Figure 1. Induction of autophagy by VPA treatment in LNCaP and PC-3 cells, but not in DU145 cells. Autophagy was measured by LC3-II western blot analysis (A–D) and LC3B immunofluorescence microscopy (E and F). Cells were treated with indicated concentrations of VPA for 24 h (A), or 10 mM VPA for 24 h in the presence or absence of 25 μM chloroquine (CQ) (B), or 10 mM VPA for indicated time lengths in the presence or absence of 25 μM CQ (C and D). Total proteins were extracted by 2× SDS-PAGE loading buffer. LC3A and LC3B were probed by western blotting, respectively. TUBB was used as loading control. The relative densitometry values under each LC3 blot is the ratio of LC3-II (16 kDa) densitometry to that of TUBB. A dash (−) indicates that no LC3-II band was observed. Data are from one of three independent experiments with similar results. (E and F) Cells were treated with 10 mM VPA and/or 25 μM CQ (E) or 2 μg/ml rapamycin (Rapa) (F) and then immunostained with LC3B antibody followed by CF568-conjugated second antibody. Fluorescent images were obtained by fluorescence microscopy with a 100 × oil objective lens. LC3B (red) fluorescent puncta were only observed in LNCaP and PC-3 cells. Rapamycin treatment was included to confirm that autophagy was deficient in DU145 cells (B and F). Nuclei (blue) were revealed by Hochest33342 staining. Arrowheads indicate 17-kDa bands of LC3B. Scale bar: 10 μm.