Fig. 6. Comparison of the contribution of Wipi3 and Wipi2 to canonical autophagy.
a Wipi3Cr MEFs were generated from WT MEFs using the CRISPR/Cas9 system. A 19-bp deletion of wipi3 (third exon) was confirmed by genomic sequencing. The deleted nucleotide sequence and the protospacer adjacent motif (PAM) are indicated below the sequence. b The indicated MEFs were starved for the indicated times, and the expression of the indicated proteins was analyzed by western blotting. Actin was included as a loading control. c The indicated MEFs expressing GFP-LC3 were starved for the indicated times, and GFP-LC3 puncta formation was analyzed. Representative images are shown. Bars = 10 µm. d The indicated MEFs were starved with or without bafilomycin A1 (10 nM) for the indicated times, and the expression of each protein was analyzed by western blotting. e Similar experiments to (d) were performed using etoposide (10 µM), instead of starvation. The deletion of wipi3 showed a minimal effect on canonical autophagy. f Flag-Wipi2 or Flag-Wipi3 were expressed in HA-LC3-expressing WT MEFs, and were treated with etoposide (10 µM) for 12 h. Then, cells were immunostained with anti-HA and anti-Flag antibodies. Bars = 10 µm. Magnified images of the dashed squares are shown in the insets. Bars = 2 µm. LC3 signals were merged with Wipi2 but not with Wipi3. g Flag-Wipi3 and mRFP-GFP were expressed in WT MEFs, and were treated with etoposide (10 µM) for 12 h. Then, cells were immunostained with an anti-Flag antibody. Bars = 10 µm. Magnified images of the dashed squares are shown in the insets. Bars = 1 µm. Red puncta (autolysosomes) were well merged with Wipi3 signals. h, i Electron micrographs of Wipi3Cr MEFs (h) and Wipi3KO MEFs (i) treated with etoposide (10 µM) for 12 h. “G” indicates the Golgi apparatus, and arrows indicate swollen rod-shaped Golgi membranes. Bars = 1 µm (h) and 0.5 µm (i). Source data are provided as a Source Data file.