Fig. 5.

Higd-1a overexpression delays cleavage of the Opa1 L isomer. (A and B) HEK293T cells were transfected with mock or Higd-1a–Flag vector for 2 d and incubated under hypoxia (0.1% O₂) (A) or with 10 µM CCCP (B) for the indicated times. Total cell lysates were subjected to Western blotting by using anti-Opa1 or anti-Flag antibodies. Hsp60 protein levels are shown as loading controls. (C) HeLa cells were transfected with Higd-1a–Flag or Higd-1a–ΔNT-Flag vectors for 3 d and treated with 10 µM CCCP for 15 min. Cells were visualized under a confocal fluorescence microscope after staining with MitoTracker (red) and FITC-tagged anti-Flag (green). Merged confocal fluorescence images are shown in Right, and enlargements of boxed areas a-d are shown in Left Bottom. (Scale bar: 10 µm.) Approximately 100 FITC-negative (FITC⁻) and 100 FITC-positive (FITC⁺) cells were counted, and the percentages of each having more than 50% tubular, less than 50% tubular, and fragmented mitochondria were calculated (Right). (D) HEK293T cells were transfected with mock or Higd-1a–Flag vectors for 2 d and treated with 10–50 µM CCCP for 1 h. Twenty-four hours after CCCP washout, total viable cells were counted by Trypan blue exclusion. (E) The Opa1 isoform 1 and its Opa1-ΔS1 mutant were overexpressed in HEK293T with or without Higd-1a siRNA for 4 d, and cell growth was assessed by 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Left). Expression of Opa1 and Opa1-ΔS1 proteins in cells not harboring Higd-1a siRNA was verified by Western blotting (Right). Results shown are representative of three independent experiments. Data are mean ± SE; ***P < 0.001.