Fig 2: hCdc73 translocates to SGs under various stress conditions. (A) Immunoblot analysis (IB) of hCdc73 from the cytosol (top), nucleus (middle), and whole-cell lysates (WCL, bottom). HeLa cells were treated with 500 µM sodium arsenite for 1 h (ARS), 50 µM thapsigargin for 1 h (TG), 10 µM MG132 for 4 h (MG132) and 46°C heat shock for 1 h (HS). Each sample was loaded next to its own control set. NT, nontreated; Mock, DMSO treated. GAPDH and LAMINB were used as marker proteins for the cytosol and nuclear fractions, respectively. (B) Confocal images of endogenous hCdc73 and G3BP1 in cells either untreated (NT) or stressed as indicated. The same conditions in A were used except for the heat-shock condition, which was 43°C for 30 min. (C) Schematic diagram of the mutation sites used to generate hCdc73NLSmut. (D) Confocal images of hCdc73NLSmut–Myc-transfected HeLa cells that were untreated (NT) or treated with 500 µM sodium arsenite for 1 h (ARS). TIAR, marker for SGs. (E) Confocal images of endogenous hCdc73 and G3BP1 in HeLa cells transfected with siRNA against hCdc73 (siCdc73) or control diRNA (siCtrl) treated with 150 µM sodium arsenite for 2 h. (F) Area (top) and number of SGs per cell (bottom) quantified from the data in E (n=51). (G) Schematic diagram of the SG maintenance and disassembly experiment. (H) Percentage of SG-positive cells measured during SG maintenance and disassembly conditions (n=50–99). (I) Representative confocal images of endogenous hCdc73 and G3BP1 tested at an additional six and 12 h of SG maintenance and disassembly conditions. Scatter plot graphs and bar graphs are presented as the mean±s.d. ***P<0.0005; n.s., not significant (unpaired two-tailed t-test). All images and blot data are representative of at least three biologically independent experiments. Insets in B, D, E and I show a magnification of the indicated area. Scale bars: 10 µm. *P<0.05; **P<0.005; ***P<0.0005; ****P<0.00005; n.s., not significant.