Journal: Nature Communications

Article Title: Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

doi: 10.1038/s41467-020-20259-0

Figure Lengend Snippet: a YB-1 was silenced in K562, K562R, and KBM5 T315I 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 KBM5 T315I 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 γH 2 AX 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 KBM5 T315I 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 γH 2 AX expression after irradiation (IR, 3 Gy) in the control and YB-1 stably knockdown KBM5 T315I cells. Band intensity (γH 2 AX 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 ex p ressed in the cell line with stably knocked down USP47 , the expression of γH 2 AX 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 γH 2 AX 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 γH 2 AX foci ( n ) was performed ( n = 3 biologically independent samples per group). Scale bars, 20 μm. Source data are provided as a Source Data file.

Article Snippet: The number of apurinic/apyrimidinic sites (AP sites) was determined using the DNA Damage Quantification Kit (DK02, DOJINDO Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction.

Techniques: Retroviral, Transduction, Transfection, Expressing, Western Blot, Knockdown, Immunofluorescence, Staining, Irradiation, Control, Stable Transfection, Cell Culture