Fig. 4

BAY 87-2243-mediated complex I inhibition increases mitochondrial and cytosolic ROS levels resulting in ROS-mediated cell death. a G-361 cells were treated with BAY 87-2243 (10 nM). Mitochondrial ROS levels were measured using the MitoSOX dye after 8 and 16 h (n = 3). b G-361 cells were treated with BAY 87-2243 (10 nM). Cytosolic ROS levels were measured using the ROS-reactive dye CM-H₂DCFDA after 8 and 16 h (n = 3). c–d Melanoma cells were treated with BAY 87-2243 (10 nM) under (c and d) low- (5 mM) and d high (25 mM) glucose conditions. Cell lysates were collected from treated melanoma cell lines (8 h) and probed with antibodies recognizing NRF2. Actin was used as a loading control. e–f Melanoma cells were treated for 16 h with BAY 87-2243 (10 nM) alone or in combination with the antioxidant vitamin E (25 μM). e Mitochondrial and f cytosolic ROS levels were analyzed using flow cytometry (n = 3). g SK-MEL-28 cells were treated with BAY 87-2243 (10 nM) alone or in combination with vitamin E (25 μM). Cell lysates collected from treated SK-MEL-28 cells (16 h) were probed with antibodies recognizing NRF2. Actin was used as a loading control. h BRAF mutant melanoma cells were treated with BAY 87-2243 (10 nM) alone or in combination with vitamin E (25 μM). Cell death was measured after 72 h using propidium iodine (n = 3). i–j Melanoma cells were treated with BAY 87-2243 (10 nM) alone or in combination with vitamin E (25 μM) under (i and j) low- and j high glucose conditions. Cell lysates collected from treated A-375 and G-361 cells (16 h) were probed with antibodies recognizing cleaved PARP. Actin was used as a loading control. k–l BRAF mutant melanoma cells were treated with BAY 87-2243 (10 nM) alone or in combination with vitamin E (25 μM). Cell lysates collected from treated cells (8 and 16 h) were probed with antibodies recognizing k phospho-p38 MAPK and total p38 MAPK and l phospho-AMPK (Thr172) and total AMPK. Actin was used as a loading control. Data are represented as the mean ± SD. *p < 0.05