Figure 3.
ANP32B interacts with and inhibits the transcriptional activity of p53 to maintain the function of HSCs. (A) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of Flag-tagged ANP32B-transfected 32D cells. (B) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of endogenous p53 in 32D cells. (C) Immunofluorescent staining of endogenous ANP32B, p53 together with restaining of 4′,6-diamidino-2-phenylindole in mouse LSK cells, followed by imaging with confocal microscopy. (D) Bacterially expressed His-ANP32B was incubated with GST or GST-tagged p53, followed by GST-tag pulldown and Western blot analysis of indicated proteins. (E) Structure schematic diagram of full-length and truncated segments of p53 and ANP32B. (F-G) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of anti-ANP32B antibody in H1299 cells transfected with Flag-p53 full-length plasmid and truncated segments. (H) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of anti-FLAG M2 beads in 293T cells transfected with Flag-ANP32B full-length plasmid and N163 segments. (I) GSEA analysis of RNA-seq data from Anp32b+/+ and Anp32b−/− LSK cells using p53-regulated gene set (n = 3 biologically independent p53+/+ and p53−/− HSPCs obtained from the GEO, accession code GSE137126). (J) Clonally derived HCT116 cell lines depleted of ANP32B (gANP32B) or not (gNS), empty vector (EV), or ANP32B-infected HCT116 cells were cotransfected with a luciferase reporter plasmid for p53 transcription (pp53-TA-luc) and Renilla luciferase reporter plasimid, and the relative luciferase activity were determined. (K) Relative mRNA expression levels of indicated genes in BM LSK cells from Anp32b+/+ and Anp32b−/− mice by quantitative RT-PCR. (L) ChIP-quantitative RT-PCR of immunoglobulin G and p53 on the promoters of the indicated genes in BM LSK cells from Anp32b+/+ and Anp32b−/− mice. (M-O) Competitive transplantation assay was conducted with Anp32b+/+p53+/+, Anp32b−/−p53+/+, Anp32b+/+p53+/−, and Anp32b−/−p53+/− BM CD45.2 cells (6 × 105) along with 6 × 105 BM cells from CD45.1 competitor. Percentages of CD45.2+ cells in PB were analyzed at the indicated time points (M). Frequencies of CD45.2+ cells (N) and CD45.2+LSK cells (O) in BM were analyzed at 16 weeks after transplant (n = 5). Error bars denote mean ± SEM. Statistical significance was determined by a 2-tailed, unpaired Student t test (J-O). The experiments in panels A-D, F-H, and J were repeated 3 times independently with similar results, and the results of 1 representative experiment are shown. The animal experiments were repeated twice with similar results, and the results of 1 representative experiment are shown.

ANP32B interacts with and inhibits the transcriptional activity of p53 to maintain the function of HSCs. (A) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of Flag-tagged ANP32B-transfected 32D cells. (B) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of endogenous p53 in 32D cells. (C) Immunofluorescent staining of endogenous ANP32B, p53 together with restaining of 4′,6-diamidino-2-phenylindole in mouse LSK cells, followed by imaging with confocal microscopy. (D) Bacterially expressed His-ANP32B was incubated with GST or GST-tagged p53, followed by GST-tag pulldown and Western blot analysis of indicated proteins. (E) Structure schematic diagram of full-length and truncated segments of p53 and ANP32B. (F-G) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of anti-ANP32B antibody in H1299 cells transfected with Flag-p53 full-length plasmid and truncated segments. (H) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of anti-FLAG M2 beads in 293T cells transfected with Flag-ANP32B full-length plasmid and N163 segments. (I) GSEA analysis of RNA-seq data from Anp32b+/+ and Anp32b−/− LSK cells using p53-regulated gene set (n = 3 biologically independent p53+/+ and p53−/− HSPCs obtained from the GEO, accession code GSE137126). (J) Clonally derived HCT116 cell lines depleted of ANP32B (gANP32B) or not (gNS), empty vector (EV), or ANP32B-infected HCT116 cells were cotransfected with a luciferase reporter plasmid for p53 transcription (pp53-TA-luc) and Renilla luciferase reporter plasimid, and the relative luciferase activity were determined. (K) Relative mRNA expression levels of indicated genes in BM LSK cells from Anp32b+/+ and Anp32b−/− mice by quantitative RT-PCR. (L) ChIP-quantitative RT-PCR of immunoglobulin G and p53 on the promoters of the indicated genes in BM LSK cells from Anp32b+/+ and Anp32b−/− mice. (M-O) Competitive transplantation assay was conducted with Anp32b+/+p53+/+, Anp32b−/−p53+/+, Anp32b+/+p53+/−, and Anp32b−/−p53+/− BM CD45.2 cells (6 × 105) along with 6 × 105 BM cells from CD45.1 competitor. Percentages of CD45.2+ cells in PB were analyzed at the indicated time points (M). Frequencies of CD45.2+ cells (N) and CD45.2+LSK cells (O) in BM were analyzed at 16 weeks after transplant (n = 5). Error bars denote mean ± SEM. Statistical significance was determined by a 2-tailed, unpaired Student t test (J-O). The experiments in panels A-D, F-H, and J were repeated 3 times independently with similar results, and the results of 1 representative experiment are shown. The animal experiments were repeated twice with similar results, and the results of 1 representative experiment are shown.

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