Extended Data Fig. 1: STING inhibits cap-dependent translation through the PERK-eIF2α axis and is independent of the UPR.
(A) Expression of STING but not MAVS inhibited new protein synthesis, as determined by immunoblotting for puromycin in HEK293 cells. The graph shows the relative protein synthesis rate normalized to that of GAPDH. n = 3 independent experiments (mean ± SEM), and P values were indicated by one-way ANOVA with the Bonferroni correction. (B) Global protein synthesis was substantially attenuated by inducing expression of a constitutively active form of STING (R281Q) in DLD1 cells via the Tet-On system, as indicated by the declined levels of puromycin. (C) Translation arrest induced by the ER and oxidative stress was typical in STING-deficient HCT116 cells. (D) Ectopic expression of both NvSTING and DrSTING distinctly induced eIF2α but not IRF3 phosphorylation in HEK293 cells. (E) Phosphorylation of endogenous eIF2α in HEK293 cells was induced by ectopic expression of hSTING, NvSTING, or DmSTING. (F-G) Immunofluorescence imaging revealed that eIF2α was mainly localized in the cytoplasm and partially associated with the ER, which was not aggregated upon STING expression. Scale bars=20 μm. (H) eIF2α phosphorylation, which STING triggered, was declined explicitly in the presence of a PERK inhibitor but not an ER stress inhibitor (TUDCA), TBK1 inhibitor (BX795), or mTOR inhibitor (rapamycin) in HCT116 cells. (I) Expression of STING led to eIF2α phosphorylation in HCT116 cells, which was abolished by PERK inhibitors (iPERK-1 and iPERK-2) but not NF-κB inhibitors (TPCA-1 and JSH-23). (J) Ectopic expression of STING R331A/R334A failed to induce the phosphorylation of both eIF2α and IRF3 in HEK293 cells. (K) Immunofluorescence imaging revealed the partial accumulation of ATF6, an indication of ATF6 activation, by the treatment of UPR activator but nut STING agonist. Scale bars=10 μm. (L) Genetic ablation of STING expression did not affect the activation of UPR signalling, as evidenced by the similar levels of phospho-PERK, phospho-eIF2α, and phospho-IRE1α in WT and STING KO MEFs upon DTT treatment. Data shown in A-L represent three independent experiments.
