(A–B) Representative images of control MRC5 cells (WT) (A, B) and VPS13 KO MRC5 cells (A’, B’) transfected with SPLICSS (A, A’) or SPLICSL (B, B’) to detect narrow (~8–10 nm) or long (~40–50 nm) distance ER-mitochondria contact sites respectively. A’, B’. Quantification of narrow and long distance contact sites in WT and VPS13A KO MRC5 cells. Error bars, mean ±s.e.m (n = 3 (A”) and n = 5 (B”)), two-tailed unpaired Student’s t-test was used (*p≤0.05, **p≤0.01). (C) Quantification of the mitochondria morphology of cells cultured under normal conditions (control) or under starved conditions (HBSS). WT and VPS13A KO MRC5 cells, were stained for the mitochondria marker TOMM20 (red) and DAPI (blue). For the quantification three cell-types with different mitochondrial appearances were pre-defined, type 1 cells with short, fragmented, densely packed mitochondria, type 2 cells with a mixture of round densely packed and more tubulated and less densely packed mitochondria and type 3 cells with tubulated dispersed and long mitochondria. Typical images and schematics are provided(C’). Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (*p≤0.05). (D) Mitophagy assay of control MRC5 (WT) and VPSA13 KO cells. The cells were transfected with FLAG-Parkin, which allows for the removal of damaged mitochondria and were treated with DMSO (control) or 20 µM CCCP (inducing mitochondria damage). After the transfection/treatment the cells were stained for the mitochondria marker TOMM20 and the mean fluorescence TOMM20 intensity was measured exclusively in FLAG-Parkin positive cells. The decrease in TOMM20 fluorescence after CCCP represents mitophagy. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (**p≤0.01, ***p≤0.001). (E,F) In control and starved (HBSS) MRC5 WT and VPS13A KO cells levels of pDRP1 and total DRP1 were determined by immunoblotting using GAPDH as a loading control (E). Quantification of protein band intensities in F was performed using ImageJ and plotted as a ratio of pDRP to GAPDH (F). Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (*p≤0.05, **p≤0.01, ***p≤0.001). Scale bars = 10 μm (A, A’, B, B’, C’).