Fisetin induced modulation of YB-1/RSK signaling is associated with decrease in MDR1 (A) Representative images (20x) showing immunofluorescence for MDR1 in fisetin-treated A375 and 451Lu melanoma cells. Scale bar, 20 µm. DAPI was used as nuclear staining control. (B) Whole cell lysates of NCI/ADR-Res ovarian cancer cells treated with fisetin (40–80 µM:24 h) were analyzed for p-YB-1, YB-1 and MDR1 expression. Equal loading was confirmed by reprobing for vinculin. (C) Histogram represents relative MDR1 levels in NCI/ADR-Res cells treated with fisetin (40–80 µM:24 h). Gene expression was measured by qPCR and normalized to GAPDH. Error bars represent mean ± SE among three independent experiments, where each experiment was performed in triplicate. (D) Analysis of MDR1 and p-RSK in fisetin-treated A375 cells at specified time points. Equal loading was confirmed by reprobing for vinculin. (E) Equal amounts of cell lysates treated with/without fisetin (60 μM:24 h) were immunoprecipitated with RSK antibody followed by western blot analysis for MDR1 and YB-1 antibodies. (F) Representative images (20x) showing immunofluorescence for MDR1 (green) and YB-1 (red) in fisetin-treated (60 µM:24 h) A375 melanoma cells. Scale bar, 20 µm. (G) WM35 melanoma cells transfected with pcDNA-HA-YB-1, treated with fisetin (60 µM:24 h) were analyzed for p-RSK and MDR1 expression. Equal loading was confirmed by reprobing for vinculin. (H) Viability studies in 451Lu melanoma cells, 24 post treatment with/without fisetin and vemurafenib, as assessed by MTT assay. (I) Intracellular vemurafenib concentration in A375 melanoma cells with/without fisetin treatment (60 μM) at 4 and 8 hours, quantified by LC-MS/MS analysis. (J) A375 cells loaded with DiOC2(3) and incubated at 37 °C with/without vinblastine or fisetin were quantified on Biotek plate reader, where DMSO at 4 °C served as positive control. Data shown are representative of three independent experiments with similar results.