Figure 4
Pharmacological inhibition of CK1 disturbs VANGL2-EGFP polarization. (A) The effect of CK1ε, DVL3, CK1 inhibitor and Wnt-C59 (porcupine inhibitor) on the electrophoretic mobility of HA-VANGL2 as determined by Western blotting. (B) Snapshots from time-lapse microscopy of MEC1 cells co-transfected with VANGL2-EGFP and Actin-RFP. All conditions are CCL19-stimulated. Cells were treated with two casein kinase 1 inhibitors: PF670462 (CK1i-I) and D4476 (CK1i-II). (C) Ratios of the EGFP signal intensity in the trailing edge divided by the EGFP signal intensity in the leading edge for all three conditions are represented in the bar graph. Statistical analysis was performed using one-way ANOVA and Kruskal-Wallis test in GraphPad (***, P < 0.001). (D,E) Dose-dependent effects of D4476 (CK1i-II) on MEC1 cell migration, viability and VANGL2 asymmetry are summarized graphically. Migration (migration index) and asymmetry (ratio of fluorescent intensity in trailing/leading edge) were analysed by nonlinear regression. 35 μM D4476 is labelled red. Data points of viability experiments are shown with a connecting line (no regression). (F,G) Asymmetric distribution of VANLG2 in MEC1 cells is disturbed neither by the mutation of VANGL2 serines phosphorylated by CK1 to alanine (VANGL2-S5,8,11,82,84A) nor by inhibition of WNT production by porcupine inhibitors Wnt-C59 (10 μM) and LGK-974 (10 μM) nor by inhibition of WNTs by sFRP1 (10 μg/ml). The Western blot in F demonstrates that overexpression of CK1/DVL3 is unable to trigger phosphorylation-dependent shift of VANGL2-S5,8,11,82,84A. (H) Transwell migration assay experiment (N = 3) shows that VANGL2-Venus MEC1 cells respond significantly better to CCL19 stimuli. Number of migrated cells in 20 μL of sample is shown. Western blot confirms VANGL2-Venus in the stable cell line. (I) MEC1 cells nucleofected by VANGL2 siRNA show decrease in migration in conditions with and without CCL19. Expression of VANGL2 was significantly decreased 36 h after transfection as determined by qPCR. Migration in CCL19-treated conditions (H,I) was compared by unpaired t-test (*, P < 0.05).