Figure 2: DYRK1A overexpression negatively affects the actin cytoskeleton.

(a) Endogenous GLI1 and DYRK1A protein expression in siRNA-transfected Ptch1−/− MEF cells (1% FBS). (b) Endogenous GLI1 and DYRK1A protein expression in siRNA-transfected Hek293T cells. (c) GLI1 and DYRK1A mRNA expression as determined by qPCR of siRNA-transfected DAOY cells. The fold induction on SAG of the Hh target gene GLI1 is indicated above the bars (mean of n=3±s.d.). (d) Western blot of different MEF cell lines stably overexpressing V5-tagged DYRK1A (1% FBS). (e) Hh pathway target gene expression of the cells shown in d (mean of n=3–5±s.d.) (1% FBS). (f) H&E staining of mouse wild type (wt) or KPC pancreata from control or from harmine-treated animals. Scale bar, 100 μm. (g) Vimentin staining (brown) of mouse wild type (wt) or KPC pancreata from control or from harmine-treated animals. Scale bar, 100 μm. (h) Keratin 17/19 staining (brown) of mouse wild type (wt) or KPC pancreata from control or from harmine-treated animals. Scale bar, 100 μm. (i) Pancreatic GLI1 protein expression in KPC control and in harmine-treated KPC mice. (j) Three-dimensional deconvolution images of F-actin fibres (phalloidin) in NIH3T3 cells transiently transfected with the indicated constructs. Transfected cells were identified through co-transfection of a small amount of nuclear histone H2B–GFP plasmid. The F-actin severing Cofilin (CFL1) was transfected as a positive control. The inset in the upper right panel depicts a cell with clearly visible fragmented F-actin and a lack of stress fibres. Scale bar, 10 μm. (k) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or DYRK1A. G, G-actin fraction; F, F-actin fraction. Jasplakinolide: 10 nM for 30 min. Shown is a representative anti β-actin western blot of three independent experiments; the mean value of the western blot band intensities of these three experiments is shown below. *P<0.05; **P<0.005; ***P<0.0005 (Student’s t-test).