Fig. 4.

Autophagy promotes androgen-mediated lipid accumulation. A, LNCaP cells were treated for 3 d with vehicle (ethanol) or 10 nm R1881. Cells were then fixed and examined by TEM as in Fig. 2. B, LNCaP cells stably expressing an eGFP-LC3B fusion were treated for 3 d with vehicle, DHT (10 nm), or R1881 (10 nm). Cells were then fixed and costained with LipidTOX (neutral lipid depots) and DAPI (nucleus). eGFP-LC3B, LipidHTX, and DAPI colocalization was visualized using immunofluorescence confocal microscopy. Scale bars, 20 μm; 5 μm for higher resolution images on far right. C, LNCaP or VCaP cells were treated as in A and subjected to immunogold TEM to determine LC3B subcellular localization (yellow arrows). D, LNCaP cells were transfected with siRNAs as in Fig. 1B and treated for 3 d with vehicle or 10 nm R1881. Intracellular TG levels were then determined using a fluorescent Nile Red-based stain (AdipoRed) and normalized to cell numbers that were determined using duplicate plates that were instead subjected to the fluorescent DNA-binding dye described in Fig. 1. Each sample was performed in triplicate, and results from a representative experiment are shown. Results are expressed as TG levels normalized to cell numbers + se (n = 4). *, Significant changes from mock-transfected cells. E, LNCaP cells were treated with vehicle or increasing concentrations of chloroquine (CQ) ± 10 nm R1881 for 3 d. Intracellular TG levels were then determined as described in D. Each sample was performed in triplicate and results from a representative experiment are shown. Results are expressed as TG levels normalized to cell numbers + se (n = 4). *, Significant changes from vehicle (no CQ)-treated cells.