hSMG-1 localizes to stress granules. (A) hSMG-1 (detected by Ab1, red) colocalizes with the SG marker TIA-1 (green) in response to H₂O₂. NFF were stained after H₂O₂ treatment (1.5 mM) for 1 h. (B) Colocalization of hSMG-1 (Ab2, red) with a second SG marker eIF4G (green) after treatment of NFF with heat (45°C) or 1 mM NaAs for 1 h. DAPI (blue) was used to stain nuclei. Colocalization (yellow) is indicated in a merged panel on the right. (C) An HA-tagged form of hSMG-1 localizes to SG. HeLa cells were transiently transfected with HA-hSMG-1 and incubated for 48 h in fresh medium prior to exposure to heat or H₂O₂, followed by staining with anti-HA or anti-eIF4G antibodies. (D) hSMG-1 (Ab1) (red) colocalizes with G3BP1 (green) in SG formed in NFF in response to heat. Hoechst was used to stain the nuclei.

Localization of NMD proteins to SG but lack of evidence for a role for hSMG-1 in mRNA decay. (A) Upf2 colocalizes with TIA-1 in SG. NFF were fixed with or without heat treatment and stained for TIA-1 (green) and Upf2 (red). Colocalization (yellow) following heat treatment is shown in the merged panel on the right. (B) Upf1 also localizes to SG. HeLa cells were transiently transfected with HA-Upf1. After 48 h, the cells were treated with H₂O₂ and NaAs and then stained with anti-HA (green) and anti-eIF4G (red) antibodies. (C) Endogenous Upf1 localizes to hSMG-1. NFF were treated with NaAs or heat and after 1 h stained for Upf1 (green) and hSMG-1 (Ab2, red). (D) Upf1 colocalizes with hSMG-1. The line profile shows overlapping fluorescence signals for Upf1 (green) and hSMG-1 (red). Image analysis was performed using the Softworx computer program. Line profiles were determined by drawing a line through the cytoplasm of SG positive fibroblasts. (E) Upf1 is not phosphorylated in heat- and NaAs-induced SG. NFF were treated with NaAs or heat and, after 1 h, stained with phospho-specific antibodies recognizing sites phosphorylated on Upf1 during NMD. P-Upf1 (green) was not detected in hSMG-1 (Ab2, red) positive SG but could be clearly seen in the nucleus. (F) Localization of a nonphosphorylatable form of Upf1, HA4SAUpf1, to SG. NFF were transiently transfected with HA4SAUpf1 and incubated for 48 h in fresh medium prior to exposure to H₂O₂ and heat, followed by staining with anti-HA and anti-Upf1 antibodies. (G) hSMG-1 (Ab1) does not localize to P bodies. NFF were transiently transfected with the P-body-specific marker DCP1-YFP and incubated for 48 h prior to treatment with H₂O₂ for 1 h and followed by the detection of YFP and staining with antibodies against hSMG-1.

Role for PIKK kinase activity in SG formation. (A) Wortmannin inhibits SG formation in response to NaAs treatment but not heat treatment. Cells were NaAs or heat treated after 2 h of incubation with the indicated inhibitor. NFF were then stained with anti-hSMG-1 (Ab2, red) and anti-eIF4G (green) antibodies, with the colocalization appearing as yellow. Nuclei were detected with DAPI (blue). Inhibition of ATM (Ku55933), DNA-PK_cs (AMA37), or mTOR (rapamycin) did not interfere with formation of SG in response to either treatment. The data are representative of two to four independent experiments. (B) Effect of ATM-specific inhibitor on ATM autophosphorylation at S1981. NFF were incubated with Ku55933 for 2 h prior to 10 Gy of IR and then incubated for 30 min or 1 h prior to the preparation of cell extracts. Immunoblotting was carried out with a phospho-specific antibody for ATM S1981. Nbs1 protein was used as a loading control. (C) Effect of different inhibitors on DNA-PK autophosphorylation at S2056. NFF were incubated with either Ku55933 (ATM inhibitor), wortmannin (general PIKK inhibitor), or AMA37 (DNA-PK_cs inhibitor) for 2 h prior to exposure to 10 Gy of IR. Extracts were prepared and immunoblotted either with the phospho-specific antibody (P2056-DNA-PK) or an anti-DNA-PK_cs antibody. Total DNA-PK_cs detection was used as a loading control. (D) Rapamycin prevents passage of NFF from G₁ to S phase. Cells were exposed to concentrations of rapamycin from 20 to 100 μM prior to analysis by flow cytometry using propidium iodide (PI) staining. (E) PCCs for cells treated with PIKK inhibitors prior to SG formation. These coefficients were determined using Softworx software for a defined region of the cytoplasm of fibroblasts. The data show the averages of at least five measurements from different cells from at least two independent experiments and the standard deviations of the measurements.

