Isolation and characterization of stable HEK-293 clones expressing GFP-DFCP1. (A) Four clones were screened for GFP-DFCP1 expression (inset shows blots of endogenous DFCP1 and GFP-DFCP1 with β-COP as loading control; the fold overexpression of tagged DFCP1 over endogenous DFCP1 is also indicated) and for a good response to starvation, as measured by translocation of GFP-MAP-LC3 to punctate structures (shown in the graph) or acquisition of endogenous LC3-II form (last lane of inset). High levels of DFCP1 inhibited starvation responses (especially evident for clone 206). Error bars show the standard deviation from three independent experiments. (B) Clone 201 cells were left untreated or starved for 45 min in the absence or presence of wortmannin (wortm) or BFA as indicated, and the distribution of GFP-DFCP1 was examined by fluorescence microscopy. Note that wortmannin inhibited translocation, whereas BFA was without effect. (C) Clone 201 cells were treated with siRNA against Vps34 or beclin-1, or with a control siRNA as shown, starved for 60 min, and examined by fluorescence microscopy. Note that the level of punctate structures is reduced in Vps34- and beclin-1–reduced cells; this is analyzed for three independent experiments in D (error bars show standard deviation). (D, inset) The levels of Vps34 and beclin-1 after siRNA treatments. Of peripheral interest for this work is that the reduction of Vps34 during treatment with siRNA for beclin-1 is reproducible. (E) Clone 201 cells were starved for 60 min. Imaging was at 1 frame per 20 s, and selected frames throughout the sequence are shown. Also see Video 2 . (F) For a selected time interval (starting approximately at 35 min after starvation), a sequence showing formation and collapse of an omegasome (arrows) is shown. Bars (A–C and E) 20 μm; (F) 2 μm.