Skip to main content
. 2024 Feb 1;134(3):e169441. doi: 10.1172/JCI169441

Figure 6. RAB7 silencing impairs mitochondrial membrane potential and mitochondrial function.

Figure 6

(A) RAB7 siRNA impaired ΔΨm as assessed by TMRE and MitoTracker Green (MitoGreen) flow cytometry. Carbonyl cyanide FCCP was used as a positive control (depolarizes mitochondrial membrane). TMREhi MitoGreenhi cells indicate cells with functional mitochondria, whereas TMRElo MitoGreenhi cells are cells with dysfunctional mitochondria. n = 8 (control) and n = 9 (RAB7). (B) Representative TMRM-stained images show overall reduction and perinuclear accumulation of functional mitochondria in RAB7 siRNA–treated PAECs. Results are representative of 3 experiments. Scale bars: 5 μm. RAB7 knockdown increased mitochondrial motility in peripheral and perinuclear regions. (C) RAB7 knockdown promoted mitochondrial ROS production as indicated by flow cytometry for mitoSOX. n = 6 per group for the representative histogram plots and quantification of MFI. (D) Clustered heatmap shows upregulated glycolysis-related DEGs and downregulated oxidative phosphorylation–related DEGs in bulk RNA-Seq from PAECs plus RAB7 siRNA. Expression is normalized log2-fold. (E) Seahorse high-resolution respirometrics show a reduced OCR with RAB7 siRNA at basal respiration, maximal respiration, and spare respiratory capacity. n = 15 (control) and n = 21 (RAB7). (F) Luminescence assay for lactate: increased lactate production in RAB7 siRNA–treated PAECs. n = 10 (control) and n = 11 (RAB7). (G) ECAR data reveal more rapid acidification (i.e., greater reliance on glycolysis) in the RAB7 siRNA–treated PAECs as shown for glycolysis and the basal and maximum glycolytic rates. n = 12 per group. All graphs show single values and the median ± interquartile range (A, E, and F) or the mean ± SD (C and G) (except B, as bar graphs in B indicate the geometric mean of a log-normal distribution). Data in A–C, E, F, and G are from 2 or more experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Mann-Whitney U test (A and F), 2-tailed Student’s t test (C), and 2-way ANOVA (E and G) with Holm-Šidák post hoc test and normality testing of residuals (D’Agostino-Pearson).