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. 2017 May 31;8:15637. doi: 10.1038/ncomms15637

Figure 1. All WIPI members fold into seven-bladed β-propeller proteins that bind PtdIns3P and co-localize at nascent autophagosomes.

Figure 1

(a) Structural homology modelling using HHpred. Propeller blades are indicated (1 to 7), and unstructured sequences are omitted. (b) Protein-phospholipid binding overlay assays using G361 cell extracts followed by the detection of endogenous WIPI1, WIPI2 or WIPI4 or using a monoclonal U2OS cell line stably expressing GFP-WIPI3 followed by anti-GFP enhanced chemiluminescence (ECL) detection (left panels). Monoclonal U2OS cell lines stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved for 3 h with nutrient-free medium. Images were acquired by confocal LSM and processed using Volocity to generate 3D-reconstruction fly-through movies (Supplementary Movies 1–4). Representative still images are presented (right panels). Scale bars: 3 μm. (c) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were fed (F) or starved (S) for 3 h with or without bafilomycin A1 (BafA1) or LY294002 (LY). The percentage GFP-WIPI puncta cells were calculated (up to 429 cells per condition, n=3). (d) U2OS cells stably expressing GFP-WIPI1, GFP-WIPI2B or GFP-WIPI3 or transiently expressing GFP-WIPI4 were starved (3 h), and endogenous ATG12, LC3, p62 (anti-ATG12, anti-LC3, anti-p62 and IgG-Alexa Fluor 546 antibodies) or transiently expressed myc-tagged ATG14 or DFCP1 detected (anti-myc/IgG-Alexa Fluor 546 antibodies). Merged confocal LSM images are presented (left panels, dashed lines: cell boundaries; right panels: magnified subsections). Scale bars: 20 μm. (e) Confocal LSM examinations of starved (3 h) U2OS cells stably expressing GFP-WIPI1 and immunostained with anti-WIPI2/IgG-Alexa Fluor 546 and anti-WIPI4/IgG-Alexa Fluor 633 antibodies. Intensity profiles of co-localizations (peaks 1 and 2, upper panel) are displayed, along with the corresponding magnified image sections. Scale bar: 2.5 μm. (f) U2OS cells stably expressing GFP-WIPI3 and transiently expressing myc-tagged WIPI1 or WIPI2B were starved (3 h) and immunostained using anti-myc or anti-WIPI4 and IgG-Alexa Fluor 546 antibodies. Scale bar: 2.5 μm. Arrows in magnified image sections indicate co-localization events (d–f). Supplementary Material is available: Supplementary Fig. 1, Supplementary Note. Statistics and source data can be found in Supplementary Data 1. Mean±s.d.; heteroscedastic t-testing; P values: *P<0.05, **P<0.01, ***P<0.001, ns: not significant.