hSMG-1 is cytoplasmic and nuclear and localizes to cytoplasmic granules in response to stress. (A) Schematic of the regions recognized by of hSMG-1 antibodies. GST fusion proteins were prepared using the regions indicated (region 1, Ab1; region 2, Ab2; region 3, Ab3) and used to generate polyclonal antibodies in sheep. (B) Specificity of detection of hSMG-1 by immunoblotting. Extracts were prepared from LCLs and NFF, and proteins were separated by SDS-PAGE prior to immunoblotting with the three different hSMG-1 antibodies. (C) hSMG-1 is present both in the cytoplasm and in the nucleus. Immunoblot analysis was performed for hSMG-1 in nuclear and cytoplasmic extracts from primary normal foreskin fibroblasts (NFF). ATM was used as a control to demonstrate fractionation. (D) hSMG-1 does not localize to sites of DNA damage. DNA damage induced nuclear foci in response to 10 Gy of IR. NFF were fixed and stained with anti-γH2AX (red) and anti-hSMG-1 (Ab2, green) antibodies. Nuclei were detected with DAPI. (E) Detection of cytoplasmic granules in NFF in response to H₂O₂. The cells were incubated for 1 h after exposure, fixed, and stained with hSMG-1 (Ab2) antibody. (F) Immunofluorescent detection of hSMG-1 with three different antibodies in NFF. hSMG-1 antibodies (red) directed to different regions of hSMG-1 show cytoplasmic granule formation in response to heat.

hSMG-1 plays a central role in SG formation in primary cells but does not localize to SG in tumor cells. (A) Appearance of SG in primary cells in response to H₂O₂ or heat treatment. The images represent merged images between hSMG-1 (Ab1) (red) and TIA-1 (green). Nuclei were visualized with Hoechst stain. Abbreviations: PTC, human proximal tubular cells; MEL, primary melanocytes; HEK, undifferentiated keratinocytes. (B) A549 and U2OS cells were treated with NaAs for 1 h and stained for hSMG-1 (Ab1) (red) and TIA-1 (green). TIA-1-positive but hSMG-1-negative SG were formed in response to NaAs.

Effect of hSMG-1 or Upf1 disruption on SG formation. (A) Knockdown of hSMG-1 in NFF by three different siRNA oligonucleotides. hSMG-1 protein was detected by immunoblotting (Ab3), and the loading control was DNA-PK_cs. (B) At 48 h after siRNA treatment, the NFF were treated with NaAs or heat for 1 h and then stained for hSMG-1 (Ab2, red) and eIF4G (green). Colocalization appears as yellow. Nuclei were detected with DAPI (blue). After siRNA treatment, the numbers of eIF4G SG-positive cells were reduced in NFF that had been treated with NaAs but not in heat-treated NFF. Control siRNA had no affect on SG formation in response to either agent. (C) Quantification of SG formation following treatment with two different anti-hSMG-1 or control siRNA. The percentage of NFF with eIF4G-positive granules was scored. The data represent the averages of three independent experiments, and error bars show the standard errors of the mean. Asterisks denote statistically significant differences in SG formation (*, P < 0.05; **, P < 0.01). (D) Knockdown of Upf1 expression in NFF using two different siRNA. Western blotting confirmed knockdown of Upf1 protein level but no affect on hSMG-1 or TIA-1 expression levels. GAPDH was probed for as a loading control. An irrelevant lane has been removed from the image, but all samples were run on the same gel. (E) Upf1 knockdown does not block stress granule formation. Approximately 64 h after anti-Upf1 siRNA treatment SG were induced with either NaAs or heat for 1 h. Fibroblasts were stained for TIA-1 (green) and Upf1 (red). SG formation could be seen in all treated samples. The data are representative of three independent experiments. (F) Cells were treated as for panel E but stained for hSMG-1 (green) and Upf1 (red). Nuclei were visualized with DAPI (blue). hSMG-1 was still recruited to SG following Upf1 knockdown. The data are representative of three independent experiments.

Detection of phosphorylated proteins in SG and importance of protein kinase activity of hSMG-1. (A) NaAs and H₂O₂ treatment of NFF induced the appearance of proteins phosphorylated at (S/T)Q sites, and these sites partially colocalize in SG with hSMG-1. Heat induced SG formation (hSMG-1 positive) without detectable phosphorylation at (S/T)Q sites. Staining in the upper panel used an older batch of rabbit anti-P(S/T)Q antibody: P(S/T)Q (green), hSMG-1 (Ab2, red), and DAPI (blue). In the lower panel, a newer batch of rabbit anti-P(S/T)Q antibody was used: P(S/T)Q (red), hSMG-1 (Ab2, green), and DAPI (blue). (B) P(S/T)Q signals associate with hSMG-1 after SG induction. The left panel shows a line profile indicating regions of overlapping fluorescence signals for P(S/T)Q (green) and hSMG-1 (red) following NaAs treatment. The right-hand panel shows greater overlap of the P(S/T)Q (green) signal with hSMG-1(red) after H₂O₂ treatment. Image analysis was performed using the Softworx computer program. Line profiles were determined by drawing a line through the cytoplasm of SG-positive fibroblasts. (C) Overexpression of kinase-deficient hSMG-1 does not inhibit SG formation. HeLa cells were transfected with either HA-hSMG-1 or HA-hSMG-1-DA (kinase deficient). The cells were treated with 1 mM H₂O₂ or NaAs for 1 h and then stained with anti-HA (green) and anti-P(S/T)Q (red) antibodies. In cells transfected with HA-hSMG-1-DA, no P(S/T)Q sites were observed in response to H₂O₂ treatment.