Fig 1: Flubendazole reciprocally controls dynamic and acetylated microtubules and affects mTOR.(a) Confocal images of HeLa cells treated with DMSO or flubendazole (1 h, 5 μg ml−1); immunofluorescence with antibody to acetylated tubulin and α-tubulin. Antibodies against α-tubulin (Abcam, ab6161; green fluorescence) and acetylated tubulin Sigma (clone 6-11B-1; red fluorescence) were imaged with constant (per fluorophore) excitation, emission, pinhole and exposure time. Scale bars, 20 μm. (b) Immunoblot analysis of acetylated tubulin (antibody: Sigma clone 6-11B-1) levels in lysates from HeLa cells treated with ethanol or 5 μg ml−1 flubendazole in ethanol (see Supplementary Fig. 3b for DMSO as a solvent). (c) Immunoblot analysis of ATAT1 and HDAC6 levels in lysates from HeLa cells treated with DMSO or 5 μg ml−1 flubendazole (in DMSO) for different durations as indicated. Graph, quantification from three different experiments; mean±s.e.; Student's t-test. *P<0.05. (d) Confocal images of HeLa cells starved (1 h) or treated with DMSO or flubendazole (1 h, 5 μg ml−1) and immunostained with antibodies to mTORC1 and LAMP1. Dashed squares, fields with tracings for colocalization analysis. Fed, full medium control where mTOR localizes on lysosomes, versus starvation or flubendazole treatment. (e) HeLa cells treated with DMSO or flubendazole (5 μg ml−1) were lysed and subjected to immunoblotting with indicated. Scale bars, 20 μm (a) and 10 μm (d).