Targeting of septins to mitochondria induces perinuclear clustering in a SEPT9-dependent manner.(A and B) Images (A) show localization of MitoTagRFP- and SEPT9-MitoTagRFP-labeled mitochondria in living COS-7 cells, which were labeled with MitoSpy as an indicator of cell and mitochondria health. Plot (B) shows the fluorescence intensity (mean ± SEM; Mann–Whitney U test) of MitoSpy per mitochondria (MitoTagRFP) surface area per cell (n = 13–19). Scale bars, 20 µm. (C and D) Images (C) show DAPI-stained COS-7 cells, which were transfected with MitoTagRFP (control) or MitoTagRFP-tagged SEPT2, SEPT6, and SEPT7. Scale bars, 20 µm. Bar graph (D) shows percentage (mean ± SEM; two-way ANOVA; n = 20–28 cells) of total mitochondria fluorescence intensity in the perinuclear and peripheral cytoplasm. (E) Images show COS-7 cells transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with antibodies against SEPT2, SEPT6, and SEPT7. Scale bars, 20 µm. (F) Images of DAPI-stained COS-7 cells after cotransfection with scramble or SEPT9 shRNAs (GFP) with MitoTagRFP-tagged SEPT2, SEPT6, or SEPT7. Scale bars, 20 µm. Bar graph shows the percentage of perinuclear and peripheral mitochondria (mean ± SEM; two-way ANOVA; n = 24–35 cells) in the indicated conditions. ns, nonsignificant (P > 0.05); ***, P < 0.001.
Figure S2.

Targeting of septins to mitochondria induces perinuclear clustering in a SEPT9-dependent manner. (A and B) Images (A) show localization of MitoTagRFP- and SEPT9-MitoTagRFP-labeled mitochondria in living COS-7 cells, which were labeled with MitoSpy as an indicator of cell and mitochondria health. Plot (B) shows the fluorescence intensity (mean ± SEM; Mann–Whitney U test) of MitoSpy per mitochondria (MitoTagRFP) surface area per cell (n = 13–19). Scale bars, 20 µm. (C and D) Images (C) show DAPI-stained COS-7 cells, which were transfected with MitoTagRFP (control) or MitoTagRFP-tagged SEPT2, SEPT6, and SEPT7. Scale bars, 20 µm. Bar graph (D) shows percentage (mean ± SEM; two-way ANOVA; n = 20–28 cells) of total mitochondria fluorescence intensity in the perinuclear and peripheral cytoplasm. (E) Images show COS-7 cells transfected with MitoTagRFP or SEPT9-MitoTagRFP and stained with antibodies against SEPT2, SEPT6, and SEPT7. Scale bars, 20 µm. (F) Images of DAPI-stained COS-7 cells after cotransfection with scramble or SEPT9 shRNAs (GFP) with MitoTagRFP-tagged SEPT2, SEPT6, or SEPT7. Scale bars, 20 µm. Bar graph shows the percentage of perinuclear and peripheral mitochondria (mean ± SEM; two-way ANOVA; n = 24–35 cells) in the indicated conditions. ns, nonsignificant (P > 0.05); ***, P < 0.001.

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