Figure 7. Autophagy‐associated PI3P synthesis is affected in cells lacking E‐Syts.

• A
  Representative confocal images of HeLa cells transfected with non‐targeting siRNA (siCTRL) or with siRNAs targeted to the three E‐Syts (siE‐Syts) grown under basal and starvation conditions with or without wortmannin (WORT, a PI3K inhibitor) and immunostained for PI3P (detected by the GST‐2xFYVE peptide), EEA1 (an endosomal marker) and DAPI (to stain nuclei). Scale bars, 10 μm.
• B, C
  Quantifications of confocal images for (B) PI3P‐positive and (C) PI3P‐ and EEA1‐positive structures per area (250 μm²). Plotted are means ± s.e.m. of 15–30 images from three independent experiments; ***P < 0.001, unpaired two‐tailed t‐test.
• D
  Western blots of protein lysates and immunoprecipitates from siCTRL‐ or siE‐Syt‐transfected HeLa cells immunostained for VPS34. Actin was used as a loading control.
• E
  Quantification of in vitro VPS34 enzymatic activity in siCTRL‐ and siE‐Syt‐transfected HeLa cells. NS, non‐significant, unpaired two‐tailed t‐test (n = 3). Means ± s.e.m. are plotted.
• F
  HeLa cells transfected with GFP‐E‐Syt2 and RFP‐Sec61β (an ER marker) were immunostained for WIPI2 (a PI3P‐binding protein) and DAPI. Empty arrowheads indicate co‐distribution of Sec61β/WIPI2/E‐Syt2. Scale bars, 5 μm.
• G, H
  Representative confocal images of (left) HeLa cells transfected with the ER‐PM markers GFP‐E‐Syt2 and mCherry‐E‐Syt3 (wt) and siCTRL; (middle) HeLa cells transfected with siE‐Syts and GFP‐E‐Syt2 and mCherry‐E‐Syt3 vectors (rescue experiment); (right) HeLa cells transfected with siE‐Syts and vectors for expression of the tethering‐defective mutants GFP‐∆C2C‐E‐Syt2 and mCherry‐∆C2C‐E‐Syt3. Cells were immunostained for WIPI2 and with DAPI, and (G) perinuclear and (H) peripheral planes are shown. Scale bars, 10 μm. WIPI2 staining is shown as glow picture (signal intensity).
• I
  Quantifications of perinuclear and peripheral WIPI2 puncta from experiments described in panels (G and H). Plotted are means ± s.e.m. of 15–30 images. ***P < 0.001, NS, non‐significant, unpaired two‐tailed t‐test.
• J
  Western blots from protein lysates of the cells from experiments described in panels (G, H) immunostained as indicated. Actin was used as a loading control.