Figure 1. Translation inhibition and IFN-β production are induced by poly I:C in MEFs.

A) Protein synthesis was monitored in poly I:C(pI:C)-stimulated MEFs using puromycin labelling followed by immunoblot with the anti-puromycin mAb 12D10. Controls are cells not treated with puromycin (No puro) and cells treated with cycloheximide (chx) 5 min prior puromycin incorporation. β-actin immunoblot is shown for equal loading control. Quantification of puromycin signal was quantified with ImageJ software and is represented above the immunoblot. Phosphorylation of eIF2α (P-eIF2α) was assessed in the same MEFs extracts. B) Immunofluorescence staining for puromycin, P-eIF2α and dsRNA of MEFs treated with poly I:C for 4 h and labeled with puromycin for 1 h. Scale bar, 10 µm. C) WT and PKR^−/− MEFs were stimulated for 8 h with poly I:C (pI:C), thapsigargin (th) or arsenite (as). PKR and P-eIF2α were detected by immunoblot. D) WT and PKR^−/− MEFs were stimulated for 8 h with poly I:C and protein synthesis was monitored like in (A). β-actin immunoblot is shown for equal loading control. E) IFN-β levels were measured, by ELISA, in cell culture supernatants of WT, PKR^−/−, eIF2αA/A and control eIF2αS/S MEFs after 4 and 8 h of poly I:C stimulation. Data are mean ± standard deviation of 3 independent experiments. F) Protein synthesis was measured in NIH3T3 cells by puromycin incorporation after 7 h of poly I:C treatment. Where indicated, a chase of 1 h with fresh media was performed prior to puromycin labeling and immunoblotting. Samples with cycloheximide (chx) and arsenite (as) added respectively 5 min and 30 min before the puromycin pulse are shown as controls. G) IFN-β was quantified by ELISA in culture supernatants in the conditions described above after 7 h of poly I:C stimulation or 7 h of poly I:C stimulation followed by 1 h with fresh media (chase). Data are mean ± standard deviation of 4 independent experiments.