Fig. 4

Pt induced autophagy in HaCaT cells. Diverse concentrations of Pt (0–5 μM, 24 h) were treated to HaCaT cells - (A) The change of LC3-I to LC3-II and p62 protein expressions were measured through the Western blot method. Data were expressed as fold differences over untreated control cells. The β-actin protein functioned as a loading control. (B–C) Pt increased AVO formation – 3-MA (1 mM, 1 h) pretreated HaCaT cells were incubated with Pt (5 μM, 24 h). A fluorescence microscope (under red filter) was used to visualize the intracellular AVOs (Bar = 50 μm). AVO number is proportional to the intensity of red fluorescence. Values were quantified using Olympus Softimage Solution software. Data were denoted as fold differences over control (untreated) cells. (D) Pt decreased gp100 levels – B16F10 cells were stimulated with α-MSH (1 μM, 72 h). After incubation, the medium was separated and centrifuged at 1200 rpm for 5 min to collect the supernatant (containing melanin). 2 mL of this supernatant was treated to HaCaT cells (grown in 60 mm dish) and incubated for 72 h. Later, HaCaT cells were treated with Pt (2.5 or 5 μM, 24 h). The Western blot technique measured the LC3-II and melanoma gp100 proteins expression. β-actin functioned as an internal control protein. LC3-II data were denoted as percentage difference over untreated control. Melanoma gp100 protein data were denoted as fold-difference over untreated control. All results were denoted as mean ± SD of three or more independent experiments. Statistical significance was considered as *p < 0.05, **p < 0.01, ***p < 0.001 compared to untreated control cells. #p < 0.05, ##p < 0.01 compared to melanin alone or Pt alone treated cells.