Figure 2.

SHISA5-depletion enhanced autophagy only under basal conditions. (A) Representative immunofluorescence images of LC3B (green) and LAMP1 (red) in WT and SHISA5 KO HeLa cells. Cells were transfected with empty vector or SHISA5-DsRed plasmid (cyan). Then cells were incubated in complete medium (NT) or EBSS 2 h. (B) The numbers of LC3 puncta in each cell were quantified. (C) The colocalization of LC3B and LAMP1 was calculated by determining the Pearson’s correlation coefficient using ImageJ (n = 10–16). (D) Representative immunofluorescence images of LC3B (green) in WT and SHISA5 KO HeLa cells. Cells were transfected with empty vector or SHISA5-DsRed plasmid (cyan). Then cells were treated with DMSO (NT) or 100 nM of Torin 1 for 2 h. (E) The numbers of LC3B puncta per cells were quantified. (F) WT and SHISA5 KO HeLa cells were untreated with DMSO or Baf A1 (100 nM) for 4 h before cell lysis. Western blot analysis for LC3B and TUBA1B/α-tubulin is shown in the upper panel. Quantification of LC3-II levels normalized to TUBA1B level is shown in the lower panel. (G) Left panel: confocal images of WT HeLa cells and SHISA5 KO cells stably expressing mRFP-GFP-LC3B. Cells were incubated in complete medium (NT) or EBSS for 2 h. See Figure S2B for the images before merge. Right panel: the number of red and yellow puncta per cell was determined using ImageJ plugins and plotted. The error bars indicate SEMs. Scale bar: 10 μm.