Figure 9.

ARH depletion affects cell cycle progression. (A) Phosphohistone-H3 (pH3), a mitosis marker, is reduced by 55–85% in ARH-depleted cells (lane 1), indicating reduced entry into mitosis. Total histone-H3 (H3) is not significantly changed. ARH siRNA leads to a 69–87% reduction in ARH. Rat-1 cells were transfected with rat ARH smartpool siRNA (ARH), scramble siRNA (ctrl), or Lipofectamine 2000 alone (lipof.) and analyzed 48 h after transfection. (B) Quantification of protein levels of three replicate blots as in A. The levels of ARH, phosphohistone-H3(pH3), and total histone (H3) are expressed as % of the scramble control (set to 100%). (C) Phosphohistone-H3 levels are partially restored upon introduction of full-length human ARH into ARH siRNA-treated cells (lane 1). The band for human ARH (asterisk, lane 2) appears weak due to the fact that the ARH antibody was made against rat ARH and reacts only weakly with transfected human ARH. Retroviral infection with hARH was performed 15 h after siRNA transfection followed by analysis 48 h later. (D) Quantification of levels of phosphohistone H3 (pH3) and total histone H3 (H3) after infection of ARH siRNA-treated cells with a control (mock) or hARH virus (rescue). The data represent the mean of three replicate analyses as in C. The levels are expressed as a ratio of pH3/H3, with the mean of the ratio of the scramble control set to 1. (E) Bar graph showing effects of ARH depletion on cell growth. Rat ARH siRNA-treated cells grow more slowly than controls treated with scramble siRNA. This effect is partially rescued by expressing full-length human ARH-GFP (hARH-GFP) in siRNA-treated cells, whereas expression of GFP alone has no significant effect. Retroviral infection (hARH) was performed 24 h after siRNA transfection, and the number of cells/well were counted 48 h later. (F) wt and Arh^−/− MEF cell numbers were counted at the indicated times after plating. Arh^−/− MEFs grow more slowly than wt cells.