<i>Vps34</i> KO MEFs show a decrease in LC3 conjugation and LC3 puncta formation upon starvation.

<p>(<b>A</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal medium (N) or HBSS (St) in the presence or absence of 50nM Bafilomycin (Nm+B or St+B, respectively) for 90 min. Right: Lysates were analyzed by immunoblotting using the indicated antibodies. Left: Relative LC3-II levels normalized to actin (n=4). (<b>B</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal media (Nm) or HBSS (St) for 30 and 90 min, fixed and immunostained. Confocal analysis of LC3, p62 and GFP-Cre fluorescence, which is artificially shown in green, red and blue colors, respectively. Arrowheads indicate LC3 and p62 colocalization (yellow). Scale bar: 10 µm. (<b>C</b>) Quantification of the number of LC3 (left panel) and p62 puncta (middle panel) per cell. Colocalization of p62 with LC3 puncta is also shown (right panel) (colocalization was defined as the number of pixels overlapping in the p62 and LC3 channels normalized per cell) (n=35-45 cells).</p

A higher affinity PI3P-binding probe, 4x-FYVE^Hrs, reveals a larger pool of intracellular PI3P in the absence of Vps34 compared to the conventional 2x-FYVE^Hrs probe.

<p>Control and <i>Vps34</i> KO MEFs were transiently transfected with both RFP-2x-FYVE^Hrs and GFP-4x-FYVE^Hrs PI3P-binding constructs for 24hr, grown in normal media and fixed. Confocal microscopy analysis of RFP-2x-FYVE^Hrs (red) and GFP-4x-FYVE^Hrs (green) is shown. Scale bar: 10 µm.</p

Silencing class II PI3Ks decreases autophagy in both control and <i>Vps34</i> null MEFs.

<p>(<b>A</b>) 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>B</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.(<b>C</b>) Control and <i>Vps34</i> 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).</p

Macroautophagy-mediated protein degradation is partially impaired in <i>Vps34</i> null MEFs.

<p>(<b>A</b>) Quantification of total [¹⁴C]-valine long-lived protein degradation induced by nutrient deprivation (HBSS) in control and <i>Vps34</i> KO MEFs. (<b>B</b>) Assessment of autophagy efficiency in control and <i>Vps34</i> KO MEFs by ¹⁴C-valine long-lived protein degradation under starvation (HBSS), starvation with 3MA or starvation with NH₄Cl conditions (see Methods) (n=8 for both A and B).</p

Recruitment of WIPI-1, a PI3P-binding protein, to sites of AP biogenesis occurs in <i>Vps34</i> KO MEFs but at diminished levels.

<p>Control and <i>Vps34</i> KO MEFs were transiently transfected with GFP-WIPI-1 for 24 hrs, cultured in HBSS for 90 min (90 min St) and fixed. Left: Confocal microscopy analysis of GFP-WIPI-1 fluorescence (green). Nuclear inactive and active Tomato-Cre is shown in red. The contrast was enhanced to reveal the WIPI puncta over the cytosolic background fluorescence. Right: Quantification of the number of GFP-WIPI-1 puncta (n=13-15 cells). Scale bar: 10 µm.</p

Ablation of Vps34 alters levels of Vps34 complex proteins.

<p>(<b>A</b>) Western blot analysis showing a time-course of Vps34 protein levels in <i>Vps34</i>^Flox/Flox MEFs infected with lentiviruses expressing either an inactive Cre (Δ) or active (CRE) full-length Cre recombinase for 7-13 days. (<b>B</b>) Western blot using an antibody directed to the NH₂-terminus of Vps34 in control and <i>Vps34</i> KO cell extracts. (<b>C</b>) Western blot analysis of Vps34 complex components and endosomal protein levels in control and <i>Vps34</i> KO MEF lysates. Quantification of protein levels after normalization to tubulin (n=3).</p