In thapsigargin-treated cells, protein synthesis was reduced in the first hour of treatment and rapidly recovered (Fig. 3B ) [54] . Poly I:C, however, nearly completely inhibited translation despite active eIF2a dephosphorylation. This was particularly obvious when poly I:C was co-administrated together with thapsigargin. Indeed, poly I:C dominated the response by preventing the translation recovery normally observed after few hours of drug treatment (Fig. 3B) . Surprisingly, in absence of functional GADD34, although eIF2a phosphorylation induction by poly I:C was augmented dramatically, no further decrease in protein synthesis was observed upon treatment of GADD34 DC/DC cells with the dsRNA mimic ( Fig. 3A and 3C ). The functionality of GADD34 in translation restoration was, however, fully demonstrated, when the same cells were treated with thapsigargin, and protein synthesis was completely inhibited by this treatment [52] control. Quantification of puromycin signal was quantified with ImageJ software and is represented above the immunoblot. Phosphorylation of eIF2a (P-eIF2a) was assessed in the same MEFs extracts. B) Immunofluorescence staining for puromycin, P-eIF2a and dsRNA of MEFs treated with poly I:C for 4 h and labeled with puromycin for 1 h. Scale bar, 10 mm. C) WT and PKR 2/2 MEFs were stimulated for 8 h with poly I:C (pI:C), thapsigargin (th) or arsenite (as). PKR and P-eIF2a were detected by immunoblot. D) WT and PKR 2/2 MEFs were stimulated for 8 h with poly I:C and protein synthesis was monitored like in (A). b-actin immunoblot is shown for equal loading control. E) IFN-b levels were measured, by ELISA, in cell culture supernatants of WT, PKR 2/2 , eIF2aA/A and control eIF2aS/S MEFs after 4 and 8 h of poly I:C stimulation. Data are mean 6 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-b 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 6 standard deviation of 4 independent experiments. doi:10.1371/journal.ppat.1002708.g001 (Fig. 3C) . Thus, cytosolic dsRNA delivery induces a type of protein synthesis inhibition, which requires eIF2a phosphorylation for its initiation, but conversely cannot be reverted by GADD34 induction and subsequent GADD34-dependent eIF2a dephosphorylation.