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. 2023 May 21;13(10):3165–3187. doi: 10.7150/thno.78292

Figure 1.

Figure 1

COX7A2L regulates complex II-dependent mitochondrial respiration. (A) Western blot analysis of COX7A2L and Myc Tag in control HEK293T cells (WT), COX7A2L-/- cells (KO), and COX7A2L-/- cells infected with lentivirus containing an empty vector (KO+Vector) or COX7A2L cDNA (KO+COX7A2L). β-Actin was used as an internal control. (B-C) Blue native PAGE (BNG)/immunoblotting analysis of respiratory chain supercomplexes in WT and KO (B) as well as KO+Vector and KO+COX7A2L (C) mitochondria solubilized with digitonin. Complex I-V were immunoblotted with anti-Grim19, anti-SDHA, anti-UQCRC2, anti-MTCOI, and anti-ATP5A antibodies, respectively. Alkaline phosphatase-conjugated or fluorescence- conjugated secondary antibodies were used for differently presenting BNG. Samples were denatured and applied to SDS-PAGE for the incubation of anti-TOM70 as a loading control. (D-E) Relative total (D) and mitochondrial ATP levels (E) in WT, KO, KO+Vector, and KO+COX7A2L cells (n = 6). (F-G) Glutamate and malate (G+M)-dependent, succinate (S)-dependent, and glutamate, malate, and succinate (G+M+S)-dependent respiration in WT and KO (F) as well as KO+Vector and KO+COX7A2L cells (G) with 6 independent replicates. (H-I) Immunofluorescence analysis of WT and KO as well as KO+Vector and KO+COX7A2L cells. Mitochondria were stained with anti-TOMM20 antibodies (H). Fragmentated and tubular mitochondria were counted (I) (100 cells per group). Scale bars, 50 μm. (J-K) Transmission electron microscopy of mitochondria in WT and KO as well as KO+Vector and KO+COX7A2L cells (J). Average mitochondrial cristae lumen widths (K) were determined with 3 cristae per mitochondria, with a total of ten mitochondria each group. Scale bars, 1 μm. Quantitative data are presented as mean ± SEM. N.S, not significant; * P ≤ 0.05, ** P ≤ 0.01.