Fig. 3
Oxidative stress in AG1522 CAFs that were cocultured with MDA-MB-231 or MCF7 breast cancer cell for 120 h. a Protein carbonylation demonstrating alterations in protein oxidation with both increased (~ 40 kDa) and decreased (~ 60 kDa) bands. b Induction of mitochondrial oxidative stress: the prevalence of superoxide anion (O2·−) was analyzed by MitoSOX™ Red and flow cytometry in AG1522 CAFs cocultured with MDA-MB-231 (light green histogram) or MCF7 (dark green histogram) breast cancer cells, and in AG1522 fibroblasts cocultured with MCF10A (purple histogram) non-tumorigenic breast epithelial cells or themselves (orange histogram) (control). Additional controls consisted of AG1522 fibroblasts without MitoSOX™ Red loading (red histogram) (negative control) and AG1522 fibroblasts treated with antimycin A (blue histogram) (positive control). AG1522 CAFs cocultured with MDA-MB-231 (light green) show a ten-fold increase in MitoSox™ levels over negative control (red). The histograms corresponding to AG1522 cocultures with MCF7 (dark green), MCF10A (purple), or AG1522 (orange) possess subpopulations of cells with a higher expression of MitoSOX™ Red (see appearance of bumps in tails of descending parts of the curves compared to the smoother lognormal shape in the negative control) than the larger population; however, no other significant changes were observed for these samples. c, d Modulation of antioxidant enzyme activity. Antioxidant enzyme activities in c AG1522 CAFs and d MDA-MB-231 cells cocultured with each other for 5, 48 or 120 h. Coculture with MDA-MB-231 resulted in an increased in MnSOD activity in AG1522 CAFs at 48 and 120 h. Additionally, although the enzyme activity remained unchanged, the catalase band appeared to run slower through the gel in AG1522 CAFs at 48 and 120 h. No changes were noted in CAF CuZnSOD or any of the antioxidant enzymes in the MDA-MB-231 lysate samples. Fold change = relative change. The results are representative of two separate experiments. In each experiment, 5 independent replicates were combined to generate enough cells for analysis