Cytoskeleton disruptors influence NMD or readthrough.

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Cytoskeleton disruptors influence NMD or readthrough. Cytoskeleton disruptors influence NMD or readthrough. (A) Cytoskeleton disruptors inhibit NMD. 6CFSMEo- cells (above) and IB3 cells (below) were incubated with DMSO (negative control), cytochalasin D (CytoD), JPK, colchicine (COL), Taxotere (TAX) or amlexanox (positive control) for 48 h. The level of CFTR mRNA was measured by qRT-PCR and normalized to the level of GAPDH mRNA. The five leftmost lanes represent two-fold serial dilutions of RNA from Calu-3 cells overexpressing CFTR mRNA. A histogram representation of the results is presented to the right of each gel (mean±s.d.; n=3). (B) Actin inhibitors activate PTC readthrough of nonsense-mutation-containing CFTR mRNA. 6CFSMEo- cells (above) or IB3 cells (below) were incubated with DMSO, CytoD, JPK, COL, TAX or amlexanox for 48 h before analysis of protein content by western blotting. The Ku80 protein was detected as a loading control. The three leftmost lanes represent two-fold serial dilutions of protein extract from Calu3 cells. (C) Actin inhibitors promote PTC readthrough on PTC-containing mRNA introduced by transfection. 6CFSMEo- cells were transfected with an expression vector encoding YFP-tagged GPx1 46 Ter or Flag-tagged SRSF7 as a reference plasmid. The transfected cells were incubated with DMSO, CytoD, JPK, COL, TAX or amlexanox for 48 h before analysis of protein content by western blotting. Flag-tagged SRSF7 protein was used as the loading control. The three leftmost lanes represent two-fold serial dilutions of protein extract from 6CFSMEo- cells transfected with an expression vector encoding GPx1 Norm (wild type). * indicates non-specific protein species. These results are representative of three independent experiments. Jieshuang Jia et al. J Cell Sci 2017;130:3009-3022 © 2017. Published by The Company of Biologists Ltd