IST1 regulates endosomal tubulation. (a and b) Wild-type HeLa cells were subjected to mock transfection or transfected with either of the two IST1 siRNA oligonucleotides indicated and then labeled with SNX1. The number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted in b. (c) Depletion of IST1 was verified by immunoblotting. (d–g) Wild-type HeLa cells (d) or HeLa cells stably expressing myc-tagged siRNA-resistant IST1 (e) were subjected to mock transfection or were transfected with an siRNA targeting endogenous IST1. The number of SNX1 tubules per cell was counted as in a. To control for any variation in the baseline number of tubules per cell in the two 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. wt, wild type. (g) Depletion of endogenous IST1 was verified by immunoblotting. (h) HeLa cells depleted of IST1 by transfection with IST1 siRNA1 were labeled with antibodies to endogenous SNX1 and α-tubulin (MT). Arrowheads in the magnified images of the boxed areas indicate an aligned SNX1 tubule and MT. (i–k) Mock-transfected cells or cells depleted of IST1 by transfection with siRNA1 were treated with vehicle (DMSO), nocodazole (i), taxol (j), or latrunculin A (k), and then, the number of SNX1 tubules per cell was counted (30 cells per condition), and the mean values of three independent experiments were plotted. KD, knockdown. Insets are magnifications of boxed regions. Bars, 10 µm. Error bars show SEMs.