Figure 7

siRNA-mediated TFEB knockdown (KD) abolishes PE-induced autophagy and protection against mitochondrial ROS stress in SY5Y cells. (a) Top: PCR analysis of TFEB mRNA expression levels in SY5Y cells after 72 h transfection with 25 μM scrambled (Scr) siRNA or TFEB siRNA. Bottom: Quantification of TFEB mRNA levels, expressed as fold change against Scr control. (b-c) Top: Immunoblots of LC3 (b) and LAMP1 (c) in Scr control and TFEB KD cells treated with DMSO or 300 μg/ml PE for 16 h. Bottom: Quantification of LC3-II (b) and LAMP1 (c) levels, expressed as fold change against Scr siRNA control cells treated with DMSO. Full-length blots are presented in Supplementary Figure S11b and c. (d) Top: Immunofluorescence images of TOMM20 and LC3 colocalization in Scr siRNA control and TFEB KD cells, untreated or treated with 10 μM CCCP supplemented with either DMSO or 300 μg/ml PE for 16 h. Bottom: Quantification of TOMM20 and LC3 colocalization per cell area, expressed as fold change against DMSO treated Scr siRNA control cells. (e) Left: Immunoblot of TOMM20 in CHX-treated Scr siRNA control and TFEB KD cells subjected to 10 µM CCCP and 300 μg/ml PE in the absence or presence of lysosomal inhibition (NL + 1 µM Vb). Right: Quantification of TOMM20 flux in Scr siRNA control and TFEB KD cells. Full-length blots are presented in Supplementary Figure S11e. (f) Top: Immunofluorescence images of TOMM20 and Mitosox in Scr siRNA control cells treated with DMSO, 10 µM CCCP or CCCP with 300 µg/ml PE, and in TFEB KD cells treated with 10 µM CCCP with 300 µg/ml PE. Bottom: Quantification of Mitosox intensity per cell area, expressed as fold change against DMSO treated Scr siRNA control cells. 20–30 cells from random fields were analyzed for TOMM20-LC3 colocalization and Mitosox levels. Nuclei were stained with DAPI. All values are mean + S.E.M (n = 3–7). Differences are significant at *p < 0.05, **p < 0.01, ***p < 0.005. N.S = Not significant. Scale bar is 10 μm, unless otherwise stated.