dilution ratio, 1:1000; SC-8422, Santa Cruz Biotechnology) were used for immunoblot analysis.

Techniques: Comparison, Gene Expression, Control, Derivative Assay, Transduction, Expressing, Plasmid Preparation, Marker, Two Tailed Test

Journal: Science Advances

Article Title: Steroid nuclear receptor coactivator 2 controls immune tolerance by promoting induced T reg differentiation via up-regulating Nr4a2

doi: 10.1126/sciadv.abn7662

Figure Lengend Snippet: ( A ) Schematic representation of the six regions on Nr4a2 promoter covered by P0 to P5 primers, the locations of identified NFAT1-binding sites, and the region deleted using CRISPR-Cas9. ( B and C ) ChIP-qPCR analysis of SRC2 binding (B) or NFAT1 binding (C) to the Nr4a2 promoter (P0 to P5 regions) in CD4 + cells from indicated genotypes under T reg polarization at the indicated time points ( n ≥ 3 per genotype per group). ( D ) Immunoblot analysis of NFAT1 among anti-SRC2 antibody immunoprecipitated (IP) SRC2 complexes from SRC2 fl/fl CD4 + cells 24 hours after T reg polarization. Bottom blots, whole-cell lysate input control. ( E ) PCR analysis of the abundance of NFAT1/SRC2 binding region on Nr4a2 promoter in CD4 + cells transduced with virus expressing nontarget control ( NTC ) or the region containing NFAT1/SRC2-binding guiding RNAs shown in (A) ( crNr4a2 ) and polarized under T reg condition for 40 hours. ( F ) Immunoblot analysis of Nr4a2 in naive CD4 + cells or CD4 + T cells transduced with virus expressing NTC and crNr4a2 shown in (E) and polarized under T reg conditions for 40 hours. The number in the bottom of the blots is the relative mean intensity of each band, and the right panel is the summary of the relative mean intensity. ( G ) Representative flow cytometric analysis of Foxp3 (left panels), percentages of Foxp3 + cells (middle panels), and MFI for Foxp3 (right panels) among GFP hi CD4 + cells transduced with virus expressing NTC, crFoxp3 , and crNr4a2 guiding RNAs and polarized under T reg condition at the indicated time points ( n ≥ 3 per treatment per group). Data are from three experiments (B, C, and F, right panels; G, middle and right panels; presented as means ± SD) or are from one representative of three independent experiments (D to G, left panels). * P < 0.05, ** P < 0.01, and *** P < 0.001 (two-tailed Student’s t test). MW, molecular weight; bp, base pairs.

Article Snippet: Antibodies against SRC2 (dilution ratio, 1:2000; A300-346A, Bethyl), rabbit IgG (P120-101, Bethyl), NFAT1 (dilution ratio, 1:1000; 5861S, Cell Signaling Technology), Nr4a2 (dilution ratio, 1:150; sc-376984, Santa Cruz Biotechnology), and β-actin (dilution ratio, 1:1000; SC-8422, Santa Cruz Biotechnology) were used for immunoblot analysis.

Techniques: Binding Assay, CRISPR, ChIP-qPCR, Western Blot, Immunoprecipitation, Control, Transduction, Virus, Expressing, Two Tailed Test, Molecular Weight

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) First, 293T cells were transfected with Flag-NCOA2 alone or HA-RTA alone or cotransfected with Flag-NCOA2 along with HA-RTA. Cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by western blotting with the indicated antibodies. (B) The 293T cells were transfected with Flag-RTA alone or HA-NCOA alone or cotransfected with Flag-RTA along with HA-NCOA2. Cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by western blotting with the indicated antibodies. (C) In vitro GST affinity binding assay. Bacterially expressed GST alone and GST-NCOA2 attached to GST-Sepharose beads were incubated with the purified His-tagged RTA, and the pull-down lysates were immunoblotted with anti-His or anti-GST antibodies. (D) Colocalization of NCOA2 and RTA in HeLa cells. Following transfection with Flag-RTA and HA-NCOA2, HeLa cells were fixed with 4% paraformaldehyde and then stained with anti-HA and anti-Flag antibodies. Secondary antibodies conjugated to FITC or Cy3 were used to visualize the stained RTA and NCOA proteins, respectively. Diamidino-2-phenylindole shows the nuclei of cells.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Affinity Binding Assay, Incubation, Purification, Staining

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) NCOA2 expression in HEK293T cells and KSHV-positive human cells (iSLK.RGB, BCBL1, JSC1 and BC3) was detected by western blotting. (B) Co-IP of endogenous RTA and NCOA2 in KSHV-positive cells. Lytic replication of KSHV in these cells was induced by dox or VPA, and cell lysates were subjected to immunoprecipitation with anti-NCOA2 antibody or rabbit IgG controls. Purified proteins, along with input samples, were detected by western blotting with the indicated antibodies. (C) Endogenous NCOA2 colocalizes with endogenous RTA in the nucleus. KSHV-positive B cells that were uninduced (Un) or induced with VPA (In) were fixed and stained with anti-NCOA2 antibody and anti-RTA antibody, followed by incubation with secondary antibodies conjugated to FITC or Cy3. The right sides show a high magnified view.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Purification, Staining, Incubation

