Extended Data Fig. 9: ER/PR/HER2 regulate USP15-PARP1 axis via distinct mechanisms. | Nature Cancer

Extended Data Fig. 9: ER/PR/HER2 regulate USP15-PARP1 axis via distinct mechanisms.

From: Loss of the receptors ER, PR and HER2 promotes USP15-dependent stabilization of PARP1 in triple-negative breast cancer

Extended Data Fig. 9

(a) Analysis of the PARP1 mRNA level in MDA-MB-231 cells overexpressing ER, n = 3 technical replicates. (b) Comparison of the USP15 mRNA level between TNBC tissues and adjacent normal mammary tissues, n = 3 technical replicates. (c) Comparison of the USP15 mRNA level between non-TNBC tissues and adjacent normal mammary tissues, n = 3 technical replicates. (d) Cells transfected with vectors encoding EGFP or PR were supplemented with 10 μM MG132 for 12 h, then subjected to co-IP experiments. (e) Analysis of USP15 enzymatic activity in the presence or absence of PR using a Ub-CHOP2-reporter deubiquitylation assay. The procedure was previously described (Lim et al.,58). n = 3 independent biological samples. (f) The analysis of the interaction between USP15 and PR in MDA-MB-231 cells using the PLA assay. (g) Analysis of the PARP1-USP15 interaction in the presence or absence of HER2 using the BiFC assay. n = 3 independent biological samples. (h) Analysis of the interaction between HER2 and USP15 in MDA-MB-231 cells. (i) HER2 did not interact with USP15 in vitro. (j) Analysis of HER2 subcellular localization in HCC1954 cells using immunostaining. Cells were transfected with control siRNA or siRNA against HER2 prior to immunofluorescence staining with an anti-HER2 antibody. n = 12 cells for each group. (k) Western blot analysis of HER2 in the cytoplasm and nucleus in BT474 and HCC1954 cells. Cytosolic and nuclear proteins were extracted as previously described (Liu et al.,58). (l) Co-IP assay of the interaction between PARP1 and HER2 in nucleus. (m) The analysis of the interaction between PARP1 and HER2 in HCC1954 cells using the PLA assay. Cells were treated with DMSO or 500 mM MMS for 2 h before PLA assay. Data are expressed as mean ± s.d. (e, g). Data are expressed as mean ± s.e.m., each point represents a cell, the cells used for analysis in each experiment were from a single replicate (j). n.s., not significant. P values are indicated (e, g, j). Statistical significance was determined by student’s t test (g, j), or two-way ANOVA test followed by Tukey’s multiple comparison (e). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a-i, k-m).

Source data

Back to article page