Fig. 5.

A low level of αSNAP is sufficient to partially promote autophagy under starvation conditions. (A, Left) HeLa cells were transfected with nontargeting (control) siRNA (non tgt) or αSNAP siRNA using DharmaFECT 1 transfection reagent. After 48 h, the cells were incubated for 4 h in normal growth medium or EBSS in the absence or presence of 0.1 µM BafA, lysed with RIPA extraction buffer, and then analyzed by Western blotting. (A, Right) At least four independent trials were quantified, using Image Studio Lite software normalized to the nontargeting control sample. Values are means ± SE. Starv, starvation conditions. (B, Left) HeLa cells were treated as in A, fixed with 100% methanol, immunostained with anti-LC3 antibodies, analyzed by a Leica confocal microscope using the same threshold for all samples and deconvolved using Huygens software. Z-stack projection was done using ImageJ software. (Scale bar, 10 µm.) Large magnification of stained cells is presented at Right. (Middle) Quantification of LC3 puncta using LAS X software, Leica Microsystems. n > 1,000, ***P < 0.0001. Values are means ± SE. (Right) HeLa cells stably expressing GFP-LC3 were treated as in A, then harvested by trypsin, fixed with 100% methanol, and stained with DAPI. Cells were then subjected to ImageStream analysis, as described in Materials and Methods. Four independent trials were analyzed and quantified by IDEAS v. 6.2 software. (C) HeLa cells were transfected as in A, homogenized, and fractionated as described in Materials and Methods. Total cell homogenates are presented on the Right.