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) Truncated versions of NCOA2 are shown schematically, including N765 (1 aa-765 aa), 765C (765 aa-1464 aa), N1007 (1 aa-1007 aa), N624 (1 aa-624 aa), 624C (624 aa-1464 aa). The N-terminal bHLH-PAS domain, the centrally located nuclear receptor (NR) boxes responsible for NR binding, the C-terminal activation domain (AD1 and AD2) and a repression domain (RD) are marked. (B) Defining the RTA-interacting domain of NCOA2. Co-IP and western blotting of 293T cells transfected with HA-tagged RTA along with a vector expressing the indicated Flag-tagged NCOA2 truncations or the full-length NCOA2. (C) Schematic diagram of the RTA protein and its deletion mutants, including N273 (1 aa-273 aa), N544 (1 aa-544 aa), RTA273-544 (273 aa-544 aa), C544-691 (544 aa-691 aa), C273-691 (273 aa-691 aa). The nuclear localization (NLS), DNA binding and dimerization and transcriptional activation domain (TAD) are marked. (D) Defining the NCOA2-interacting domain of RTA. Co-IP and western blotting of 293T cells transfected with HA-tagged NCOA2 along with Flag-tagged RTA truncations or full-length RTA. An empty vector was used as a negative control. (E) In vitro GST affinity binding assay. Bacterially expressed GST alone and GST-NCOA2 attached to GST-Sepharose beads were incubated with the purified His-tagged RTA mutation (His-C544-691). The pull-down lysates were immunoblotted with anti-His or anti-GST antibody. (F) Schematic diagram of a PARS II region deletion mutant of RTA. (G) Co-IP and western blotting of 293T cells transfected with HA-NCOA2 along with Flag-tagged PARS II region deletion mutants.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Binding Assay, Activation Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Negative Control, In Vitro, Affinity Binding Assay, Incubation, Purification, Mutagenesis

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) Effect of NCOA2 on RTA expression. First, 293T cells were cotransfected with 1 μg of RTA expression plasmid and increasing amounts of NCOA2 expression vector (0, 0.5, 1, 2 μg). The expression of RTA proteins was examined by immunoblotting with the indicated antibodies. (B) The 293T cells were treated as in (A). RTA mRNA was detected using RT-qPCR with the indicated primers. (C) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then, cells were transfected with the Flag-tagged RTA expression plasmid. The expression of NCOA2 and RTA was determined by immunoblotting with the indicated antibodies. (D) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then cells were transfected with the Flag-tagged RTA-△PARS-II expression plasmid. The expression of NCOA2 and RTA-△PARS-II was determined by immunoblotting with the indicated antibodies. (E) The N-terminal truncation of NCOA2 regulates the stability of RTA. First, 293T cells were cotransfected with HA-tagged RTA expression vector with or without NCOA2 and its truncations. Then, RTA expression levels were examined by western blotting using anti-HA antibodies. (F) Effect of NCOA2 on the expression of the RTA mutant with deletion of the PARS II region. First, 293T cells were cotransfected with 1 μg of Flag-tagged RTA-△PARS-II expression plasmid and increasing amounts of NCOA2 expression vector (0, 0.5, 1, 2 μg). Then, the expression of RTA-△PARS-II proteins was examined by immunoblotting with the indicated antibodies. (G) The 293T cells were treated as in (F), and the mRNA level of RTA-△PARS-II was detected using RT-qPCR with the indicated primers. (H) Measurement of RTA stability in the absence and presence of NCOA2. First, 293T cells were transfected with Flag-tagged RTA with or without the NCOA2 expression plasmid for 36 h. Then, cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA. Tubulin was used as a control for equivalent sample loading. (I) The relative levels of RTA from immunoblots (H) were quantified by densitometry and normalized to the Tubulin level. The band intensities on the exposed film were plotted graphically. (J) Measurement of RTA-△PARS-II stability in the absence and presence of NCOA2. First, 293T cells were transfected with Flag-tagged RTA-△PARS-II with or without the NCOA2 expression plasmid for 36 h. Then, cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA-△PARS-II. Tubulin was used as a control for equivalent sample loading. The band intensities on the exposed film are plotted graphically (K). (L) First, 293T cells were transfected with RTA expression plasmid with sh-NCOA2 or sh-GFP. Then, the cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA. Tubulin was used as a control for equivalent sample loading. The band intensities on the exposed film were plotted graphically (M).

