A Schematic overview of the experimental design. Dormant MMK1 cells were isolated by treating parental MMK1 cell population with 0.5 μM of colchicine (Colch) for 14 days. Dormant cells were then cultured in drug-free medium (drug holidays, DH) or treated with DYRK1A inhibitor ALGERNON (ALG). Scale bar: 10 μm. B Immunoblot analysis of DYRK1A (DYR) and retinoblastoma (RB) expression in parental and dormant MMK1 cells. Representative images of two independent experiments are shown. C, D RT-PCR analysis of cell cycle and dormant genes in dormant MMK1 cells. E Immunofluorescence images and quantification of Ki67-positive MMK1 cells. Dormant cells were treated ± ALG (1 μM, 7 days), then stained to detect Ki67 (pink), tubulin (green) and DAPI (blue). Scale bar: 5 μm. F Nuclear staining quantification of dormant MMK1 cells treated ± ALG (1 μM) for 7 days. G RT-PCR analysis of cell cycle genes in MMK1 cells treated as outlined in panel (E). H Immunoblot analysis and densitometric quantification of cyclin B (Cyc B) expression in dormant MMK1 cells treated ± ALG (1 μM) for 72 h. I, J Images and nuclear staining quantification of cells treated with Colch ± ALG (1 μM) for 14 days. Scale bar: 10 μm. K Images and nuclear staining quantification of HW1 cells treated with Colch ± ALG (1 μM) for 14 days. Prior to drug treatments, cells were transfected with scramble (si Ctr) and RB (si RB) targeting siRNA (24 h). Scale bar: 10 μm. L Summary of the proposed role of DYRK1A in glioblastoma stem cell proliferation. Bar graphs represent mean ± SEM from at least three independent experiments (two-tailed unpaired t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).