AR blockade and metabolic stresses induce TNTs in PCa cells. (A) Exponentially growing PC3 and LNCaP cells were fixed and stained with Alexa Fluor 488-labeled phalloidin (for F-actin) to visualize TNTs under fluorescent confocal microscope or under the bright field (40x lens). Arrows indicate TNTs. (B) Lengths of TNTs in PC3 and LNCaP cells were measured and presented as average +/− standard deviation (SD). (C) LNCaP xenografts were stained with Alexa Fluor 594-labbeled phalloidin. Right panel is cropped from the white box of the left panel. (D) PC3 and (E) LNCaP cells receiving stress treatments were stained for TNTs as in (A). TNTs were quantified as described in Methods. *p < 0.001 vs. ctrl of the respective group, **p = 0.01 vs. ctrl. Data is presented as average +/− SD from 3 independent experiments. (F) LNCaP cells labeled with MitoTracker Deep Red FM (for mitochondria) were treated with 10 μM ENZA for 24 hours and then stained with phalloidin. Co-localization of mitochondria and TNTs was captured by confocal microscopy. Right panel is cropped from the white box of the left panel. (G) LNCaP cells treated with 10 μM ENZA for 24 hours were stained for phalloidin and lysosome protein LAMP1. Co-localization of lysosome and TNTs was captured by confocal microscopy. Right panel is cropped from the white frame from the left panel. All scale bars: 20 μm.