Research Paper Volume 8, Issue 12 pp 3356—3374

Figure 6. Overexpression of NonO activates G2/M arrest-associated signaling. (A) Cos7 cells were stably transfected with either truncated GFP-NonO(1-394) or full length (FL) NonO, harvested at the indicated time points, and analyzed by SDS-PAGE/western blotting with anti-Rb mAb; anti-Actin mAb served as a loading control. (B) Expression of Cyclin A1 (lower panel) and p53 (upper panel) in GFP-nonO-transfected Cos7 cells. (C) Activation of DNA damage-associated punctate γH2AX formation 12 days following overexpression (OE) of FL or NonO(1-394). In pre-senescence nuclei, γH2AX clusters overlap with NonO-containing paraspeckles (middle panels), whereas in senescent nuclei, NonO(1-394) localizes to the cytoplasm while γH2AX foci are retained in condensed nuclei (lower panels). (D) Overexpression (OE) of NonO(1-394) (right panels) induces hypophosphorylation of ppRb to pRb and upregulation of p53 more rapidly than OE of FL NonO (left panels). Actin served as a loading control (bottom panel). (E) NonO or PSF overexpression (OE) (lanes 4 and 6) or antisense (AS) knockdown (lanes 5 and 7) do not activate telomerase activity in NIH 3T3 fibroblasts. TRAP assays as well as negative (-) and positive (+) cell extract controls (Lanes 1 and 3) are described in Materials and Methods.