antibodies against src2  (Bethyl)

Journal: Cell Death & Disease

Article Title: HNF4A mitigates sepsis-associated lung injury by upregulating NCOR2/GR/STAB1 axis and promoting macrophage polarization towards M2 phenotype

doi: 10.1038/s41419-025-07452-z

Figure Lengend Snippet: A The expressions of TNF-α and Arg-1 in alveolar lavage fluid are detected by flow cytometry. pro-inflammatory macrophages are F4/80 + TNF-α + cells, whereas anti-inflammatory macrophages are F4/80 + Arg-1 + cells. B Total cells, neutrophils, and macrophages in the alveolar lavage fluid are counted by Wright-Giemsa staining. C The expression of NCOA2 is assayed by real-time PCR in alveolar macrophages. D The expression of NCOA2 is assayed by WB in alveolar macrophages. E The expression of NCOA2 in lung tissues is observed by IF staining (×400) of F4/80 and NCOA2. F The expression of NCOA2 in alveolar macrophages is observed by IF staining (×400). The data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies were as follows: HNF4A antibody (1:500, bs-3828R, Bioss, Beijing, China), NCOA2 antibody (1:1000, bs-20558R, Bioss, Beijing, China) and STAB1 antibody (1:500, bs-7510R, Bioss, Beijing, China).

Techniques: Flow Cytometry, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: HNF4A mitigates sepsis-associated lung injury by upregulating NCOR2/GR/STAB1 axis and promoting macrophage polarization towards M2 phenotype

doi: 10.1038/s41419-025-07452-z

Figure Lengend Snippet: A Isolation and culture of mouse bone BMDMs. M2-type polarization is induced by treating BMDMs with IL-4 for 24 h. Schematics are obtained from SciDraw. Mouse: 10.5281/zenodo.3926105; Adenovirus: doi.org/10.5281/zenodo.3926233. B The expression of HNF4A in cells is detected by real-time PCR and WB. C Ad-HNF4A infected BMDMs for 48 h, followed by inducing M2 polarization with IL-4 for 24 h. D The expression of HNF4A in BMDMs is detected by real-time PCR and WB. E The expressions of CD206, CD163, and Arg-1 are detected by real-time PCR in BMDMs. F The expression of NCOA2 is measured by real-time PCR in BMDMs. G Dual luciferase reporter assays are performed with 293 T cells co-transfected with pGL3-basic luciferase reporter containing NCOA2 promoter (pGL3-NCOA2 pro) and the HNF4A overexpression or empty vector. Luciferase activities are measured and normalized to the Renilla activity. H ChIP assay is performed to show that HNF4A binds to the NCOA2 promoter. The data are presented as mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies were as follows: HNF4A antibody (1:500, bs-3828R, Bioss, Beijing, China), NCOA2 antibody (1:1000, bs-20558R, Bioss, Beijing, China) and STAB1 antibody (1:500, bs-7510R, Bioss, Beijing, China).

Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Infection, Luciferase, Transfection, Over Expression, Plasmid Preparation, Activity Assay

Journal: Cell Death & Disease

Article Title: HNF4A mitigates sepsis-associated lung injury by upregulating NCOR2/GR/STAB1 axis and promoting macrophage polarization towards M2 phenotype

doi: 10.1038/s41419-025-07452-z

Figure Lengend Snippet: A BMDMs infected with Ad-HNF4A are infected with Ad-shNCOA2 or Ad-shNC for 48 h, and then cells are cultured with IL-4 and Dex for 24 h followed by transcriptome analysis. B Principal-component analysis (PCA) analysis. C Volcano plot of differentially expresses genes (DEGs). D The heatmap of DEGs. E GO and KEGG enrichment analysis of DEGs. F Venn diagram shows that overlapping peak is identified between anti-GR and anti-NCOA2 binding peaks in ChIP-seq-GSE99887 in the left panel. The middle Venn diagram shows that overlapping genes between overlapping genes of left Venn diagram and DEGs in mRNA-seq. The right Venn diagram shows that overlapping genes between the top 10% of Genecards-retrieval involved in macrophage polarization and overlapping genes of middle Venn diagram. The data are presented as mean ± SD.

Article Snippet: The primary antibodies were as follows: HNF4A antibody (1:500, bs-3828R, Bioss, Beijing, China), NCOA2 antibody (1:1000, bs-20558R, Bioss, Beijing, China) and STAB1 antibody (1:500, bs-7510R, Bioss, Beijing, China).

