Figure 4.

Rescue of HSF2 expression reverses the invasive phenotype of PrCa organoids. (a) Western blot of PC3 cells growing in 2D, 48 h after transfection. Cells were transfected with shRNA-producing plasmids against a scrambled sequence (Ctrl) or HSF2, and the HSF2 expression was restored by overexpression of an RNAi-resistant HSF2 construct (Mut HSF2). The exogenous HSF2 is marked with an asterisk. Tubulin was used as a loading control. (b) Live-cell imaging of invasive PC3 cells grown in 3D and transfected with the indicated shRNA constructs at day 3 in organotypic culture. Representative images from days 0, 3, 5 and 7 in 3D culture are shown. Scale bar, 100 μm. (c) Confocal microscopy images of immunofluorescence stainings in PC3 cells cultured in 3D for 8 days, and transfected as in (b). Upper panel: Alexa488-labeled phalloidin was used to detect filamentous actin (F-actin; green), and DAPI (4',6-diamidino-2-phenylindole) to label nuclei (blue). Lower panel: HSF2 staining was used to detect the silencing and rescue efficacy in PC3 organoids. No antibody staining (No Ab Ctrl) was used as a negative control. Scale bar, 50 μm.