Loss of NLRX1 increases ROS production and apoptosis after hypoxia. Primary isolated TECs obtained from WT (white bars) or NLRX1 KO (black bars) mice were subjected to hypoxia and (A) labeled with MitoSOX Red for 45 min during reoxygenation and (B) stained for cleaved caspase-3 at 24 h of reoxygenation. (C) Western blot for NLRX1 on parental IM-PTEC, cEmpty, and cNLRX1 cells. WT and NLRX1 KO kidney was used as control. B actin was used as loading control. Protein molecular weight is indicated in kilodaltons. Digital image analysis of cEmpty (white bars) or cNLRX1 (black bars) cells subjected to hypoxia and (D) labeled with MitoSOX Red for 45 min during reoxygenation and (E) stained for cleaved caspase-3 at 24 h of reoxygenation. Representative images for (G) MitoSOX Red labeling (red) and a nuclear counterstain (blue) as analyzed in D and for (H) cleaved caspase-3 (green) with counterstains for F actin (red) and nuclei (blue) as analyzed in E. (F) Analysis of cleaved caspase-3 after 48 h in a hypoxic chamber and (I) representative images for cleaved caspase-3 (green) with counterstains for F actin (red) and nuclei (blue). (J) Digital image analysis of cells incubated for 3 h with the indicated compounds and labeled with MitoSOX Red. DDC, diethyldithiocarbamate; DEM, diethyl maleate. (K) Representative images with MitoSOX Red labeling (red) and a nuclear counterstain (blue) used for analysis presented in J. Data are expressed as mean ± SEM and were analyzed using an unpaired t test or ANOVA (nephrotoxicants; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. respective normoxic control; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. cEmpty control). All conditions were determined in triplicate; representative data from three independent experiments are shown. Bars, 50 µm (G–I) and 10 µm (K).