Ai600 imager

Control and patient fibroblasts were seeded onto T25 flasks and allowed to grow overnight at 37°C and 5% CO₂. Cells were then treated with 100 nM SS-31 or vehicle control for 24-h. Subsequently, cells were harvested, cell pellets washed and lysed with RIPA buffer containing protease inhibitors (Amersco, cat. M250). Total cell lysates (20 μg) were resolved on SDS-PAGE gels and transferred onto PVDF membranes. Blots were probed with antibodies against OPA1 (BD Bioscience, cat. 612606) at 1:1000 final dilution followed by horseradish peroxidase-conjugated secondary antibodies. Blots were finally incubated with Clarity ECL substrate (Biorad) according to manufacturer's instructions and imaged on an Amersham Imager AI600. Densitometric analysis of band intensities were performed using ImageJ and normalized to a loading control (HSP60). Data was plotted using Prism 7 (GraphPad Software) and significance was determined using an one-way ANOVA followed by a Tukey correction for multiple comparisons.

For Western analyses, 3 × 10⁵ cells were seeded in 100 mm plates, allowed to grow for 2–3 days, harvested by trypsin digestion, washed with 1X phosphate-buffered saline (PBS) and lysed with RIPA buffer containing protease inhibitors. Total cell lysates (50 µg) were resolved on SDS-PAGE gels and transferred onto PVDF membranes. Blots were subsequently probed with the following antibodies (1:1000 dilution unless otherwise indicated): anti-MSTO1 (Genetex, GTX105110) (1:500), anti-V5 (Millipore, AB3792), anti-Mitofusin1 (Cell Signalling, 14739), anti-Mitofusin2 (Abnova, H00009927-M03), anti-Opa1 (BD Bioscience, 612606), anti-Actin (Sigma A5316), anti-HSP60 (Cell Signalling, 12165), VDAC1 (Abcam, ab14734) and the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000). Blots were incubated with Clarity ECL substrate (Biorad, 1705061) and imaged on an Amersham Imager AI600.

Whole-cell extracts were prepared by freeze and thaw lysis (three cycles) in 600 mM NaCl, 20 mM Tris-HCl pH 7.8, 20% glycerol. After SDS–PAGE, proteins were transferred onto PVDF membrane in semi-dry conditions. The membrane was then blocked in 5% non-fat dry milk buffer and incubated with mouse anti-γH2AX (Clone JBW301, Upstate, 1:5,000). Immunoblots were stained with corresponding HRP-conjugated secondary antibodies (GE Healthcare, 1:20,000) and detected with the enhanced chemiluminescence detection system (Amersham Biosciences). Quantification was performed using ImageJ.
For the validation of antibody specificity and cross-reactivity, a dilution series of synthetic peptides (CKATQASQEY; Peptide Specialty Laboratories GmbH), with the underlined serine in either phosphorylated or non-phosphorylated form, was immobilized on a nitrocellulose membrane at the indicated concentrations and probed with anti-γH2AX and anti-H2AX as described above.
CTCF knockdown western blots were developed using a rabbit anti-CTCF (#D31H2, Cell Signaling, 1:700) and a mouse anti-actin (AC-40, Sigma-Aldrich, 1:1,000) and overnight incubation at 4 °C, followed by a direct immunofluorescence detection using anti-rabbit-IgG-Cy5 (#711-175-152, Jackson, 1:1,000) and an anti-mouse-IgG-Alexa488 (A11029, Invitrogen, 1:1,000). Images were recorded using a AI600 Imager (Amersham) and quantified using ImageJ.