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR, shRNA, Transfection, Mutagenesis, Control

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) NCOA2 inhibits the degradation of RTA. First, 293T cells were cotransfected with the indicated expression constructs for 36 h and then treated with 0.5 μM MG132 or 0.1% DMSO for another 6 h. The cells were lysed and used for western blotting with the indicated antibodies. The band intensities of RTA were plotted graphically. Data are from a minimum of three experimental replicates with a standard deviation. (B) NCOA2 inhibits the ubiquitination of RTA. The same lysates as in (A) were subjected to immunoprecipitation using anti-Flag antibody. Purified proteins, along with input samples, were analyzed by western blotting with anti-ubiquitin antibodies. (C) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then cells were transfected with the Flag-tagged RTA expression plasmid for 24 h and then treated as in (A). Cells were lysed and used for western blotting with the indicated antibodies. The band intensities of RTA were plotted graphically. Data were from a minimum of three experimental replicates with a standard deviation. (D) Same lysates as in (C) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. (E) First, 293T cells were cotransfected with NCOA2 and the RTA-△PARS-II mutant, and then, they were treated as in (A). The expression of the RTA-△PARS-II mutant was analyzed by western blotting. The band intensities of RTA were plotted graphically. (F) The same lysates as in (E) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. (G) 293T cells were transfected with shRNA-NCOA2 or shRNA-GFP plasmids, and then cells were transfected with RTA-△PARS-II mutant and treated as in (A). The expression of RTA-△PARS-II mutant was analyzed by western blotting. The band intensities of RTA were plotted graphically. (H) The same lysates as in (G) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. Statistical significance was analyzed with a two-tailed Student’s t -test (*P < 0.05 or **P < 0.01).

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Expressing, Construct, Western Blot, Standard Deviation, Ubiquitin Proteomics, Immunoprecipitation, Purification, shRNA, Transfection, Plasmid Preparation, Mutagenesis, Two Tailed Test

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A-C) NCOA2 disrupts the interaction between RTA and MDM2. 293T cells were transiently transfected with Flag-RTA and HA-MDM2 together with an increasing amount of Myc-NCOA2 (A), Myc-N624 (B) or Myc-624C (C) (0, 0.5, 1, 2 μg). Forty-eight hours after transfection, the cell lysates were collected and subjected to immunoprecipitation using an anti-Flag antibody. Purified proteins, along with input samples, were detected by western blotting with the indicated antibodies. (D) NCOA2 inhibits the proteasome-mediated degradation of RTA induced by MDM2. 293T cells were transiently transfected with Flag-RTA and HA-MDM2 together with an increasing amount of Myc-NCOA2 (0, 0.5, 1, 2 μg) for 36 h, and then, they were treated with 0.5 μM MG132 or 0.1% DMSO for another 6 h. The cells were lysed and used for western blots with the indicated antibodies. (E) NCOA2 inhibits the ability of MDM2 to increase the ubiquitination of RTA. The same lysates as in (D) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. (F-G) 293T cells were transiently transfected with Flag-RTA and HA-MDM2 together with an increasing amount of Myc-N624 (F) or Myc-624C (G) (0, 0.5, 1, 2 μg) for 36 h, then cells were treated with 0.5 μM MG132 for another 6 h. Cells were lysed and subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Transfection, Immunoprecipitation, Purification, Western Blot, Ubiquitin Proteomics

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) iSLK.RGB cells were stably transfected with lentiviruses containing a Flag-tagged NCOA2 expression plasmid or an empty vector plasmid, named iSLK.RGB-NCOA2 and iSLK.RGB-Vector, respectively. The overexpression of NCOA2 was detected by western blotting. (B) iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells were treated with dox at different time points as indicated. Fluorescence microscopy images of EGFP-positive cells among iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells. (C) Flow cytometry analysis of EGFP-positive cells among iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells. (D) Quantitation of the percent of EGFP-positive cells from (C). (E) NCOA2 overexpression increases the transcription of viral genes. RNA was extracted from dox-induced cells at 48 hours post-induction (hpi) to investigate the transcriptional level of NCOA2 and several KSHV genes: RTA, PAN, ORF57, ORF65 and LANA. (F) NCOA2 overexpression increases the expression of viral genes. The expression levels of RTA protein and ORF64 protein were determined by immunoblotting with the indicated antibodies. (G) NCOA2 overexpression increases virus production. Culture supernatants from dox-induced iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells at 48 hpi were quantified by qPCR for KSHV copy numbers. (H) BCBL1 cells were stably transfected with lentiviruses containing a NCOA2 expression plasmid or an empty vector plasmid, named BCBL1-NCOA2 and BCBL1-Vector, respectively. The overexpression of NCOA2 was detected by western blotting. (I) NCOA2 overexpression increases virus production in BCBL1 cells. BCBL1-NCOA2 and BCBL1-Vector cells were treated with VPA for 24 h, and the KSHV copy numbers from culture supernatants were quantified by qPCR. Data in D, E, G, and I represent the mean +/- SD of 3 replicates pooled from three independent experiments. Data were analyzed with a two-tailed Student’s t -test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot, Fluorescence, Microscopy, Flow Cytometry, Quantitation Assay, Virus, Two Tailed Test

