FIG. 8.

A putative human Atg13 homologue is required for autophagy. (A) 293/GFP-LC3 cells were transfected with control siRNA (Ctrl) or siRNA targeting ULK1 or a SMARTpool targeting KIAA0652 (Atg13) for 72 h. Cells were then left unstarved or starved in EBSS-leupeptin for 2 h before fixation and morphological analysis. The quantification represents the total GFP-LC3 spot intensity per cell (arbitrary units), and each bar represents the average of 60 image fields (from six independent cell samples) ± the standard deviation. **, P < 0.0007 in pairwise comparisons with Ctrl knockdown starved cells. (B) 293/GFP-LC3 cells were transfected for 72 h with control siRNA, the Atg13 SMARTpool, or individual duplexes (#1 or #2) specific for Atg13. Cells were left unstarved or starved as described for panel A and then lysed for immunoblot analysis of GFP-LC3 lipidation, as described in the legend for Fig. 2. *, P < 0.02 in pairwise comparisons with siRNA Ctrl starved cells. (C) HEK293A cells were transfected with control siRNA or Atg13 duplex 2 for 72 h. Cells were then left unstarved (Unst) or starved (st) for 2 h in EBSS (with or without leupeptin [Leu]) before lysis for immunoblot analysis of endogenous LC3 lipidation. The quantified ratio of LC3-II/LC3-I is shown below each sample. βTub, β-tubulin. (D) 293/GFP-LC3 cells were transfected for 72 h with control siRNA (siCtrl) or siRNAs targeting ULK1 (siULK1) or Atg13 (duplex 2) (siAtg13), starved in EBSS-leupeptin for 2 h, and then fixed and immunostained to detect endogenous mAtg9 localization (red). After starvation in siCtrl-treated cells, mAtg9 becomes redistributed predominantly to a diffuse cytoplasmic pool. After knockdown of Atg13, mAtg9 redistribution is blocked and mAtg9-positive vesicles remain in juxtanuclear clusters (arrows) to an extent similar to that after knockdown of ULK1. Inhibition of starvation-induced GFP-LC3 autophagosome formation after knockdown of ULK1 and Atg13 is shown as a control. Bar = 10 μm.