Figure 6: Downregulation of VRK1 causes G1 arrest and increases sensitivity to PARP inhibitor treatment.

(a) Lung cancer cell lines (H460 and H1299) were analysed by immunoblot assay to measure VRK1 protein levels after vector and VRK1 shRNA transfection. Reduced protein expression of VRK1 was observed in VRK1 shRNA cell lines. (b) The same cell lines were used for FACS analysis to check cell cycle status. While normal cell cycle progression was found in vector-transfected cells, G1 arrest was observed in VRK1 shRNA-transfected H460 cells (P<0.0001) and H1299 cells (P=0.03). Experiments were carried out three times independently. V, vector-transfected cell lines; VRK1sh, VRK1 shRNA-transfected cell line. (c) Vector and VRK1 shRNA-transfected H1299 cell lines were treated with PARP inhibitor (ABT-888, 20 μM) and DMSO to measure cell viability. A synergistic increase in sensitivity (decreased cell viability) was observed in PARP inhibitor-treated VRK1 shRNA cell lines compared with vector-transfected cells (P=0.001). Experiments were done three times independently. The data are represented as the mean±s.d. (d) A colony-formation assay was done in H1299 lung cancer cell line with VRK1 shRNA and control vector transfection. H1299 cells (2,400 cells per well in the left and 600 cells per well in the right panel) were treated with ABT-888 (PARP inhibitor, 20 μM) in serum-free medium for 48 h. Regular medium was then given to cells after two times washing with PBS. After a week in regular medium, each plate was fixed with 100% methanol and stained with a 0.25% crystal violet staining solution. All experiments were done in triplicate. A synergistic reduction in colony number was identified in VRK1 shRNA-transfected cells treated with the PARP inhibitor (P=0.003 for 2,400 cells and P=0.003 for 600 cells per well). The P-values were calculated using Student’s t-test.