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A) iSLK.RGB cells were transfected with control siRNA and two NCOA2-specific siRNAs. The knockdown efficiency was determined by western blotting. At 24 h after transfection, cells were induced by dox for another 24 h. (B) EGFP-positive cells were analyzed by fluorescence microscopy. (C) The KSHV gene transcription level was analyzed by qPCR. (D) The expression levels of RTA and ORF64 were examined by western blotting. (E) The progeny viruses from culture supernatants were analyzed by qPCR. (F) BCBL1 cells were also transfected with control siRNA and two NCOA2-specific siRNAs. The knockdown efficiency was determined by western blotting. At 24 h after transfection, cells were induced by VPA for another 24 h. The KSHV gene transcription level (G), the expression levels of RTA and ORF64 (H) and the progeny viruses (I) were analyzed by the same approaches that were used in iSLK.RGB stable cell lines. Data in C, E, G, and I represent the mean +/- SD of 3 replicates pooled from three independent experiments. Data were analyzed with a two-tailed Student’s t -test (*P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Transfection, Control, Knockdown, Western Blot, Fluorescence, Microscopy, Expressing, Stable Transfection, Two Tailed Test

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: (A and C) The expression kinetics of NCOA2 and RTA were analyzed in iSLK.RGB cells (A) or iSLK cells (C) at indicated time points after dox treatment. (B and D) NCOA2 mRNA expression level in cells from (A) or (C). (E) Effect of RTA on NCOA2 expression. 293T cells were cotransfected with 1 μg of NCOA2 expression plasmid and increasing amounts of RTA expression vector (0, 0.5, 1, 2 μg). The protein level of NCOA2 was examined by immunoblotting. (F) NCOA2 mRNA expression level in cells from (E). (G) RTA inhibits the degradation of NCOA2. 293T cells were cotransfected with the indicated expression constructs for 36 h and then treated with 0.5 μM MG132 or 0.1% DMSO for another 6 h. Cells were lysed and used for western blots with the indicated antibodies. The band intensities of NCOA2 were plotted graphically (H). Data were from a minimum of three experimental replicates with a standard deviation. Data were analyzed by unpaired t -test. *, P < 0.05; **, P < 0.01. (I) Measurement of NCOA2 stability in the absence and presence of RTA. 293T cells were transfected with HA-tagged NCOA2 with or without Flag-tagged RTA expression plasmid for 36 h. Cells were treated with 100 μg/ml of CHX and analyzed at indicated time points by immunoblotting for NCOA2. Tubulin was used as a control for equivalent sample loading. (J) The relative levels of NCOA2 from (I) were quantified by densitometry and normalized to the Tubulin level.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Expressing, Plasmid Preparation, Western Blot, Construct, Standard Deviation, Transfection, Control

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: After interacting with RTA, MDM2 promotes the ubiquitination and degradation of RTA, which inhibits KSHV lytic replication. NCOA2 competes with MDM2 to interact with RTA, which blocks the interaction between RTA and MDM2 and then inhibits the degradation of RTA. The abundance of RTA is increased by NCOA2. In turn, RTA promotes the expression of NCOA2, which forms a positive feedback loop between NCOA2 and RTA and promotes KSHV lytic replication.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Ubiquitin Proteomics, Expressing

Journal: PLoS Pathogens

Article Title: NCOA2 promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus by enhancing the expression of the master switch protein RTA

doi: 10.1371/journal.ppat.1008160

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The following primary antibodies were used: anti-NCOA2 rabbit monoclonal antibody (CST, #96687), anti-RTA (prepared in our laboratory), anti-ORF64 (a kind gift from Yan Yuan, University of Pennsylvania, Philadelphia, Pennsylvania USA), anti-ubiquitin antibody (ABclonal, A3207), anti-GST (ABclonal, AE001) anti-His (ABclonal, AE003), anti-Flag antibody (Sigma, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GAPDH (Sigma-Aldrich, G8795), and anti-α-Tubulin antibody (Sigma-T6199).

Techniques: Sequencing, Cloning, PCR Cloning

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: Schematic diagrams of HEY1, NCOA2, and HEY1–NCOA2 chimeric proteins and their intracellular localization, as well as the induced expression of FLAG‐tagged HEY1, NCOA2, and HEY1–NCOA2 in the iPSC‐MSCs cell models. (A) Schematic diagrams of HEY1, NCOA2, and HEY1–NCOA2 proteins. Dashed lines represent exon–exon borders. Only coding exons are presented. (B) The mEGFP‐HEY1 , mEGFP‐NCOA2 , and mEGFP‐HEY1–NCOA2 constructs as well as the empty vector were introduced into iPSC MSC cells transiently and photographed at 24 h. The first row shows the direct EGFP fluorescence, the second row shows Hoechst staining of DNA, and the third row shows the merged image indicating the intracellular localization. Bars, 20 μm. (C) Immunoblot using anti‐Flag antibody showed the induced expression of FLAG‐tagged HEY1–NCOA2, HEY1, and NCOA2 in stably transduced iPSC‐MSCs.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Expressing, Construct, Plasmid Preparation, Fluorescence, Staining, Western Blot, Stable Transfection

