Figure 1.

Loss of WIPI1 alters the endo-lysosomal compartment. (A) Representative images of control (CTR), WIPI1-KD and WIPI1 KO HK2 cells immuno-stained with an anti-EEA1 and with anti-LAMP1 antibodies (scale bars: 10 μm). (B-C) Quantification of cells from (A). The average number per cell of enlarged (diameter >2 μm²) EEA1-positive (B) or LAMP1-positive (C) structures, and of all LAMP1- or EEA1-positive structures was determined for CTR and WIPI1 KD cells. The integrated fluorescence intensity per cell was also measured (right panels). Mean values ± s.d. are shown (D) Rescue of WIPI1 knockdown. Experiment as in (A), with cells expressing an siRNA-resistant allele of wild-type (WIPI1) or of a version with substitutions in the lipid binding sites (WIPI1[FAAG]). EEA1 (red), LAMP1 (magenta) and EGFP-WIPI1 (green) are shown. Scale bars: 10 μm. (E) Quantification of EEA1- and LAMP1-fluorescence from (D). Images were acquired and analyzed as in (B). For (B-C) and (E), 180 cells per condition, pooled from 3 independent experiments, were analyzed. Mean values ± s.d are shown