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. 2012 Dec 18;27(2):280–295. doi: 10.1210/me.2012-1260

Fig. 3.

Fig. 3.

Androgens increase autophagic flux in prostate cancer cells. A, LNCaP (representative pictures shown on left) or VCaP (representative pictures shown in Supplemental Fig. 2A) cells stably expressing an eGFP-LC3B fusion were treated for 3 d with vehicle or 10 nm R1881. Cells were then fixed and stained with LysoTracker to identify acidic organelles. eGFP-LC3B and LysoTracker colocalization was determined using immunofluorescence confocal microscopy. Scale bars, 10 μm. Quantification of colocalization (right) was done using ImageJ software. B, LNCaPs (representative pictures shown in Supplemental Fig. 2B) or VCaPs (representative pictures shown on left) were transfected with an mCherry-GFP-LC3B construct and treated for 3 d with vehicle, DHT (10 nm), or R1881 (10 nm) to assess autophagic flux. Cells were then fixed and visualized using a confocal immunofluorescence microscope. Scale bars, 10 μm. Right, Quantification of average total, GFP+mCherry+ (yellow, early autophagy/autophagosome) and GFP−mCherry+ (red, late autophagy/autolysosomes) punctae/cell ± se for both LNCaP and VCaP cells (n > 10 cells each condition). *, Significant changes (P < 0.05) from vehicle-treated cells. **, Significant changes (P < 0.01) from vehicle-treated cells. C, LNCaP cells were treated for 3 d ± 10 μg/ml each of E-64-d and pepstatin A (specific lysosomal protease inhibitors) and ± 10 nm R1881. Cells were then subjected to Western blot analysis as in Fig. 2.