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: HEY1–NCOA2 fusion protein DNA‐binding pattern in the genome. (A) Genomic context of HEY1 and HEY1–NCOA2 binding peaks, with a breakdown into three groups: (1) Peaks that are overlapping between HEY1 and HEY1–NCOA2 fusion protein, (2) HEY1‐only binding peaks, and (3) HEY1–NCOA2‐only binding peaks. (B) Signal enrichment heatmap for the peaks specific for HEY1 only (blue bar), HEY1–NCOA2 only (red bar), and overlapping peaks (purple bar). (C) Normalized enrichment of HEY1‐binding targets, comparing HEY1‐only peaks and HEY1‐binding peaks overlapping with HEY1–NCOA2‐binding peaks. (D) E‐box sequences bound by the HEY1 and HEY1–NCOA2 fusion protein. Distribution of the distance between binding peaks and the closest E‐box motif(s); the distance equals 0 for the direct overlap between peak and motif, which fraction is visualized with pie plots.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Binding Assay

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: Gene expression profile associated with HEY1–NCOA2. (A) Heatmap showing row‐normalized expression level of genes differentially regulated by HEY1‐NCOA2 in comparison to wildtype HEY1 [MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+)]. (B) GSEA Enrichment plot of the expression of genes that were identified as downregulated by HEY1 in MSC‐HEY1(+) versus MSC‐HEY1 ctrl . The gene rank was based on the log2(fold‐change) of the expression of genes in MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1‐NCOA2 ctrl . (C) GSEA enrichment plot for the expression of genes of which their promoters had binding peaks of both HEY1 and HEY1–NCOA2. The gene rank was based on the log2(fold‐change) of the gene expression in MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+). (D) Boxplots visualizing the enrichment of the 674 HEY1–NCOA2 direct‐target‐and‐transactivating genes identified using the iPSC‐MSCs cell models in sarcoma tumor samples. The enrichment was visualized across patients' samples from a total of eight sarcoma entities. GSEA, Gene Set Enrichment Analysis; NES, Normalized Enrichment Score; FDR, False Discovery Rate; MCS, Mesenchymal Chondrosarcoma; GIST, Gastrointestinal Stromal tumor; RHB, Rhabdomyosarcoma; IFS, Infantile Fibrosarcoma; ASPS, Alveolar Soft Part Sarcoma; SS, Synovial Sarcoma; DSRCT, Desmoplastic Small Round Cell Tumors; EWS, Ewing's Sarcoma.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Gene Expression, Expressing, Comparison, Binding Assay

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: Functional pathways enriched in HEY1–NCOA2 upregulated genes. (A) Bar plot showing the −log10(FDR) of the top‐10 enriched KEGG pathways, computed for 1,563 HEY1–NCOA2 genes upregulated by HEY1–NCOA2 as compared with HEY1 [MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+)]. (B) Heatmap summarizing the GSEA (Gene Set Enrichment Analysis) of selected gene sets. Each row represents one gene set. Row #4–#8, gene signatures were selected based on the KEGG pathway enrichment analysis. The values in the heatmap indicate the −log10 of p value, multiplied by −1 if the NES score was indicating the enrichment biased toward the negative phenotype. (C) GSEA enrichment plot of MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1‐NCOA2 ctrl and MSC‐HEY1‐NCOA2(+) versus MSC‐HEY1(+). The gene set ‘Chondrocyte Differentiation’ is from Gene Ontology (GO). The false discovery rate (FDR), nominal p value and normalized enrichment score were calculated by GSEA.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Functional Assay

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: HEY1–NCOA2 target gene expression validation. (A) RT‐qPCR validation of genes regulation by inducible HEY1, NCOA2, or HEY1–NCOA2 expression in iPSC MSC. The graph represents the fold change between the induced and uninduced condition. (B) Immunoblotting was performed to further confirm the expression of BCL2, CCND1, and HES1 after HEY1–NCOA2 induction in stably transduced iPSC‐MSCs. GAPDH was used as a loading control.

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Targeted Gene Expression, Biomarker Discovery, Quantitative RT-PCR, Expressing, Western Blot, Stable Transfection, Control

Journal: The Journal of Pathology

Article Title: Genomic profiling identifies genes and pathways dysregulated by HEY1–NCOA2 fusion and shines a light on mesenchymal chondrosarcoma tumorigenesis

doi: 10.1002/path.5899

Figure Lengend Snippet: HEY1‐NCOA2 significantly increases cell proliferation in iPSC‐MSC. (A) 1 × 10 5 of FACS sorted GFP+ iPSC‐MSCs were seeded with or without doxycycline (50 ng/ml). Cell numbers were counted on days 6 and 12; the graph shows the mean ± SD ( n = 3). (B) GFP+ iPSC‐MSCs were cultured with or without doxycycline (50 ng/ml) for 7 days and labeled with 10 μM of EdU for 3 h. Cells were then harvested, fixed, and stained with fluorescent dye 647 picolyl azide. EdU was detected by flow cytometry and the percentage of cells in S‐phase was analyzed using FlowJo software. The graph shows the mean ± SD ( n = 2). (C) FACS‐sorted GFP+ iPSC‐MSCs were cultured as in panel A except without addition of rh‐FGF basic and rh‐IGF‐1 to the culture medium. Cell numbers were counted on day 7; the graph shows the mean ± SD ( n = 3).

