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. 2022 Sep 16;18(4):849–855. doi: 10.4103/1673-5374.353495

Figure 6.

Figure 6

CypD deficiency and CsA treatment protect against OH-induced mitochondrial injury in vitro.

(A) Immunofluorescence images showing mitochondrial calcein fluorescence (green in PC12 cells loaded with calcein-AM and CoCl2 in each group). The calcein fluorescence in the OH + CypD shRNA and OH + scrambled shRNA + CsA groups was increased compared with that in the OH + scrambled shRNA group. (B) Representative immunofluorescence images showing JC-1poly (magenta), JC-1mono (green) and Hoechst (blue) staining for mitochondrial membrane potential measurement and nuclei in each group. The JC-1poly/JC-1mono fluorescence ratio in the OH + CypD shRNA and OH + scrambled shRNA + CsA groups were significantly increased compared with that in the OH + scrambled shRNA group. (C) Representative immunofluorescence images showing TMRM (magenta), MitoTracker green (green) and Hoechst (blue) staining for mitochondrial membrane potential measurement, mitochondrial structure, and nuclei in each group. The fluorescent absorbance of TMRM in the OH + CypD shRNA and OH + scrambled shRNA + CsA groups were significantly increased compared with those in the OH + scrambled shRNA group. Scale bar: 10 μm, 6 μm (enlarged part in B), 8 μm (enlarged part in C). (D) Representative western blot and quantification of CypD and VDAC in PC12 cells in the Scrambled shRNA and CypD shRNA groups. (E) Quantification of calcein fluorescence at 30 minutes in each group. (F) Quantification of the fluorescence ratio of JC-1poly/JC-1mono in each group. (G) Quantification of the fluorescence absorbance of TMRM in each group. Data are shown as the mean ± SEM (n = 6 independent cell cultures). ###P < 0.001, vs. scrambled shRNA group; *P < 0.05, ***P < 0.001, vs. OH + scrambled shRNA group (one-way analysis of variance followed by Tukey’s post hoc test). CsA: Cyclosporin A; CypD: cyclophilin D; JC1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; OH: oxyhemoglobin; TMRM: tetramethylrhodamine methyl ester; VDAC: voltage-dependent anion channel.