Cells were cultured in chamber slides for 24 h in the absence or presence of the three HDACi. After fixation with 0.3% paraformaldehyde (#P6148, Sigma-Aldrich, St. Louis, MO, USA) and permeabilization with with 0.1% triton (#X100, Sigma-Aldrich, St. Louis, MO, USA), cells were incubated with anti-TOM20 antibody (#42406S, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Afterward, cells were incubated for 1 h at 25 °C with the Alexa-546-conjugated anti-rabbit antiserum (#A11010, Invitrogen, USA) as a secondary antibody.
Immunofluorescence confocal laser scanner microscopy (CLSM) imaging was carried out using a laser scanning spectral confocal microscope TCS SP2 AOBS (Leica, Germany), equipped with Argon ion, He–Ne 543 nm, and He–Ne 633 nm lasers. Images were acquired through an HCX PL APO CS 40×/1.25 oil UV objective and processed with Leica. Images were acquired as single transcellular optical sections.
To evaluate the mitochondrial network shape, fibroblasts were scored depending on the morphology of most of their mitochondrial population as elongated or intermediated/short following the method described in [59 (link)].
Immunofluorescence confocal laser scanner microscopy (CLSM) imaging was carried out using a laser scanning spectral confocal microscope TCS SP2 AOBS (Leica, Germany), equipped with Argon ion, He–Ne 543 nm, and He–Ne 633 nm lasers. Images were acquired through an HCX PL APO CS 40×/1.25 oil UV objective and processed with Leica. Images were acquired as single transcellular optical sections.
To evaluate the mitochondrial network shape, fibroblasts were scored depending on the morphology of most of their mitochondrial population as elongated or intermediated/short following the method described in [59 (link)].