Article Snippet: Human HEY1 (CAT#: RC200257) and NCOA2 (CAT#: RC212235) cDNA clones, from OriGene Technologies (Rockville, MD, USA), were used to construct N‐terminal Flag‐tag full‐length coding sequences, i.e.

Techniques: Cell Culture, Labeling, Staining, Flow Cytometry, Software

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Nuclear receptor coactivator 2 (NCOA2) knockout slightly affects homeostatic hematopoiesis. (A) Single‐cell RNA sequencing (scRNA‐seq) analysis of LSKs freshly sorted from the bone marrow (BM) of normal wild‐type (WT) mice. Hematopoietic stem/progenitor cell (HSPC) clustering was shown by the t‐distributed stochastic neighbor embedding (tSNE) plot. (B) Violin plots revealing NCOA2 expression in HSPC populations from (A). (C, D) The total cell numbers of (C) spleen and (D) thymus in WT and NCOA2 −/− mice at steady state ( n = 6). (E) Representative flow cytometric plots revealing the percentages of T cells, B cells, and myeloid cells in the peripheral blood (PB) of WT and NCOA2 −/− mice at steady state. (F) The percentages of T cells, B cells, and myeloid cells in the PB of WT and NCOA2 −/− mice at steady state ( n = 6). (G) The BM numbers of WT and NCOA2 −/− mice at steady state ( n = 6). (H) Representative flow cytometric plots revealing the percentages of myeloid progenitors (MPs, Lin − Sca1 − c‐Kit + ), LSKs (Lin − Sca1 + c‐Kit + ), long‐term hematopoietic stem cells (LT‐HSCs, Lin − Sca1 + c‐Kit + CD34 − Flk2 − ), short‐term HSCs (ST‐HSCs, Lin − Sca1 + c‐Kit + CD34 + Flk2 − ), multipotent progenitors (MPPs, Lin − Sca1 + c‐Kit + CD34 + Flk2 + ), and signaling lymphocyte activation molecules‐labeled HSCs (SLAM‐HSCs, Lin − Sca1 + c‐Kit + CD150 + CD48 ‐ ) in the BM of WT and NCOA2 −/− mice at steady state. (I, J) The numbers of (I) MPs, LSKs, (J) LT‐HSCs, ST‐HSCs, MPPs, and SLAM‐HSCs in the BM of WT and NCOA2 −/− mice at steady state ( n = 6). (K) Representative flow cytometric plots revealing the percentages of common myeloid progenitors (CMPs, Lin − Sca1 − c‐Kit + CD16/32 − CD34 + ), megakaryocyte erythroid progenitors (MEPs, Lin − Sca1 − c‐Kit + CD16/32 − CD34 − ), granulocyte monocyte progenitors (GMPs, Lin − Sca1 − c‐Kit + CD16/32 + CD34 + ), and common lymphoid progenitors (CLPs, Lin − CD127 + Sca1 med c‐Kit med ) in the BM of WT and NCOA2 −/− mice at steady state. (L) The numbers of CMPs, MEPs, GMPs, and CLPs in the BM of WT and NCOA2 −/− mice at steady state ( n = 6). NS, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Knock-Out, Single Cell, RNA Sequencing, Expressing, Activation Assay, Labeling

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Nuclear receptor coactivator 2 (NCOA2) deficiency leads to hematopoietic stem cell (HSC) pool exhaustion following irradiation (IR). (A) Immunofluorescence analysis of NCOA2 nuclear translocation in HSCs at the indicated time points after 5.0 Gy IR. (B) Quantitative analysis of the ratio of nuclear/whole‐cell fluorescence intensity in (A) by Image J ( n = 20 cells). “Day 0” in (A) and (B) represents the unirradiated control group. (C–E) The counts of (C) white blood cell (WBC), (D) red blood cell (RBC), and (E) platelet (PLT) in the peripheral blood (PB) of wild‐type (WT) and NCOA2 −/− mice at the indicated time points after 5.0 Gy IR ( n = 10). (F) The bone marrow (BM) numbers of WT and NCOA2 −/− mice at Day 28 following 5.0 Gy IR ( n = 6). (G) Representative flow cytometric plots revealing the percentages of myeloid progenitors (MPs), LSKs, long‐term HSCs (LT‐HSCs), short‐term HSCs (ST‐HSCs), multipotent progenitors (MPPs), and signaling lymphocyte activation molecules‐labeled HSCs (SLAM‐HSCs) in the BM of WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR. (H, I) The numbers of (H) MPs, LSKs, (I) LT‐HSCs, ST‐HSCs, MPPs, and SLAM‐HSCs in the BM of WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). (J) Representative flow cytometric plots revealing the percentages of common myeloid progenitors (CMPs), megakaryocyte erythroid progenitors (MEPs), granulocyte monocyte progenitors (GMPs), and common lymphoid progenitors (CLPs) in the BM of WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR. (K) The numbers of CMPs, MEPs, GMPs, and CLPs in the BM of WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Irradiation, Immunofluorescence, Translocation Assay, Fluorescence, Control, Activation Assay, Labeling

