Fig. 7. p62 is not an essential factor in starvation-induced autophagy. (A) HeLa cells were transfected with either transfection reagent (mock), non-targeting siRNA (control siRNA), or p62 siRNA using DharmaFect reagent. After 48 hours, the cells were incubated under control or starvation conditions in the absence or presence of 0.1 µM Baf A, lysed using RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-LC3 antibodies. (B) Quantification of LC3 lipidation and the level of p62 protein were performed as described in Materials and Methods. (C) The cells were transfected as in A, starved for 3 hours in the presence of 0.1 µM Baf A, immunostained with anti-p62 and anti-LC3 antibodies, and analyzed using confocal microscopy. Scale bar: 10 µm. (D) The cells were transfected as in A, starved for 3 hours in the presence of 0.1 µM Baf A, immunostained with anti-LC3 and anti-LAMP-1 antibodies, and analyzed using confocal microscopy (left panels). Scale bar: 10 µm. Quantitative colocalization analysis of endogenous LC3 with LAMP-1 (right) was performed as described in the Materials and Methods. Data are means ± s.d. of three independent experiments. (E) Following 48 hours of transfection, the rate of degradation of long-lived proteins was measured in siRNA-transfected cells incubated in either MEM or EBSS medium, in the absence or presence of 3-MA (10 mM) or Baf A (0.1 µM). Values express the percentage of cellular proteins degraded in 4 hours represented as the mean ± s.d. of six determinations.