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. 2014 Feb 18;111(11):4031–4036. doi: 10.1073/pnas.1314482111

Fig. 1.

Fig. 1.

OGFOD1 knockdown inhibits proliferation in a range of cell lines and induces stress granules and translational stress in U2OS cells. (A) Effect of OGFOD1 siRNA on proliferation of cell lines. Proliferation was assessed by MTS assay upon transfection with a nontargeting siRNA pool (siCON) or equivalent targeting OGFOD1 (siOGFOD1). Data are presented as the ratio of absorbance (A490) at endpoint relative to day 1. siDeath served as a transfection control. Efficacy of OGFOD1 knockdown was confirmed by immunoblotting. Data (n = 3) are expressed as the mean; error bars ± SD. **P < 0.01; n.s., not significant. (B) shRNA-mediated knockdown of OGFOD1 inhibits proliferation of U2OS cells. U2OS clones encoding dox-inducible shRNAs against OGFOD1 (OGF) (clones 7 and 12) or control (CON) (clone 1) were assayed in the presence or absence of dox. MTS data are expressed as the A490 ratio at endpoint over start. OGFOD1 knockdown was confirmed by immunoblotting. Data are n = 6 and expressed as the mean; error bars ± SD. **P < 0.01. (C) shRNA-mediated knockdown of OGFOD1 induces stress granule formation in U2OS cells. U2OS shRNA clones were cultured in the presence (48 h) or absence of dox before arsenite stress (1 mM, 30 min). G3BP1 stress granules were detected by indirect immunofluorescence (green). Nuclei were stained with DAPI. Representative confocal images of dox-treated cells are depicted. Quantitation data (percent age of cells with G3BP1 foci) are from three independent experiments; values represent the mean; error bars ± SD. *P < 0.05. (D) Temporal induction of p–eIF2α following knockdown of OGFOD1. Immunoblot of U2OS shRNA clones treated with dox for the indicated time. (E) OGFOD1 knockdown inhibits protein synthesis rate. shRNA clones were labeled with [35S]-methionine for the indicated time. 35S incorporation was monitored by scintillation counting. Data (n = 4) represent the mean; error bars ± SD. **P < 0.01. (F) OGFOD1 knockdown induces expression of stress response targets, ATF4 and CHOP. Immunoblot detection of indicated proteins in U2OS shRNA cells treated with dox for 48 h.