Techniques: Infection, Cell Culture, Binding Assay, ChIP-sequencing

Journal: Cell Death & Disease

Article Title: HNF4A mitigates sepsis-associated lung injury by upregulating NCOR2/GR/STAB1 axis and promoting macrophage polarization towards M2 phenotype

doi: 10.1038/s41419-025-07452-z

Figure Lengend Snippet: A BMDMs are infected with Ad-HNF4A for 48 h, and M2-type polarization of BMDMs is induced followed by the addition of 100 nM Dex. After 24 h, the expression of STAB1 in cells is detected by real-time PCR and WB. B BMDMs are infected with Ad-shNC, Ad-shNCOA2, or Ad-shGR for 48 h, and M2-type polarization of BMDMs is induced followed by the addition of 100 nM Dex. After 24 h, the expression of STAB1 in cells is detected by real-time PCR and WB. C M2-type polarization of BMDMs is induced followed by the addition of 100 nM Dex. ChIP assays to show that GR and NCOA2 bind to STAB1 intron 1. D pGL3-basic luciferase reporters containing STAB1 promoter and/or STAB1 intron1 are constructed. Dual luciferase reporter assays are performed. Luciferase activities are measured and normalized to the renilla activity. E After being infected with Ad-shNC or Ad-shSTAB1, M2-type polarization of BMDMs is induced. The expression of STAB1 is measured by real-time PCR and WB. F The expression of CD206 in BMDMs is measured by flow cytometry. G A possible mechanism for the treatment of sepsis is that HNF4A activates NCOA2 through transcription and promotes the transcriptional activation mediated by the NCOA2/GR complex, thereby promoting STAB1 expression. Schematics are drawn via SciDraw and Pinclipart. The lung and arrows are from Pinclipart; DNA: doi.org/10.5281/zenodo.3926245. The data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies were as follows: HNF4A antibody (1:500, bs-3828R, Bioss, Beijing, China), NCOA2 antibody (1:1000, bs-20558R, Bioss, Beijing, China) and STAB1 antibody (1:500, bs-7510R, Bioss, Beijing, China).

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Construct, Activity Assay, Flow Cytometry, Activation Assay

Journal: Cell Death & Disease

Article Title: HNF4A mitigates sepsis-associated lung injury by upregulating NCOR2/GR/STAB1 axis and promoting macrophage polarization towards M2 phenotype

doi: 10.1038/s41419-025-07452-z

Figure Lengend Snippet: primer sequences used in real-time PCR.

Article Snippet: The primary antibodies were as follows: HNF4A antibody (1:500, bs-3828R, Bioss, Beijing, China), NCOA2 antibody (1:1000, bs-20558R, Bioss, Beijing, China) and STAB1 antibody (1:500, bs-7510R, Bioss, Beijing, China).

Techniques:

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

Journal: International Journal of Biological Sciences

Article Title: Apigenin Suppresses Bladder Cancer via the SIRT6-NCOA2-PPARα Axis

doi: 10.7150/ijbs.128177

Figure Lengend Snippet: Apigenin enhances SIRT6-NCOA2 interaction, promotes NCOA2 deacetylation, and is associated with PPARα signaling activation. (A) Quantification summary of acetylated proteomics data. Paired bar graphs display identified and quantified acetylation sites, peptides, and proteins. (B) The number of modification sites in a protein. (C) Clustering heatmap of differential acetylation sites. Each row represents a lysine acetylation modification site (labeled as "protein name_K position"), and each column represents a sample (including the control group and various treatment doses). The right panel presents the p value for the comparison between the highest dose group (100) and the control group (0). The color reflects the relative acetylation levels of each site across the samples (Z-score) red indicates higher acetylation, while blue indicates lower acetylation. (D) Protein-Protein Interaction (PPI) Network of 19 genes. (E) WikiPathways enrichment analysis of differentially expressed proteins. Each circle represents an enriched pathway, with circle size proportional to the number of differentially expressed proteins in that pathway. The color corresponds to the statistical significance of the enrichment (warmer colors indicate more significant FDR values). (F) Schematic representation of post-translational modification sites in the NCOA2 protein. (G) Proteomic acetylation analysis reveals that apigenin treatment reduces acetylation of NCOA2 at lysine residues K780 and K785 (n=3). (H) Heatmap of deacetylase family protein expression. The upper graph shows proteomic data, and the lower graph shows transcriptomic data. Rows represent classical HDAC enzymes (classified as Class I, IIa, IIb, and IV) and Sirtuin family proteins (SIRT1-7), and columns correspond to different treatment group samples (T24+0 control and 20, 40, 100 µM treatment groups). The color represents the relative abundance of gene expression as a Z-score (red indicates higher expression compared to the control average, blue indicates lower expression). (I-J) Apigenin (100 μM) enhances the interaction between SIRT6 and NCOA2, as demonstrated by co-immunoprecipitation. (K) Acetylation levels of NCOA2 in T24 cells following apigenin treatment were detected using a pan-acetyl lysine antibody. (L) Pan-acetyl lysine antibody was used to assess acetylation levels of wild-type NCOA2 and its K780R, K785R, K780Q, K785Q single and double mutants following apigenin stimulation. (M) Molecular docking simulation of apigenin with SIRT6, showing a binding score of -7.6 kcal/mol. (N) Dual-luciferase reporter assays were conducted to evaluate PPARα transcriptional activity in T24 cells stably expressing NCOA2 K780R/K785R, K780Q/K785Q, or wild-type NCOA2 under apigenin treatment (n=4). (O-P) Apigenin enhances the expression of key enzymes involved in fatty acid β-oxidation, including FASN, ACC, CPT1A, and ACOX1, through deacetylation of NCOA2 at K780 and K785 sites (n=3). Statistical comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. Data are presented as mean ± SD. P values are indicated as follows: * p < 0.05 , ** p < 0.01, *** p < 0.001 , **** p < 0.0001; ns = not significant, compared to the control group.

