Fig. 8

CARS2-dependent mitochondrial morphogenesis and bioenergetics. a Mitochondrial morphological analyses with MitoTracker Red fluorescent mitochondrial stain: morphometric analysis of mitochondrial length in HEK293T cells (WT and CARS2 KO; CARS2 WT and mutants added back). AU, arbitrary unit. The data are means ± s.d. (n = 3). **P < 0.01. b Transmission electron microscope (TEM) images of the cells in a. Scale bars, 1 μm. c Identification of Drp1 activity in HEK293T cells (WT and CARS2 KO; CARS2 WT and mutants added back). The GTP-agarose pulldown assay was performed. The data are means ± s.d. (n = 3). **P < 0.01. d Drp1 expressed in extensively polysulfidated (biotin-PEG-MAL capture method) HEK293T cells. Drp1 was markedly suppressed and nullified by CARS2 KO and CARS1/2 knockdown. Lanes 1 and 2 show duplicate determinations with each siRNA. Supplementary Fig. 23 provides full blot images. e A schematic drawing of Drp1 activity as regulated by protein polysulfidation and depolysulfidation, as affected by polysulfides vs. electrophiles and Trx/TrxR system. f Changes in membrane potential as assessed by using JC-1 staining of HEK293T cells (WT and CARS2 KO; CARS2 WT and mutants added back). The data are means ± s.d. (n = 3). *P < 0.05; **P < 0.01. g Assessment of mitochondrial electron flow in HEK293T CARS2 KO cells with or without adding back WT and C78/257D, K124/127A, and K317/320A mutants, as analyzed by measuring OCR using an extracellular flux analyzer. Time dependence of oxygen consumption before/after inhibition of mitochondrial respiration at complexes I and III by rotenone/antimycin A, and its statistical summary; the data are means ± s.d. (n = 3). ***P < 0.001