Spastin regulates endosomal tubulation. (a and b) HeLa cells were subject to mock transfection or transfection with a spastin siRNA pool (spastin knockdown [KD]) and labeled versus endogenous SNX1 (a) or transfected SNX4-mCherry (b). The insets in a and b are magnified images of the boxed regions indicated. (c) The mean number of SNX1 and SNX4 tubules per cell was quantified (n = 5 independent experiments for SNX1, and n = 3 for SNX4, 30 cells counted per experimental condition in each experiment). (d) The length of the longest tubule per cell was measured in 30 cells, and the mean values were plotted. (e) Depletion of spastin was confirmed by immunoblotting. Note that M1 spastin is not routinely seen in immunoblots such as this, in which the exposure is optimized to show the much stronger M87 band. Actin labeling is shown to verify equal sample loading. (f) Complex structures, defined as having at least three tubules emanating from a central punctum (arrowheads), were commonly seen in spastin-depleted cells. (g) The mean number of complex structures per cell was quantified (n = 3, 30 cells counted per condition in each experiment). Bars, 10 µm. Error bars show SEMs.