Figure 3: UVRAG^FS inhibits cellular autophagy in a dominant-negative manner.

(a) NIH3T3 cells stably expressing vector, UVRAG^WT, and UVRAG^FS were transfected with GFP–LC3 and treated with rapamycin (100 nM). GFP–LC3 puncta per cell were quantified as shown in representative images shown. Data represent the means±s.d. (n=6). *P<0.05. Scale bar, 10 μm. (b) Western blot analysis and densitometric quantification (underneath the blot) of the LC3-II/LC3-I ratios in NIH3T3 cells treated with rapamycin in the presence or absence of Bafilomycin A₁ (100 nM). N, normal condition; R, rapamycin; R+B, rapamycin+Bafilomycin A₁. (c) Schematic depiction of the dominant-negative action of UVRAGFS on the UVRAG-Beclin1 interaction by sequestering both. (d) UVRAG^FS inhibits Beclin1-associated VPS34 kinase activity. NIH3T3 cells from (a) were transfected with p40(phox)-PX-GFP (to monitor phosphatidylinositol 3-phosphate formation). At 16 h post-transfection, cells were subjected to confocal microscopy and p40(phox)-PX-GFP puncta per cell were quantified. Data represent the means±s.d. (n=3). **P<0.01. Scale bar, 10 μm. (e) UVRAG^FS promotes cell proliferation in Atg5-knockout iMEFs. Atg5^+/+ and Atg5^−/− iMEF cells stably expressing vector and UVRAG^FS were seeded and counted in triplicate on day 8. Values are mean±s.d. (n=3). UVRAG and Atg5 expression was assessed by western blot with actin serving as a loading control. *P<0.05; **P<0.01. (f–h) Anchorage-independent growth of Atg5-knockout iMEFs expressing UVRAG^FS. Note the larger and greater number of colonies in UVRAG^FS-expressing cells. Colony diameters (g) and numbers (h) were quantified from 20 random HPFs. Data are the means±s.d. (n=3). *P<0.05; ***P<0.001. Scale bar, 50 μm.