Extended Data Fig. 6: Mild KD of Fis1 mimics Rab24’s effect on mitochondrial connectivity and respiration.

(a) Relative expression of Fis1 in cultured primary hepatocytes after RNAi treatment. 9 wells pooled into N=3 replicates per condition. (b) Seahorse measurements of the oxygen consumption rate (OCR) in primary hepatocytes (N=10 wells per time point) and their corresponding metabolic rates in (c-e) after 3 days of KD. N=3 time points with 10 wells/time point (c, d). N=8 (CTR) and N=9 (KD) wells per condition from the first time point after FCCP treatment, representing maximal respiration (e). (f) Representative confocal images (single confocal section) of cultured primary hepatocytes stained with dapi (blue) and Tom20 (grey) as single section, deconvolved, zoomed and skeletonized with Fiji. The images are representative of three independent wells of a 24-well plate. (g-j) Quantification of mean Tom20 intensity per cell, number of branches, number of junctions per area and mean length of the branches of (f) with Fiji analyzed from N=14 (CTR) and N=17 (KD) for (g), N=10 (CTR) and N=11 (KD) for (h, i), N=9 (CTR) and N=10 (KD) for (j) cellular regions (multiple cells per region). Scale bar 20 µm. All measured after 3 days of RNAi (0.1 nM). (mean +/- SEM). *P<0.05, **P<0.01, ****P<0.0001, *****P<0.00001 by two-tailed unpaired Student’s t-test.