Stress-associated proteins co-localize with and are transported via TNTs. (A) PC3 cells treated with SS for 24 hours were co-stained for TNTs (F-actin) and CLU, YB-1, Hsp27 or LC3. Images were taken using confocal fluorescent microscopy (60x). The bottom panel is cropped from the white box of the top panel. (B) PLA staining for F-actin and the respective stress-associated proteins (red) was combined with TNT staining (Phalloidin, green) in SS-treated PC3 cells. The right panel is cropped from the white box of the left images. (C) LNCaP cells were co-transfected with plasmids expressing mCherry-CLU and eGFP and treated in CSS for 24 hours. Live imaging assay was performed to capture CLU transport along TNTs. Still images created from live cell imaging are shown. In the top panel, red arrows indicate movement of a CLU-positive particle (red) along the TNT (green) from the bottom cell to the top cell. In the bottom panel, white arrows highlight movement of a CLU-positive particle transported in the opposite direction from the top cell to the bottom one. All scale bars: 20 µm.