Fig. 4: Increase of WIPI1 protein enhances autophagic flux.

a Immunoblot analysis of LC3B lipidation in U2OS cells after transfection with control plasmids (9E10) or plasmids encoding 9E10-tagged WIPI1. U2OS cells were transfected with the indicated amounts of plasmids for 48 h before protein extraction and immunoblotting against LC3B, 9E10-tagged WIPI1 or GAPDH, n = 3. Additional immunoblots provided in Supplementary Fig. 5a. b U2OS Cas9 control or U2OS WIPI1 KO cells were transfected with control plasmids (9E10) or plasmids encoding 9E10-tagged WIPI1 for 48 h, followed by treatment with bafilomycin A1 (BafA1) in fed conditions for 3 h. Immunoblotting was conducted against LC3B, 9E10 and tubulin (n = 6, mean ± SD, Two-way ANOVA with Tukey’s multiple comparisons test). WIPI1 deficiency control Taqman qPCR is presented in Supplementary Fig. 6b, and representative Western blots in Supplementary Fig. 6c. c U2OS cells stably expressing GFP-WIPI2 were transfected with control plasmids or plasmids encoding mCherry-tagged WIPI1 for 48 h. The numbers of GFP-WIPI2 puncta cells were assessed by fluorescence microscopy in transfected cells. Welch’s t test, mean ± SD, up to 1272 analysed cells from n = 4 in duplicates. d U2OS Cas9 control or U2-OS WIPI1-KO cells were seeded into 96-well glass bottom plates and transfected with siABL1/2, siDDR1 or nontargeting siRNA (siControl) for 48 h, followed by treatment with either DMEM/FBS (fed) or EBSS (starved) for 3 h. After fixation, cells were stained with DAPI and anti-WIPI2/AF488. By automated confocal LSM, 20 images per well were acquired and between 621 to 2563 single cells (from n = 3) subjected to automated CellProfiler-based image analysis (threshold-based puncta segmentation). For statistical analysis, a two-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD). e G361 cells were transfected with plasmids encoding GFP or GFP-WIPI1 and were fed or starved for 3 h in the presence or absence of bafilomycin A1, followed by anti-WIPI2/AF546 immunofluorescence staining. Confocal LSM stacks were acquired, and the numbers of WIPI2 puncta-positive cells per acquired image (individual data points represent the result derived from each image) were counted (left panel: two-way ANOVA with Tukey’s post-hoc test, mean ± SD, up to 215 single cells from n = 3 for each condition). Indicative of the presence of overexpressed GFP-WIPI1 are elongated, perinuclear autophagic membranes found to colocalize with WIPI2 (right panel: Scale bar: 5 μm, extended image presentation in Supplementary Fig. 5b). Supplementary material is available (Supplementary Fig. 6d; Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.