Mitofusin 2 Promotes Apoptosis of CD4⁺ T Cells by Inhibiting Autophagy in Sepsis

Figure 3

Overexpression of Mfn2 inhibited autophagy and induced cell apoptosis in Jurkat T cells. Transfected Jurkat T cells were cultured in RPMI-1640 with 10% FBS for more than 72 hours. (a) Representative dot plots of Annexin-V staining. (b) Representative histograms of TUNEL staining. (c) Percentage of apoptotic cells determined by Annexin-V or TUNEL staining. (d) Representative histograms of LC3-II-positive cells based on intracellular staining. (e) Cyto-ID was used to assess autophagosomes and autolysosomes. (f) Percentage of LC3-II- or Cyto-ID-positive cells. (g, h) Jurkat T cells were cultured with or without bafilomycin A1 (0.1 μM) for 4 hours, and Western blot analysis was used to determine the expression levels of Beclin1, p62, and LC3-II protein. β-Actin served as an internal control for Beclin1 and p62 protein levels. (i, j) Representative images of immunofluorescence staining. DAPI and FITC were used to stain nuclei and endogenous LC3, respectively. The arrows indicate LC3 puncta. Quantification of the average number of LC3 puncta per cell ( well, 3 independent experiments, and >50 cells examined per experiment). The results are shown as the mean ± SD from three independent experiments. Bar 1, the LV-Mfn2 group; bar 2, the LV-mCherry group; bar 3, the LV-Mfn2 RNAi group; bar 4, the LV-RFP group; bar 5, the LV-Mfn2 + B group; bar 6, the LV-mCherry + B group; bar 7, the LV-Mfn2 RNAi + B group; bar 8, the LV-RFP + B group. Baf A1 or B: bafilomycin A1. , significant difference versus the control group. ^#, significant difference versus the LV-mCherry group. ^##, significant difference versus the LV-mCherry group or the LV-mCherry + B group. ^$$, significant difference versus the LV-RFP group. ^&&, significant difference versus the corresponding group without bafilomycin A1 treatment group.

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