Research Paper Volume 8, Issue 12 pp 3356—3374

Figure 3. NonO-induced senescence is accompanied by cell cycle-regulated cytoplasmic localization within paraspeckles. (A) Subcellular localization of NonO and PSF in Cos7 cells as determined by Immunostaining with anti-NonO or anti-PSF antibodies. (a,b) Endogenous NonO and PSF accumulation colocalize within the nucleus in asynchronous cultures; (c) OE of NonO in asynchronous cultures increases NonO:PSF paraspeckle nuclear localization; (d) NonO knockdown reduces NonO:PSF paraspeckle nuclear abundance; (e). NonO and PSF localize both in the nucleus and cytoplasm of mitotic cells within asynchronous cultures. Cell nuclei are visualized by DAPI staining. (B) Cell synchronization of ~3 x 10⁶ 293T cells by sequential treatment with thymidine (16 h) and nocodazole (16 h) after an intermediate release step of 8 h as assessed by DAPI staining at 10X and 100X magnification. Untreated 293T cells (upper panels) grow asynchronously to confluency, whereas thymidine and nocodazole-treated cells (lower panels) loose adherence and arrest at G2/M with frequent condensed chromatin events. (C) Nuclear to cytoplasmic relocalization of NonO:PSF containing paraspeckles as identified by immunostaing with anti-NonO and anti-PSF antibodies. (a) Untreated asynchronous cultures; G2/M arrested cultures stained for NonO (b) and PSF (c); (d) Mitotic cells within asynchronous cultures follow overexpression (OE) of NonO.