(A and B) USP21 expression was highly correlated with ATR and COL1A1 expression. The COL1A1, ATR, and USP21 levels were determined by IHC. (C) Representative IHC images for Figure 1, C and D, and for this figure, A and B. Scale bars: 1 mm (left), 100 μm (right). (D) The interaction between USP21 and ATR in MDA-MB-231 cells was assessed by IP using ATR antibody. (E) MDA-MB-231 cells were infected with lentivirus encoding either vector or USP21, treated with MG132 for 6 hours, and harvested for ubiquitination analysis. (F) Statistical analysis of the Western blot results in E by 1-way ANOVA (n = 3). (G) MDA-MB-231 cells were transfected with either control or USP21-specific siRNA, cultured on surfaces with different stiffnesses, and harvested for IP using ATR antibody to assess ubiquitination. (H) MDA-MB-231 cells were infected with lentivirus encoding either vector, WT USP21, or C221A USP21 mutant and seeded on surfaces with different stiffnesses. After 48 hours, the cells were harvested and IP was performed using ATR antibody to assess ubiquitination. (I and J) Statistical analyses of the Western blot results in G and H by 2-way ANOVA (n = 3). Data are presented as mean ± SD. Western blot and co-IP experiments were performed using at least 3 independent biological replicates, yielding similar results.