M1 and M87 spastin are recruited to endosomes and regulate endosomal tubulation. (a and b) GFP-VPS4-E235Q was transiently transfected into cell lines stably expressing myc-tagged M87 spastin (a) or myc-tagged M1 spastin M87A (b), and then, the cells were labeled with an anti-myc antibody. (c–f) HeLa cells (c), HeLa cells stably expressing myc-tagged M87 spastin (d), or HeLa cells expressing myc-tagged M1 spastinM87A (e) were subjected to mock transfection, transfected with an siRNA oligonucleotide directed against endogenous spastin (spastin 1, to which the myc-tagged transcripts were resistant), or with a combination of two siRNA oligonucleotides that together targeted endogenous and transfected spastin (spastin 1 and 6). The cells were labeled with an antibody to endogenous SNX1, and the number of SNX1 tubules per cell was counted (30 cells per condition). To control for any variation in the baseline number of tubules per cell in different cell lines, for each cell line, the mean tubule count per cell for siRNA-treated cells was normalized by subtracting the mean tubule count per cell in the corresponding mock-transfected cells. The resulting values for the mean increase in tubule number per cell in siRNA-treated cells were then plotted in f; n = 3 independent experiments. Cellular depletion of exogenous and/or endogenous spastin in these experiments was verified by immunofluorescence and immunoblotting (Fig. S3). Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.