Figure 2

Proteome re-wiring of carboplatin-resistant 468-R cells protects from carboplatin-induced oxidative and genotoxic stress. (A,B) Hierarchical clustering of differentially expressed proteins (DE) (A) and Ingenuity Pathway Analysis (B) of the proteome alterations in MDA-MB-468 and 468-R cells treated with vehicle or 2 µM carboplatin for 5 days. Protein expression was analyzed by LC/MS–MS, and DE proteins were identified by ANOVA followed by Tukey’s HSD test with an adjusted P value < 0.05. The top 10 significantly enriched pathways are shown. P value < 0.05 was used to determine significantly enriched pathways; the –log10 (P value) was not shown if the pathway was not significantly enriched. N = 3. (C,D) ROS levels in MDA-MB-468 and 468-R cells treated with vehicle or 2 µM carboplatin for 5 days. Total ROS levels (C) were measured by the cellROX assay, and mitochondrial specific superoxide levels (D) were measured by the mitoSOX assay. Median flow of cytometric intensities were normalized to MDA-MB-468 vehicle condition. Data are shown as Mean ± SEM. P values were calculated by two-way ANOVA with Geisser–Greenhouse and Tukey’s correction. N = 3. (E) NAD/NADH ratio as measured by NAD/NADH-Glo kit. Data are shown as Mean ± SEM. P values were calculated by two-way ANOVA with Geisser–Greenhouse and Tukey’s correction. N = 3. (F) γ-H2AX staining of MDA-MB-468 and 468-R cells treated with vehicle or 2 µM carboplatin for 5 days as analyzed by flow cytometry. P values were determined by two-way ANOVA with Geisser–Greenhouse and Tukey’s corrections. N = 3. Data are shown as Mean ± SEM.