Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis (original) (raw)
Figure 8
Silencing class II PI3Ks decreases autophagy in both control and Vps34 null MEFs.
(A) Control MEFs were transfected with mock or PI3K-C2α/β siRNA as well as GFP-WIPI-1 for 48 hrs, cultured in normal media or HBSS for 90 min and fixed. Right: Confocal microscopy images of GFP-WIPI-1 fluorescence in mock or PI3K-C2α/β siRNA-treated control cells after HBSS starvation for 90 min. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of GFP-WIPI-1 puncta observed after 90 min HBSS starvation (n=11-15 cells). (B) Cells prepared as in (A) were fixed and immunostained. Right: Confocal microscopy images showing endogenous LC3 (green) in cells cultured in HBSS in the presence of 50 nM Bafilomycin (St+B) for 30 min. DAPI is shown in blue. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of LC3 puncta observed under normal media (Nm), HBSS (St) and HBSS in the presence of Bafilomycin (St+B) conditions (n=12-19, 23-40 and 26-53 cells for Nm, St and St+B conditions, respectively). Scale bars: 10 µm.(C) Control and Vps34 KO MEFs were transfected for 48 hrs with mock or PI3K-C2α/β siRNA, cultured in normal medium (N), HBSS (St) or HBSS with 50 nM Bafilomycin (St+B) for 30 min, lysed and analyzed by immunoblotting using the indicated antibodies (n=3).