Figure 7.

Induction of reductive stress (a, b) and changes in iron-mediated adaptive responses (d) in drug-induced senescent breast cancer cells treated with encapsulated Fe₃O₄ NPs. The number of gene mutations involved in the regulation of stress responses in studied cell lines (in red) is also shown (c). The senescence program was activated using etoposide treatment. Senescent breast cancer cells were treated with 100 μg/mL NPs for 4 h. (a) Levels of total ROS and mitochondrial superoxide were analyzed in live cells using dedicated fluorogenic probes and imaging cytometry. (b) Levels of FOXO3a, SOD1, SOD2, and GPX4 were analyzed in fixed cells using immunostaining and imaging cytometry. The levels of total ROS, mitochondrial superoxide (a), and antioxidant proteins, namely, nuclear FOXO3a and cytoplasmic SOD1, SOD2, and GPX4 (b), are presented in relative fluorescence units (RFU). (c) Gene mutation raw data were downloaded from the DepMap portal (https://depmap.org/portal/). Set intersections in a matrix layout were visualized using the UpSet plot. Total, shared, and unique gene mutations in genes involved in the regulation of stress responses across eight breast cancer cell lines are presented. Blue bars in the y-axis denote the total number of gene mutations in each cell line. Black bars in the x-axis denote the number of mutations shared across cell lines connected by the black dots in the body of the plot. (d) Levels of ACSL4, TfR, and ferritin were analyzed in fixed cells using immunostaining and imaging cytometry. The levels of ACSL4, TfR, and ferritin are presented in relative fluorescence units (RFU). (a, b, d) Box and whisker plots are shown, n = 3, ***p < 0.001, **p < 0.01, and *p < 0.05 compared to untreated control (ANOVA and Dunnett’s a posteriori test); ^###p < 0.001, ^##p < 0.01, and ^#p < 0.05 compared to Fe₃O₄@Dex treatment (ANOVA and Tukey’s a posteriori test). CTRL, untreated control; Fe₃O₄@Dex, dextran-based coated iron oxide nanoparticles; and Fe₃O₄@aC, glucosamine-based amorphous carbon-coated iron oxide nanoparticles.