Fig. 2

SHCBP1 is mainly located in the nucleus, with increased expression during the G2 and M phases of the cell cycle. A Representative western blot images of SHCBP1, Cyclin B1 and PLK1 protein in total (T), cytoplasmic (Cyto), and nuclear (Nuc) extracts of A549, H1299 and Hela cells after treating with low-dose ETOP (A549, 1μΜ; H1299, 5 μM; Hela, 3 μM) or vehicle for 3 h and 24 h. GAPDH and Lamin B1 were used as internal controls for cytoplasmic and nuclear extracts, respectively. B, C Immunofluorescence (IF) staining (B) and western blot densitometric analysis (C) of SHCBP1 and PLK1 throughout the cell cycle of HBE, A549, H1299 and HeLa cells (cells were synchronized to G0, G1/S, and G2/M phase by serum starvation, double thymidine and RO3306 block, respectively). In the IF staining (B), cells were co-stained with anti-SHCBP1 antibody (red), anti-α-tubulin antibody (green), and Hoechst (blue); Scale bars, 100 μm. Representative Western blot images of panel (C) are shown in Supplementary Fig. 4, data are expressed as mean ± SD (n = 3 independent experiments) and analyzed by the unpaired Student’s t test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Note: asyn, asynchronized. PLK1: well-known G2–M phase-associated proteins. D, E Western blot densitometric analysis of SHCBP1 and PLK1 in A549 and HeLa cells analyzed at indicated time points after nocodazole (NOCO) (D) and double thymidine (TdR) release (E), respectively. Data are expressed as mean ± SD (n = 3 independent experiments) and analyzed by the unpaired Student’s t test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). F Immunofluorescence staining of SHCBP1 at indicated time points after RO3306 release in A549 and HeLa cells. Cells were co-stained with anti-SHCBP1 antibody (red), anti-γ-tubulin antibody (green), and Hoechst (blue). Scale bars, 100um. G, H Representative immunofluorescence images (G) showing co-localization of SHCBP1 (green) with PHH3 (Ser10) (red) and the corresponding statistical analysis (H) of SHCBP1 mean fluorescence intensity between PHH3 (Ser10) positive and negative cells. The PHH3 (Ser10) positive cells (mitotic cells) are highlighted by the white arrows; Scale bars, 20 μm. Data in panel (H) are expressed as mean ± SD (n ≥ 4, with at least 200 cells per analysis) and analyzed by the unpaired Student’s t test (****, P < 0.0001)