Extended Data Fig. 4: USP15 interacts with and deubiquitinates PARP1 in TNBC cells.
(a) Co-IP analysis of the PARP1-USP15 interaction in different cell lines. (b) USP15 deubiquitinated PARP1 in MDA-MB-231 cells. (c) PARP1 mRNA levels did not change in USP15-depleted MDA-MB-231 cells, n = 3 technical replicates. (d, e) Immunofluorescence analysis of PARP1 in control and USP15-depleted MDA-MB-231 cells. And quantitative analysis was performed with ImageJ software. n = 20 for each group. Representative images are shown in (e). (f, g) Immunofluorescence analysis of PAR in control or USP15-depleted MDA-MB-231 cells. Cells were treated with MMS prior to immunostaining with the antibody against PAR. And quantitative analysis was performed with ImageJ software. n = 20 for each group. Representative images are shown in (g). (h) Analysis of BER efficiency in MDA-MB-231 cells with USP15 and/or PARP1 depletion. n = 3 independent biological samples. (I, j) Analysis of PARP1 and USP15 levels in PARP1-KO, USP15-KO, or PARP1 & USP15 both KO (dKO) MDA-MB-231 cells. (k, l) Analysis of PARP1 and USP15 levels in different groups of MDA-MB-231 cells. (m, n) Analysis of genomic stability in MDA-MB-231 cells in the presence or absence of 200 μM MMS by an alkaline comet assay. At least 50 cells per group were included for analysis. (o, p) Analysis of PARP1 and USP15 levels in different groups of HCC70 cells. (q, r) Analysis of genomic stability in HCC70 cells in the presence or absence of 200 μM MMS by an alkaline comet assay. Different groups of cells as indicated were treated with 200 μM MMS or DMSO and were then harvested for the alkaline comet assay. At least 50 cells per group were included for analysis. All experiments were repeated at least three times. Data are expressed as mean ± s.d. (h). Data are expressed as the mean ± s.e.m., each point represents a cell, the cells used for analysis in each experiment were from a single replicate. (d, f, m-n, q-r). n.s., not significant. P values are indicated (d, f, h, m-n, q-r). Statistical significance was determined by Student’s t test (D, F), or one-way ANOVA test followed by Tukey’s multiple comparison (h, m-n, q-r). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a-c, h-p).
