ICG-001 blocks KHOS, MG63 and 143B osteosarcoma cell lines in the G0/G1 phase. (a) Cell cycle analysis by flow cytometry in cells treated with ICG-001 (10 µM) or vehicle (Vh) for 24 h. Histograms represent means ± SD of three independent experiments. * p ≤ 0.05. (b) Cell cycle regulators protein expression in KHOS cells synchronized in early S phase with thymidine treatment, released in cell cycle during 0, 2, 8, 12 and 24 h, and treated with ICG-001 (10 µM) or vehicle (Vh). Representative images of three independent experiments. Histograms represent means ± SD of relative CYCLIN D1, CYCLIN D3 and P21 protein quantification relativized with the value of the vehicle (DMSO for each time) and normalised with VINCULIN and relative quantification of RB phosphorylated form protein expression relativized with the value of the vehicle (DMSO for each time) and normalized with total RB of three independent experiments. The dashed line represents the vehicle value set to 1 for each time. * p ≤ 0.05 vs. vehicle for each time. (c) Regulation of G1 to S phase transition implicating cyclins and cyclin dependent kinases (CDK) as well as Retinoblastoma protein (Rb) and p21.