Figure 1

Androgen treatment blocks serum starvation-induced autophagy. (a) Expression levels of androgen receptor (AR) and PSA in LNCaP cells maintained for 24 h in medium containing 10% fetal bovine serum (FBS), serum free (SF) medium or SF+10 ng/ml mibolerone (SF+mib). Cell lysates were western blotted as indicated, with actin as a loading control. (b) Representative confocal images of cells transiently expressing GFP-LC3 maintained as described in (a). (c) Quantification of GFP-LC3 puncta-positive cells depicted in (b). A positive cell contained five or more punctae. Fifty cells per condition per experiment were analysed. (d) Cells were treated for 0, 2 or 6 h with SF medium with or without bafilomycin and/or mibolerone. Lysates were western blotted with an anti-LC3 antibody, with actin as a loading control. The lipid-conjugated (LC3-II) and -unconjugated (LC3-I) forms are indicated by arrows. Graph depicts densitometric analysis of LC3-II band intensity, normalized to actin levels and expressed as fold change from untreated control. (e) Representative confocal images of cells stably expressing mRFP-GFP-LC3 treated as described in (a). Merged images depict overlay of GFP (green) and mRFP (red) images. (f) Quantification of mRFP+ and mRFP+/GFP+ punctae in cells shown in (e), 25 cells per condition per experiment. For (c), (d) and (f), n=3, error bars indicate mean S.E. ^*P<0.05 and ^**P<0.01, as indicated. For (b) and (e), scale bar=20 nm