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. 2021 Dec 10;24(12):103484. doi: 10.1016/j.isci.2021.103484

Figure 3.

Figure 3

Senp5L/5S and Drp1-SUMOylation regulate mitochondrial morphology

(A) Mitochondrial morphologies in 293T cells expressing EGFP, EGFP-Senp5L, or EGFP-Senp5S were analyzed after deconvolution of the confocal projection images. pMT-mKO1 (red) was co-transfected to visualize mitochondria. Right column is higher magnification of the boxed areas, depicting individual mitochondrial morphology (white). Arrowheads denote fragmented mitochondria.

(B) Overexpression of SUMO3 enhances the mitochondrial fragmentation. Cells were co-transfected with HA-SUMO3 along with EGFP, EGFP-Senp5L, or EGFP-Senp5S. HA-SUMO3 expression was confirmed by immunostaining with anti-HA (magenta).

(C) Quantified comparison of the effect of Senp5L, Senp5S, and SUMO3 overexpression on mitochondrial length. Box and whisker plots summarize the length (μm) of mitochondria. Numbers in parentheses indicate the number of cells examined in multiple independent experiments in (A) and (B). ∗∗, p < 0.01; ∗∗∗, p < 0.001; Welch's t tests with Holm-Bonferroni correction.

(D) Confocal projection images of HEK293T cells transfected with YFP-Drp1-WT or YFP-Drp1-4KR (green). pMT-mKO1 (red) was co-transfected to visualize mitochondria. Right column: magnified views of the boxed areas showing individual mitochondria (white). Arrowheads indicate fragmented mitochondria.

(E) Quantification of mitochondrial length in cells expressing Drp1-WT and Drp1-4KR. Box and whisker plots summarize the mitochondrial length (μm). Numbers in parentheses indicate the number of cells measured for mitochondrial length determination. ns, not significant, ∗, p < 0.05; Welch's t tests with Holm-Bonferroni correction.

(F) Mitochondrial morphologies in Drp1-KO HeLa cells expressing EGFP, EGFP-Senp5L, or EGFP-Senp5S along with HA-Drp1-WT or -4KR were analyzed after deconvolution of the confocal images. HA-Drp1 expression was confirmed by immunostaining with anti-HA (magenta). pMT-mKO1 (red) was co-transfected to visualize mitochondria. The second column from the right is higher magnification of the boxed areas, depicting individual mitochondrial morphology (gray scale). Arrowheads denote the fragmented mitochondria.

(G) Quantified comparison of the effect of EGFP, Senp5L, or Senp5S on mitochondrial length in Drp1-KO HeLa cells expressing either Drp1-WT or Drp1-4KR. Box and whisker plots summarize the length (μm) of mitochondria. Numbers in parentheses indicate the number of cells examined in multiple independent experiments in (F). ns, not significant; ∗, p < 0.05; ∗∗∗, p < 0.001; Welch's t tests with Holm-Bonferroni correction. Scale bars, 5 μm in (A), (B), (D), and (F), and 1 μm in the magnified view.