Figure 6.

Cisplatin induced autophagic flux in SCC cells. ΔNp63α-wt (A) and ΔNp63α-S385G (B) cells were cultured in the presence (CIS) or absence (Con) of the 10 µg/ml of cisplatin for 16 h and protein levels for LC3B-I, -II, SQSTM1 (p62) and α-tubulin (loading control) were examined using immunoblotting with antibodies to MAP1LC3B (#3868), α-tubulin (#2125) and SQSTM1 (#8025) from Cell Signaling. In some samples, the 100 nM of bafilomycin A₁ (BAF A₁, #B1793, Sigma) was added for 12 h. (C) Quantitative analysis of LC3B-I/-II conversion. Immunoblots were scanned using Phosphorimager (Molecular Dynamics) and quantified by ImageQuant software version 3.3 (Molecular Dynamics). Values of LC3B-II were expressed as a portion of LC3B-I values defined as 1. The LC3B-II/LC3B-I ratios were plotted as bars using the Microsoft Excel software with standard deviations (±SD) resulting from three independent experiments and three individual measurements of each experiment. (p < 0.05, t-test). (D) ΔNp63α-wt cells (upper parts) and ΔNp63α-S385G cells (lower parts) were cultured in the presence (Cisplatin) or absence (Control) of the 10 µg/ml of cisplatin for 16 h. Cells were trypsinized, fixed and embedded in spur resin. the 90-nm thick sections were cut and examined with a JEOL 1200EX transmission electron microscope (46). Arrows indicate autophagic vacuoles. (E and F) Cell viability assay. ΔNp63α-wt cells (E) and ΔNp63α-S385G cells (F) were cultured in the presence (CIS) or absence (Con) of the 10 µg/ml of cisplatin for 72 h in the presence or absence of BAF A₁ (100 nMm 12 h). 104 cells/well in 96-well plates were then incubated in serum-free medium with 5 µg/ml of the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (American Tissue Culture Collection) in the dark for 4 h at 37°C. Cells were lysed and incubated for 2 h at 37°C, and the measurements (A570 nm to A650 nm) were obtained on a Spectra Max-250 plate reader (Molecular Devices), as described in reference ⁴⁴. Each assay was repeated at three times in triplicate. Diagrams indicated the extent of cell viability expressed as a portion of control represented as 1. The bars are the mean ± SD of triplicate; p < 0.05, t-test.