Fig. 6. YB-1 contributes to USP47-mediated DNA damage repair in CML cells.
a YB-1 was silenced in K562, K562R, and KBM5T315I cells with a retroviral transduction system, and viable cells were counted in the transfected cell lines at different times (n = 3 biologically independent samples per group). Data are mean ± s.d. p-values were analyzed by two-way analysis of variance (ANOVA). ****p < 0.0001. b DNA damage-related protein expression was measured by western blot after USP47 knockdown in the K562 and KBM5T315I cells. c GFP+ BM mononuclear cells were collected from Usp47+/+ and Usp47−/− CML mice, and the indicated proteins were examined by western blot. d Immunofluorescence staining of γH2AX foci in the USP47 or YB-1 knockdown K562 cells. Scale bars, 20 μm. e The number of AP sites (DNA damage quantification) was detected in genomic DNA after USP47 knockdown in the K562 and KBM5T315I cells (n = 3 biologically independent samples per group). Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA). ***p < 0.001. f Time-course analysis of the USP47 knockdown-induced DNA damage response in K562 cells and cleaved-PARP1 by western blot. g YB-1, PCNA, and TOPO IIα mRNA levels after YB-1 knockdown at day 7 in K562 cells (n = 3 biologically independent samples per group). Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA). *p < 0.05; **p < 0.01; ****p < 0.0001. h DNA damage-related protein expression was measured by western blot after YB-1 knockdown in the K562 cells. i The time-course of DNA damage is shown by γH2AX expression after irradiation (IR, 3 Gy) in the control and YB-1 stably knockdown KBM5T315I cells. Band intensity (γH2AX relative to β-actin) is shown by the histogram (n = 3 biologically independent samples per group). Data are mean ± s.d. p-values were analyzed by two-way analysis of variance (ANOVA). ***p < 0.001; ****p < 0.0001. j Exogenous YB-1 protein was expressed in the cell line with stably knocked down USP47, the expression of γH2AX and exogenous YB-1 (GFP tag) was detected by western blot. k The number of AP sites was measured as described above (n = 3 biologically independent samples per group). Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA). ***p < 0.001. l γH2AX expression in Usp47−/− MEFs was measured by western blot after the reintroduction of YB-1. m, n USP47 was knocked down in primary CML CD34+ cells, then the cells were cultured in a stem cell colony formation medium. The colonies (m) were counted on day 14 (n = 3 biologically independent samples per group). Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA). ***p < 0.001. Immunofluorescence staining of γH2AX foci (n) was performed (n = 3 biologically independent samples per group). Scale bars, 20 μm. Source data are provided as a Source Data file.