Figure 4

SMAR1 favors G2/M cell cycle arrest through Chk2 phosphorylation. (a) Metaphase spread analysis of SMAR1 overexpressed (Ad-SM) and knockdown (sh3) HCT116 cells upon IR (10 Gy). Red arrows indicate chromosomal aberrations. The data are representative of >50 images (n>50), which were acquired in various fields from three independent experiments. (b) In vivo NHEJ assay upon SMAR1 overexpression (Ad-SM) and knockdown (sh3) in HCT116 cells cotransfected with a linearized pGL2-Luc plasmid that was previously digested with either HindIII (purple bar) or EcoRI (black bar), and pRL-SV40 renilla luciferase vector. The luciferase enzyme activity was normalized by dividing the Luc signal with the renilla signal. Error bars represent S.D. from three independent experimental repeats. (c) PI staining to analyze the effect of SMAR1 on IR-induced cell cycle progression. Statistical representation of different cell cycle phases upon irradiation (10 Gy, 48 h) in control, and SMAR1 overexpressed (Ad-SM) or knockdown (sh3) HCT116 cells. Error bars represent S.D. from three independent experiments. (d) Western blot analysis of indicated proteins in control and SMAR1 overexpressed (Ad-SM) HCT116 cells that were treated either with or without IR (10 Gy, 4 h) and KU-55933 (10 μM, 2 h). (e) Chk2 kinase activity assay in the presence and absence of SMAR1 in control (green bar) and irradiated (brown bar) HCT116 cells (10 Gy, 4 h). Error bars represent S.D. from three independent experimental repeats