Article Snippet: Lysates were incubated with Protein A+G magnetic beads (Beyotime, China, P2179M) pre-coupled with NCOA2 (CST, USA, 96687), FLAG (Proteintech, China, 66008-4-Ig), or SIRT6 (Abcam, UK, ab191385) antibodies.

Techniques: Activation Assay, Modification, Labeling, Control, Comparison, Histone Deacetylase Assay, Expressing, Gene Expression, Immunoprecipitation, Binding Assay, Luciferase, Activity Assay, Stable Transfection

Journal: International Journal of Biological Sciences

Article Title: Apigenin Suppresses Bladder Cancer via the SIRT6-NCOA2-PPARα Axis

doi: 10.7150/ijbs.128177

Figure Lengend Snippet: NCOA2 Acetylation and its role in bladder cancer progression, metastasis. (A-B) JC-1 staining analysis of mitochondrial membrane potential in T24 cells overexpressing wild-type NCOA2, the deacetylation-mimetic mutant (K780R/K785R), or the acetylation-mimetic mutant (K780Q/K785Q), with or without apigenin treatment. (A) Representative flow cytometry analysis of JC-1 fluorescence. (B) Quantitative analysis of red/green fluorescence intensity ratio (n=3). (C-D) Colony formation assay evaluating proliferation of T24 cells overexpressing wild-type or mutant NCOA2 under apigenin stimulation. (C) Representative colony images. (D) Quantification of colony numbers (n=3). (E-F) Wound healing assay assessing cell migration in T24 cells expressing wild-type or mutant NCOA2 following apigenin treatment. (E) Representative images at 0 h and 24 h. (F) Quantitative analysis of wound closure percentage (n=8). (G) Tumor growth evaluation in CDX mice implanted with NCOA2 K780R/K785R or K780Q/K785Q-mutated T24 cells (n=6). (H-L) In vivo lymph node metastasis model using T24 cells stably expressing wild-type NCOA2, K780R/K785R, or K780Q/K785Q. (H) Schematic diagram of the animal experiment protocol. (I-J) Representative images and quantitative analysis of tumor size in inguinal lymph nodes (n=4). (K-L) Representative images and quantitative analysis of tumor size in popliteal lymph nodes and Inguinal lymph nodes (n=4). All quantitative data are presented as mean ± SD from at least three independent experiments. Statistical comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. Data are presented as mean ± SD. P values are indicated as follows: * p < 0.05 , ** p < 0.01, *** p < 0.001 , **** p < 0.0001; ns = not significant, compared to the control group. ## p < 0.01, ### p < 0.001 , #### p < 0.0001; ns = not significant, compared to the vehicle group.

Article Snippet: Lysates were incubated with Protein A+G magnetic beads (Beyotime, China, P2179M) pre-coupled with NCOA2 (CST, USA, 96687), FLAG (Proteintech, China, 66008-4-Ig), or SIRT6 (Abcam, UK, ab191385) antibodies.

Techniques: Staining, Membrane, Mutagenesis, Flow Cytometry, Fluorescence, Colony Assay, Wound Healing Assay, Migration, Expressing, In Vivo, Stable Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Apigenin Suppresses Bladder Cancer via the SIRT6-NCOA2-PPARα Axis

doi: 10.7150/ijbs.128177

Figure Lengend Snippet: Clinical relevance of NCOA2 acetylation and its role in tumor progression and metastasis in bladder cancer. (A-B) Immunohistochemical (IHC) staining of NCOA2, SIRT6, TOMM20, and ACOX1 in tumor samples from patients with primary, recurrent, and metastatic bladder cancer, as well as matched adjacent normal tissues (n=6). (A) Representative IHC images. (B) Quantitative analysis of staining intensity. (C) Quantitative PCR (qRT-PCR) analysis of NCOA2 mRNA expression in tumor tissues from patients at different clinical stages compared with adjacent normal tissues (n=3). All quantitative data are presented as mean ± SD from at least three independent experiments. Statistical comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. Data are presented as mean ± SD. P values are indicated as follows: * p < 0.05 , ** p < 0.01, *** p < 0.001 , **** p < 0.0001; ns = not significant, compared to the control group. ## p < 0.01, ### p < 0.001 , #### p < 0.0001; ns = not significant, compared to the vehicle group.

Article Snippet: Lysates were incubated with Protein A+G magnetic beads (Beyotime, China, P2179M) pre-coupled with NCOA2 (CST, USA, 96687), FLAG (Proteintech, China, 66008-4-Ig), or SIRT6 (Abcam, UK, ab191385) antibodies.

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Control