Figure 8. FLCN overexpression in basal-like breast cancer cells restores TFE3 cytoplasmic localization and attenuates tumor growth.

(A) Immunoblot analysis of empty vector (EV) and FLCN-overexpressing (FLCN^OE) MDA-MB-436 and Hs578T basal-like breast cancer cells. β-Actin was used as a loading control. (B) Quantitative analysis of the immunofluorescence data showing the percentage of TFE3 nuclear localization in EV and FLCN^OE MDA-MB-436 and Hs578T cells. Data represent the average ± SEM of n = 4 independent experiments. Statistical significance was determined using 2-way ANOVA with Bonferroni’s multiple-comparison correction. *P < 0.05; ***P < 0.001. (C) Relative TFE3, PGC-1α, and HIF-1α downstream target gene mRNA levels measured by RT-qPCR in EV and FLCN^OE MDA-MB-436 and Hs578T cells. Data represent the average ± SEM of at least n = 3 independent experiments, each performed in triplicate. Statistical significance was determined using 2-way ANOVA with Bonferroni’s multiple-comparison correction. **P < 0.001; ****P < 0.0001. (D) The percentage proliferation of EV and FLCN^OE MDA-MB-436 and Hs578T cells over 5 days, as monitored and analyzed by an IncuCyte Live Cell Analysis System. Data represent the average ± SEM of at least n = 3 independent experiments, each performed in triplicate. Significance was determined using repeated-measures 1-way ANOVA. ****P < 0.0001. (E and F) Growth of mammary tumors in mice injected with WT (EV) (blue) or FLCN^OE cells (red) in MDA-MB-436 (E) and Hs578T (F) cell models over the course of 5 to 6 weeks. Data represent the mean tumor volumes ± SEM of each cohort measured each week (n = 10 mice in each cohort). Significance was determined using repeated-measures 1-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant.