Immunofluorescence studies and colocalization analysis

Cells were seeded in 12-well plates with 0.17-mm coverslips (40 × 10³ cells/well) and treated as indicated in each experiment. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 2% BSA, all in PBS. Samples were incubated with primary antibodies in 2% BSA overnight at 4 °C. Antibody dilutions were: anti-Calnexin (ER marker, Abcam ab75801) 1:200 and anti-mtHsp70 (mitochondrial marker, Thermo Fisher Scientific MA3–028) 1:500. Following incubation for 2 h with anti-mouse Alexa 568 or anti-rabbit Alex 488-conjugated secondary antibodies (dilution 1:600), coverslips were mounted on glass slides using mounting medium ProLong with DAPI (Life technologies). For the colocalization analysis, only one focal plane was analyzed in a confocal microscope (LSM 700, Carl Zeiss). Images obtained were deconvolved, and colocalization between proteins was quantified using the Manders’ algorithm and the Image J software (NIH, USA), as previously described [8, 9].