TFEB induces autophagy. (A) Inverted color micrograph of control and stably overexpressing TFEB HeLa cells transfected with a GFP-LC3 plasmid and treated as follows: normal medium (normal), 2 hours of bafilomycin 400 nM (bafilomycin) and 2 hours in medium without nutrients (starvation); ~100 cells were analyzed for each treatment. Graph shows means of GFP-positive vesicles per cell. (B) Immunoblot analysis of LC3 in stable TFEB-overexpressing cells starved (Starv) for the indicated time (h, hours) represented as quantification of LC3-II intensity (relative to actin). (C) Cellular lysates isolated from TFEB-RNAi (+) and from scrambled RNAi-treated cells (–) cultured in normal medium, starved medium, or starved mediumsupplemented with bafilomycin (baf, 400 nM for 4 hours) as the quantification of LC3-II intensity (relative to actin). (D) TFEBmRNA levels from cells transfected with siRNA oligomers targeting TFEB or a scrambled sequence (ctr). (E) Representative confocal images of fixed HeLa cells stably expressing GFP-mRFP-LC3 transfected with empty (control) or TFEB vector shown as the average of vesicles per cell relative to the control (%). Aminimum of 2000 cells was counted. AL (autolysosomes) = (mRFP-positive vesicles)/(GFP-negative vesicles); total, mRFP-positive vesicles. All values are means TSEM of at least three independent experiments. Student’s t test (unpaired); *P < 0.05, **P < 0.01.