We monitored protein synthesis in MEFs and NIH-3T3 cells after poly I:C stimulation, using puromycin labeling followed by immunodetection with the anti-puromycin mAb 12D10 [47] . Poly I:C delivery to MEFs and NIH-3T3, rapidly and durably inhibited protein synthesis, concomitant with increased eIF2a phosphorylation (P-eIF2a) ( Fig. 1A and Fig. S1A ). In MEFs, a strong eIF2a phosphorylation was observed after 4 h of poly I:C treatment, followed by a steady dephosphorylation at later times (Fig. 1A) . Protein synthesis arrest was confirmed in individual cells by concomitant imaging of poly I:C delivery, mRNA translation and P-eIF2a ( Fig. 1B and Fig. S1B ), and with a wide range of dsRNA concentrations (Fig. S1C ). Poly I:C-induced eIF2a phosphorylation and subsequent translation arrest were not observed in PKRdeficient MEFs ( Fig. 1C and 1D ), while eIF2a phosphorylation induced by the UPR-inducing drug thapsigargin (th) (an inhibitor of SERCA ATPases) or arsenite (as) was unchanged in PKR2/2 cells (Fig. 1C) . PKR is therefore necessary to induce protein synthesis inhibition in response to cytosolic poly I:C.