Fig. 2.

OGFOD1 enzyme function rescues growth and protects against arsenite-induced stress granule formation. (A) Ras-transformed Ogfod1^−/− MEFs display reduced proliferation rates relative to WT counterparts. Immortalized (SV40T) and ras-transformed (K-ras^G12V) Ogfod1^fl/fl MEFs were transduced with control (WT) or cre-recombinase (KO) to delete Ogfod1 alleles. Proliferation rates were determined by MTS assay and expressed as A₄₉₀ ratio at endpoint relative to day 1. Data (n = 4) represent the mean; error bars ± SD. *P < 0.05; **P < 0.01. (B) Introduction of WT human OGFOD1 into transformed KO MEFs confers a proliferative advantage over hydroxylase mutant and null lines as determined by CyQUANT assay. Proliferation rates of transformed MEFs reexpressing either an EV, WT human OGFOD1, or D157A mutant (MUT) OGFOD1. Data are presented as the ratio of relative luminescence (RLU) at endpoint versus day 1. Data (n = 4) represent the mean; error bars ± SD. **P < 0.01; n.s., not significant. (C) Reexpression of WT OGFOD1 in transformed Ogfod1^−/− MEFs confers a proliferative advantage over the D157A catalytic mutant and null lines in a spheroid culture model. Data are n = 36 spheroids per time point and expressed as mean values; error bars ± SD. **P < 0.01; n.s., not significant. (D) Low expression of p–eIF2α in unstressed transformed Ogfod1^−/− MEFs. Immunoblot analysis of eiF2α and p–eIF2α in ras-transformed rescue MEFs. +, arsenite control for p–eIF2α immunoblot. (E) WT OGFOD1 suppresses arsenite-induced G3BP1 stress granule formation. Stress granules were detected and quantified by indirect immunofluorescence of G3BP1 (red) in transformed rescue MEFs. Nuclei were stained with DAPI. Cells were treated with arsenite (1 mM, 30 min) and representative confocal microscopy images are presented. Quantitation data (percent age of cells with G3BP1 foci) are from three independent experiments and represent mean values; error bars ± SD. **P < 0.01; n.s., not significant.