Figure 5.
ANP32B-mediated transcriptional repression of p53 enables maintenance of CML LSCs. (A) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of endogenous ANP32B in KU812 cells. (B) Immunofluorescent staining of endogenous ANP32B and p53 together with restaining of 4′,6-diamidino-2-phenylindole in KU812 cells, followed by imaging with confocal microscopy. (C) Proliferation curves of KU812 cells infected with shNC or shANP32B. Cell numbers were counted at the indicated days (n = 3). (D) Colony-forming assay for KU812 cells infected with shNC or shANP32B. Colony numbers were evaluated at day 10 (n = 3). (E) GSEA analysis of RNA-seq data from KU812 cells with shNC and shANP32B infection using p53-regulated gene set (data obtained from CML CD34+ cells treated with RITA or not, fold change >2, false discovery rate (FDR) < 0.05, data were deposited in the European Nucleotide Archive under accession number PRJEB9942). (F) Relative mRNA expression levels of indicated genes in BM GFP+LSK cells sorted from recipients receiving BCR-ABL1–transduced Anp32b+/+ and Anp32b−/− Lin− BM cells at 12 days after transplant by quantitative RT-PCR. (G) ChIP-quantitative RT-PCR of immunoglobulin G and p53 on the promoters of the indicated genes in BM GFP+LSK cells sorted from recipients receiving BCR-ABL1–transduced Anp32b+/+ and Anp32b−/− Lin− BM cells at 12 days after transplant. (H) Survival curves and analysis of median survival from recipients transplanted with BCR-ABL1–transduced Anp32b+/+p53+/+, Anp32b+/+p53+/−, Anp32b−/−p53+/+, and Anp32b−/−p53+/− Lin− BM cells (n = 6). (I) Representative FACS plots (i) and the percentage (ii) of BM GFP+LSK cells at 14 days after transplant (n = 5). (J) Cell cycle analysis of BM GFP+LSK cells at 14 days after transplant. Percentages of cell cycle distributions are shown (n = 5). (K) Apoptosis analysis of BM GFP+LSK cells at 14 days after transplant. Percentage of apoptotic GFP+LSK cells are shown (n = 5). Error bars denote mean ± SEM. Statistical significance was determined by 2-way analysis of variance (C), 2-tailed, unpaired Student t test (D,F-G,I-K), or log-rank test (H). The experiments in panels A-D were repeated 3 times independently with similar results, and the results of 1 representative experiment are shown. All animal experiments were repeated at least twice with similar results, and the results of 1 representative experiment are shown.

ANP32B-mediated transcriptional repression of p53 enables maintenance of CML LSCs. (A) Western blot analysis of indicated proteins in the inputs and immunoprecipitates of endogenous ANP32B in KU812 cells. (B) Immunofluorescent staining of endogenous ANP32B and p53 together with restaining of 4′,6-diamidino-2-phenylindole in KU812 cells, followed by imaging with confocal microscopy. (C) Proliferation curves of KU812 cells infected with shNC or shANP32B. Cell numbers were counted at the indicated days (n = 3). (D) Colony-forming assay for KU812 cells infected with shNC or shANP32B. Colony numbers were evaluated at day 10 (n = 3). (E) GSEA analysis of RNA-seq data from KU812 cells with shNC and shANP32B infection using p53-regulated gene set (data obtained from CML CD34+ cells treated with RITA or not, fold change >2, false discovery rate (FDR) < 0.05, data were deposited in the European Nucleotide Archive under accession number PRJEB9942). (F) Relative mRNA expression levels of indicated genes in BM GFP+LSK cells sorted from recipients receiving BCR-ABL1–transduced Anp32b+/+ and Anp32b−/− Lin− BM cells at 12 days after transplant by quantitative RT-PCR. (G) ChIP-quantitative RT-PCR of immunoglobulin G and p53 on the promoters of the indicated genes in BM GFP+LSK cells sorted from recipients receiving BCR-ABL1–transduced Anp32b+/+ and Anp32b−/− Lin− BM cells at 12 days after transplant. (H) Survival curves and analysis of median survival from recipients transplanted with BCR-ABL1–transduced Anp32b+/+p53+/+, Anp32b+/+p53+/−, Anp32b−/−p53+/+, and Anp32b−/−p53+/− Lin− BM cells (n = 6). (I) Representative FACS plots (i) and the percentage (ii) of BM GFP+LSK cells at 14 days after transplant (n = 5). (J) Cell cycle analysis of BM GFP+LSK cells at 14 days after transplant. Percentages of cell cycle distributions are shown (n = 5). (K) Apoptosis analysis of BM GFP+LSK cells at 14 days after transplant. Percentage of apoptotic GFP+LSK cells are shown (n = 5). Error bars denote mean ± SEM. Statistical significance was determined by 2-way analysis of variance (C), 2-tailed, unpaired Student t test (D,F-G,I-K), or log-rank test (H). The experiments in panels A-D were repeated 3 times independently with similar results, and the results of 1 representative experiment are shown. All animal experiments were repeated at least twice with similar results, and the results of 1 representative experiment are shown.

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