14-3-3τ Regulates Beclin 1 and Is Required for Autophagy
Figure 5
A role for E2F1 in autophagy and the regulation of Beclin 1 by 14-3-3τ.
(A–C) HeLa cells were transiently transfected with 3 µg GFP-LC3 and 20 µg pSUPER-siE2F1 or pSUPER-siScr using Lipofectamine 2000. 24 hr later, the cells treated with 100 nM rapamycin for 24 hr to induce autophagy. (A) Cells were fixed in 1% paraformaldehyde, and nuclei were stained with Hoechst 33258. Images were taken with 200× magnification in a Zeiss Axioplan 2 digital fluorescence microscope. (B) More than 500 GFP-positive cells per sample were scored for GFP-LC3 puncta. The data are derived from three independent experiments. The data shown represent the means ± standard deviations. The p values are based on a paired two-tailed t test. (C) Some cells were harvested and the cellular lysates were subject to Western blot analysis as indicated. (D) The E2F1 siRNA or GFP siRNA in the U2OS cell lines as described in Fig. 4A–B was induced by doxycycline (1 µg/ml) for 3 days, and then the cells were infected with AdCMV or 14-3-3τ at MOI 300. 48 hr later, cells were harvested for Western blot analysis. (E) Primary E2F1+/+ or E2F1−/− mouse embryonic fibroblasts (MEFs) were infected with AdCMV or 14-3-3τ at MOI 1000. 48 hr later, cells were harvested for Western blot analysis (Right panel). Left panel: Genotyping of the MEFs. The 172-bp and 227-bp products were amplified from the wild type and mutant alleles respectively. Mock represents no template input control.