Figure 5. Carnosol induces Autophagy in MDA-B231 cells.

(A) Carnosol induces the formation of autophagic vacuoles MDA-MB 231 cells observed after 24 h post treatment. MDA-MB-231 cells were seeded at a density of 5×10⁴ cells per well into 12-well plate followed by treatment with DMSO (vehicle) or 50 and 100 µM carnosol. Following treatment cells were washed and stained for autophagic vacuoles using the autophagy detection kit according to manufacturer's instructions. Fluorescent autophagic vacuoles were examined under Nikon Ti U fluorescence microscope. (B) Western blotting analysis of LC3II, p62(SQSTM1), Beclin-1 and pERK1/2 in carnosol-treated MD-MB 231 cells. Cells were treated with DMSO or increasing concentration of carnosol for 24 h, then whole cell proteins were extracted and subjected to Western blot analysis, as described in Materials and Methods, for LC3II, 62(SQSTM1), Beclin1, pERK1/2 and β-actin (loading control) proteins. (C) carnosol induced autophagy is independent of Beclin1. MDA-MB-231 cells were transiently transfected with siRNA against Beclin1 for 72 hours followed by exposure to carnosol (50 µM) for 24 hours. Whole cell lysates were then probed for LC3II. (D) Time-course analysis, by Western blotting, of PARP cleavage and LC3II accumulation in carnosol-treated MDA-MB-231 cells. Cells were treated with 100 µM carnosol and proteins were extracted at the indicated time-point (3, 6, 24 and 48 h) as described in Materials and Methods.