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Loss of nuclear receptor coactivator 2 (NCOA2) decreases the return of hematopoietic stem cells (HSCs) to quiescence and inhibits their survival after irradiation (IR). (A) Cell cycle analysis of LSKs and long‐term HSCs (LT‐HSCs) in the bone marrow (BM) of wild‐type (WT) and NCOA2 −/− mice at Day 28 following 5.0 Gy IR ( n = 6). Representative flow cytometric plots are shown on the left. (B) The percentage of bromodeoxyuridine (BrdU) + cells in LSKs and LT‐HSCs from the BM of WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). Representative flow cytometric plots are shown on the left. (C) Quantitative polymerase chain reaction (qPCR) analysis of the mRNA expression of the cell cycle‐associated genes in LT‐HSCs from the BM of WT and NCOA2 −/− mice at Day 28 following 5.0 Gy IR ( n = 3). (D) The apoptosis rate of LSKs and LT‐HSCs in the BM of WT and NCOA2 −/− mice at Day 28 following 5.0 Gy IR ( n = 6). Representative flow cytometric plots are shown on the left. (E) The survival rates of WT and NCOA2 −/− mice after subjected to 7.5 Gy IR ( n = 10). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Irradiation, Cell Cycle Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Nuclear receptor coactivator 2 (NCOA2) ablation aggravates the impairment in hematopoietic stem cell (HSC) long‐term reconstitution ability after exposure to irradiation (IR). (A) The schematic of a noncompetitive transplantation assay. (B, C) The survival rates of recipient mice after (B) primary and (C) secondary transplantation ( n = 10). (D) The schematic of the competitive transplantation assay. (E) Representative flow cytometric plots revealing the percentage of donor‐derived cells in the peripheral blood (PB) of recipient mice at 16 weeks after primary and secondary transplantation. (F) The percentage of donor‐derived cells in the PB of recipient mice at 4, 8, 12, and 16 weeks after primary and secondary transplantation ( n = 6). (G, H) The percentages of donor‐derived bone marrow (BM) cells, Lin − , LSKs, and long‐term HSCs (LT‐HSCs) in the recipients at 16 weeks after (G) primary and (H) secondary transplantation ( n = 6). (I) The strategy of serial replating assay. (J) Serial colony‐forming analysis of LT‐HSCs (1 × 10 2 ) isolated from wild‐type (WT) or NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). (K) The strategy of reciprocal BM transplantation (BMT). (L) The percentage of donor‐derived cells in the PB of recipient mice at 16 weeks after reciprocal BMT ( n = 6). **P < 0.01, ***P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Irradiation, Transplantation Assay, Derivative Assay, Isolation

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Nuclear receptor coactivator 2 (NCOA2) deletion results in the accumulation of abnormal mitochondria in hematopoietic stem cells (HSCs) post irradiation (IR). (A) The strategy of RNA sequencing (RNA‐seq) analysis. (B) Heatmap and (C) volcano plots of the differentially expressed genes (DEGs) in long‐term HSCs (LT‐HSCs) from wild‐type (WT) and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 3). (D) Gene set enrichment analysis (GSEA) of hematopoiesis, HSC signature, quiescence, proliferation, mitochondria metabolism, and oxidative stress‐associated gene sets in LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR. (E) Flow cytometric analysis of mitochondrial mass in the LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR by MitoTracker Green (MTG) staining ( n = 6). Representative flow cytometric plots are shown on the left. MFI, mean fluorescence intensity. (F) Flow cytometric analysis of reactive oxygen species (ROS) levels in LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR by dichlorodihydrofluorescein diacetate (DCFH‐DA) staining ( n = 6). Representative flow cytometric plots are shown on the left. (G) The ratio of MFI of tetramethylrhodamine methyl ester (TMRM) to MTG in LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). **P < 0.01, ***P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Irradiation, RNA Sequencing, Staining, Fluorescence

