Figure S1. Septin localization and... | Rockefeller University Press

Figure S1.

$Septin localization and effects on early endosomes and lysosomes.(A) Spinning-disk confocal image shows the distribution of SEPT9-mCherry, which was expressed under the PGK promoter, with respect to GFP-EEA1 in living COS-7 cells. Scale bar, 20 µm. Perinuclear area is shown in higher magnification. Scale bar, 5 µm. (B) Quantification shows the fraction (mean ± SEM; Mann–Whitney U test) of SEPT9-mCherry puncta colocalizing with LAMP1-mGFP and GFP-EEA1 in COS-7 cells (n = 6). (C and D) PNSs of HEK-293 cell extracts were loaded on an iodixanol OPTIPREP density gradient (17%, 20%, 23%, 27%, and 30%), and fractions were blotted (C) for LAMP-1, Rab7, Rab5, GM130, BIP, SEPT9, SEPT2, SEPT6, and SEPT7. Plots (D) show quantification of indicated proteins in each fraction as percentage of total proteins across all fractions. Horizontal dashed lines provide a reference point for the percentage of septin protein in the top fraction. (E and F) Images (E) show the distribution of early endosomes (EEA1) in COS-7 cells that overexpress mCherry or SEPT9-mCherry (insets). Scale bars, 20 µm. Quantification (F) shows the ratio (mean ± SEM; unpaired t test) of perinuclear to peripheral fluorescence intensity of EEA1 per cell (n = 20). (G and H) Images (G) of LAMP1-stained COS-7 cells expressing mCherry, SEPT2-mCherry, or mCherry-SEPT6 (insets). Scale bars, 20 µm. Plot (H) shows the ratio (mean ± SEM; Kruskal–Wallis one-way ANOVA) of perinuclear to peripheral fluorescence intensity of LAMP1 per cell (n = 30–34). (I and J) Images (I) of COS-7 cells, which were transfected with plasmids that coexpress GFP and scramble or SEPT9 shRNA and stained with anti-SEPT9 antibody. Scale bars, 20 µm. Bar graph (J) shows fluorescence intensity (mean ± SEM; Mann–Whitney U test) of SEPT9 per shRNA-expressing cell (n = 27–35). A.U., arbitrary units. (K and L) Images (K) of axon segments in hippocampal neurons (DIV4), which were cotransfected with rat SEPT9-mCherry and GFP-Rab5A or LAMP1-mGFP. Scale bars, 5 µm. Plot (L) shows percentage (mean ± SEM; Mann–Whitney U test) of GFP-Rab5A (n = 13 axons) and LAMP1-mGFP (n = 17 axons) puncta with SEPT9-mCherry per cell. ns, nonsignificant (P > 0.05); **, P < 0.01; ***, P < 0.001.$

Septin localization and effects on early endosomes and lysosomes. (A) Spinning-disk confocal image shows the distribution of SEPT9-mCherry, which was expressed under the PGK promoter, with respect to GFP-EEA1 in living COS-7 cells. Scale bar, 20 µm. Perinuclear area is shown in higher magnification. Scale bar, 5 µm. (B) Quantification shows the fraction (mean ± SEM; Mann–Whitney U test) of SEPT9-mCherry puncta colocalizing with LAMP1-mGFP and GFP-EEA1 in COS-7 cells (n = 6). (C and D) PNSs of HEK-293 cell extracts were loaded on an iodixanol OPTIPREP density gradient (17%, 20%, 23%, 27%, and 30%), and fractions were blotted (C) for LAMP-1, Rab7, Rab5, GM130, BIP, SEPT9, SEPT2, SEPT6, and SEPT7. Plots (D) show quantification of indicated proteins in each fraction as percentage of total proteins across all fractions. Horizontal dashed lines provide a reference point for the percentage of septin protein in the top fraction. (E and F) Images (E) show the distribution of early endosomes (EEA1) in COS-7 cells that overexpress mCherry or SEPT9-mCherry (insets). Scale bars, 20 µm. Quantification (F) shows the ratio (mean ± SEM; unpaired t test) of perinuclear to peripheral fluorescence intensity of EEA1 per cell (n = 20). (G and H) Images (G) of LAMP1-stained COS-7 cells expressing mCherry, SEPT2-mCherry, or mCherry-SEPT6 (insets). Scale bars, 20 µm. Plot (H) shows the ratio (mean ± SEM; Kruskal–Wallis one-way ANOVA) of perinuclear to peripheral fluorescence intensity of LAMP1 per cell (n = 30–34). (I and J) Images (I) of COS-7 cells, which were transfected with plasmids that coexpress GFP and scramble or SEPT9 shRNA and stained with anti-SEPT9 antibody. Scale bars, 20 µm. Bar graph (J) shows fluorescence intensity (mean ± SEM; Mann–Whitney U test) of SEPT9 per shRNA-expressing cell (n = 27–35). A.U., arbitrary units. (K and L) Images (K) of axon segments in hippocampal neurons (DIV4), which were cotransfected with rat SEPT9-mCherry and GFP-Rab5A or LAMP1-mGFP. Scale bars, 5 µm. Plot (L) shows percentage (mean ± SEM; Mann–Whitney U test) of GFP-Rab5A (n = 13 axons) and LAMP1-mGFP (n = 17 axons) puncta with SEPT9-mCherry per cell. ns, nonsignificant (P > 0.05); **, P < 0.01; ***, P < 0.001.

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