Cbl186

WT and VPS13C^KO HeLa cells were plated on glass coverslips and fixed in a prewarmed (37°C) solution of 4% PFA in PBS for 15 min at room temperature, permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 10 min at room temperature, and blocked using filtered PBS containing 1% (wt/vol) BSA for 1 h at room temperature. Coverslips were then incubated with antibodies against DNA (CBL186, 1:150; EMD Millipore) and HSP60 (12165S, 1:1,000; CST) and diluted in filtered PBS containing 1% BSA at 4°C overnight, followed by 3× 5-min washes in PBS. Secondary antibodies (1:1,000, Alexa Fluor 488 and 555; Invitrogen) were incubated in PBS containing 1% BSA for 1 h at room temperature and removed by 3× 5-min washes in PBS. Finally, coverslips were mounted onto slides using ProLong Gold Antifade Mountant with DAPI (P36935; Thermo Fisher Scientific) and allowed to cure overnight at room temperature before imaging. A detailed protocol for immunofluorescent staining can be accessed on protocols.io at dx.doi.org/10.17504/protocols.io.14egn741mv5d/v1.

Cells were labelled with 50 nM MitoTracker Orange CMTMRos (Life Technologies, M-7510) for 45 minutes at 37 °C in culture media. Cells were then fixed in 4% paraformaldehyde (Electron Microscope Science), and permeabilized for 10 minutes with 0.1% Triton-X100. Cells were blocked with 4% normal goat serum before being labelled with 0.17 µg/ml anti-DNA antibody (EMD Millipore, CBL186) overnight at 4 °C. Fixative and all subsequent solution were based on phosphate buffered solution. Excess primary antibody was removed with three PBS washes before staining with Goat anti-IgM Alexa-488 (2 µg/ml¸ Life Technologies, A21042). Nuclei were labelled with Hoechst-33342 (1 µg/ml) for 20 minutes before mounting in ProLong Gold (Life Technologies). Cells were imaged on a Nikon TiE inverted microscope with a 100x CFI Plan Apo Lambda 1.45 NA objective using a Zyla 5.5 CMOS camera (Andor). Images were analyzed using ImageJ^{18 (link)}.

Detached skin fibroblasts were counted (MOXI Z Mini Automated Cell Counter; ORFLO Technologies, Ketchum, ID) and seeded on three autoclaved coverslips per subject at 5.0 × 10⁴ cells per slip using a 24 well-plate. Cells were incubated at 37°C overnight before being fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at 37°C for 15 min. Coverslips were quenched with NH₄Cl for 15 min and washed with PBS to minimize autofluorescence. Cells were permeabilized with 0.5 mL 0.2% Triton X-100 (Sigma; Oakville, ON) in PBS for 15 min, washed with PBS, and then blocked with 10% FBS (Life Technologies) in PBS for 30 min. Cover slips were simultaneously incubated with primary antibodies for the outer mitochondrial membrane protein TOMM20 (FL-145, Santa Cruz; Dallas, TX) and DNA (CBL-186, EMD Millipore; Etobicoke, ON) diluted to 1:1,000 in 5% FBS, 95% PBS (Life Technologies) at 37°C for 1 h. Cells were washed 3 x with PBS and then incubated with secondary antibodies (1:5,000) at RT for 1 h. The following secondary antibodies conjugated to fluorescent dyes were used: Alexa Fluor 488 goat anti-mouse IgG (TOMM20, Molecular Probes, Eugene, OR) and Alexa Fluor 647 goat anti-rabbit IgG (DNA, Molecular Probes). Cells were mounted with DakoCytomation fluorescent mounting medium (Agilent Technologies; Santa Clara, CA) and stored at 4°C until microscopic examination.