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Nuclear receptor coactivator 2 (NCOA2) regulates PINK1 expression in irradiated hematopoietic stem cells (HSCs) by coactivation of FOXO3a. (A) Gene set enrichment analysis (GSEA) of FOXO3a target genes in long‐term HSCs (LT‐HSCs) from wild‐type (WT) and NCOA2 −/− mice at Day 28 after 5.0 Gy IR. (B) Heatmap analysis of FOXO3a target genes in LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR. (C) Flow cytometric analysis of the protein expression of FOXO3a in LT‐HSCs from the bone marrow (BM) of WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). Representative flow cytometric plots are shown on the left. (D) Co‐immunoprecipitation (Co‐IP) analysis of the interaction of NCOA2 and FOXO3a protein in Lin − cells from the BM of WT mice at Day 28 after 5.0 Gy IR. Whole‐cell lysate (WCL) served as a loading control. IB, immunoblotting. (E) Quantitative polymerase chain reaction (qPCR) analysis of the mRNA expression of mitophagy‐related gene in LT‐HSCs from WT and NCOA2 −/− mice at Day 28 post 5.0 Gy IR ( n = 3). (F) Flow cytometric analysis of the protein expression of PINK1 in LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 6). Representative flow cytometric plots are shown on the left. (G) Chromatin immunoprecipitation (ChIP)‐qPCR analysis of the binding of FOXO3a to PINK1 promoter region in the LSKs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 3). Immunoglobulin G (IgG) served as a negative control ( n = 3). (H) Flow cytometric analysis of mitophagy in the LT‐HSCs from WT and NCOA2 −/− mice at the indicated time points after 5.0 Gy IR by Mtphagy Dye staining ( n = 6). Representative flow cytometric plots at Day 28 post 5.0 Gy IR are shown on the left. (I) The colocalization of TOMM20 and LAMP1 in the LT‐HSCs from WT and NCOA2 −/− mice at Day 28 after 5.0 Gy IR ( n = 20 cells). Representative immunofluorescence images are shown on the left. (J) LT‐HSCs were sorted from WT or NCOA2 −/− mice at Day 28 after 5.0 Gy IR and then were transduced with lentivirus carrying PINK1 or control. Then, the mitophagy levels in LT‐HSCs were detected by Mtphagy Dye staining ( n = 5). Oe, overexpression; Ctrl, control. **P < 0.01, ***P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Expressing, Irradiation, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Negative Control, Staining, Immunofluorescence, Transduction, Over Expression

Journal: HemaSphere

Article Title: NCOA2 promotes the return of hematopoietic stem cells to quiescence after irradiation stress by regulating FOXO3a‐dependent mitophagy

doi: 10.1002/hem3.70334

Figure Lengend Snippet: Inhibition of oxidative phosphorylation (OXPHOS) or elimination of reactive oxygen species (ROS) rescues the function of nuclear receptor coactivator 2 (NCOA2)‐deficient hematopoietic stem cells (HSCs) suffering from irradiation (IR). (A–G) Wild‐type (WT) and NCOA2 −/− mice were subjected to 5.0 Gy IR and subsequently administered metformin or vehicle by oral gavage every 2 days for 28 days. (A) The strategy of metformin treatment. (B–G) Flow cytometric analysis of (B) ROS levels, (C) tetramethylrhodamine methyl ester (TMRM)/MTG ratio, (D) apoptosis, (E) cell cycle in long‐term HSCs (LT‐HSCs) from irradiated WT and NCOA2 −/− mice with or without metformin treatment ( n = 6). (F) The number of LT‐HSCs in the bone marrow (BM) of irradiated WT and NCOA2 −/− mice with or without metformin treatment ( n = 6). (G) At Day 28 after IR, LT‐HSCs (5 × 10 2 ) from WT or NCOA2 −/− mice with or without metformin treatment, together with BM cells (5 × 10 5 ) from CD45.1 mice, were transplanted into lethally irradiated CD45.1 recipients. The percentage of donor‐derived cells in the peripheral blood (PB) of recipient mice was detected at the indicated time points after transplantation ( n = 6). (H–L) WT and NCOA2 −/− mice were subjected to 5.0 Gy IR and subsequently administered N ‐acetyl‐L‐cysteine (NAC) or vehicle by intraperitoneal injection once a day for 28 days. (H) The strategy of NAC treatment. (I, J) Flow cytometric analysis of (I) apoptosis and (J) cell cycle in LT‐HSCs from WT and NCOA2 −/− mice after NAC treatment ( n = 6). (K) The number of LT‐HSCs in the BM of irradiated WT and NCOA2 −/− mice after NAC treatment ( n = 6). (L) At Day 28 after IR, LT‐HSCs (5 × 10 2 ) from WT or NCOA2 −/− mice with or without NAC treatment, together with BM cells (5 × 10 5 ) from CD45.1 mice, were transplanted into lethally irradiated CD45.1 recipients. The percentage of donor‐derived cells in the PB of recipient mice was detected at the indicated time points after transplantation ( n = 6). (M) Schematic diagram describing the role of NCOA2 in regulating the return of HSCs to quiescence after IR via the FOXO3a‐PINK1‐mediated mitophagy axis. NS, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001; # P < 0.05, ## P < 0.01, and ### P < 0.001; and † P < 0.05, †† P < 0.01, and ††† P < 0.001.

Article Snippet: NCOA2 knockout mice (NCOA2 −/− ) were commercially acquired from Shanghai Model Organisms (China), and littermate wild‐type (WT) mice were used as controls.

Techniques: Inhibition, Phospho-proteomics, Irradiation, Derivative Assay, Transplantation Assay, Injection