Fig 1: ALM induces HIF-1α-dependent apoptosis in PC3 cells. (A) Cell growth curve of PC3 cells exposed to vehicle or 30 nM ALM for 3 days under normoxia and hypoxia. (B) PC3 cells were treated with ALM for 24 h and 48 h at 0.1 μM under normoxia and hypoxia, followed by Western blot for caspase-3. (C,D) PC3 cells were treated with ALM for 24 h, 48 h, 72 h at 0.1 μM and 1 μM under normoxia and hypoxia, followed by staining with FITC-conjugated Annexin V and PI for flow cytometric analysis. Representative result of 48 h was presented as Figure 3C (C). The flow cytometry profile represents Annexin V-FITC staining in X axis and PI in Y axis. The number represents the percentages of cells to each of the four quadrants (viable cells in the third quadrant, necrotic or dead cells in the second quadrant, early apoptotic cells in the fourth quadrant and late apoptotic cells in the first quadrant). Then, apoptosis population was quantified (D). (E) PC cells were infected with lentivirus expressing WT-HIF-1α, DM-HIF-1α (doble mutant, P402A/P564A), and cell lysates were subjected to Western blot for HIF-1α and β-actin. (F) PC3 cells stably expressioning EV, WT-HIF-1α, DM-HIF-1α were treated with 0.1 μM ALM for 48 h, followed by staining with FITC-conjugated Annexin V and PI for flow cytometric analysis. The bars show mean ± SD (n = 3 each). (G) The apoptosis population was calculated based on the flow cytometric analysis of Figure 3F; * P < 0.05 as compared with EV, 0 μm ALM group; # P < 0.05 as compared with EV, 0.1 μm ALM group.