SEPT7 knockdown abolishes septin filaments without disrupting GFP-CD63 association with microtubules. (A) Widefield microscopy images of MDCK cells which were stained for endogenous SEPT7 (inverted forest green) after 48 h transfection with plasmids that express mCherry (inverted blue) and scramble control or SEPT7 shRNAs. Scale bars, 10 μm. (B) Mean (±SEM, error bars) SEPT7 fluorescence intensity per cell area after staining of MDCK cells (n = 6), which were transfected for 48 h with plasmids that express mCherry (inverted blue) and scramble control or SEPT7 shRNAs. Data were statistically analyzed with a Mann-Whitney test. **** P < 0.0001. (C) Maximum intensity projections of super-resolution confocal microscopy z-stack images of MDCK-GFP-CD63 cells (inverted magenta), which were stained for endogenous α-tubulin (inverted forest green) after 48 h transfection with plasmids that express mCherry (inverted blue) and scramble control or SEPT7 shRNAs. Scale bars, 10 μm. (D) Quantification of the mean percentage (±SEM, error bars) of GFP-CD63 endosomes that overlap with microtubules in MDCK cells (n = 6) after 48 h transfection with control and SEPT7 shRNAs. Data were statistically analyzed with a Mann-Whitney test. n.s., not significant.