Fig 2: IFN Signaling Antagonizes RIPK in the Activation of eIF4E- and mTORC1-Mediated Translation(A) TNF-a and CXCL-1 protein levels after indicated stimulations ± treatment with eIF4E inhibitor 4EGi-1 in B6, Ifnb-/-, and RIP1 Ki Ifnb-/- BMDMs.(B) p-eIF4E and total 4E-BP levels in B6, Ifnb-/-, and RIP1 Ki BMDMs stimulated as indicated ± 5 IU IFNß priming overnight.(C) TNF-a and CXCL-1 protein levels after indicated stimulations ± treatment with mTORC1 inhibitor Torin 2 in B6, Ifnb-/-, and RIP1 Ki Ifnb-/- BMDMs.(D and E) Cell death as measured by propidium iodide incorporation over 6 h in B6, Ifnb-/-, and RIP1 Ki Ifnb-/- BMDMs stimulated with LZ ± treatment with 4EGi-1 (D) or Torin 2 (E).(F) TNF-a and CXCL-1 protein levels after indicated stimulations ± overnight treatment with IFNAR-blocking antibody (aIFNAR) in Akt1-/- and wild-type (WT) littermate BMDMs.In all panels, BMDMs were stimulated with LPS, LZ, LZNs, Z, Ns, or LZN as indicated. ELISA and qPCR data are shown as ±SD from three independent experiments compared using two-way ANOVA: n.s. (p > 0.05), *p < 0.05, and ****p < 0.0001. Western blot and kinetic cell death experiments are representative of three or more independent experiments. Kinetic data are presented as the mean ± SD of triplicate wells.See also Figures S4 and S5.

Fig 3: Gene expression analysis of eIF4E1 and eIF4E3 translatome.(a) Venn diagram illustrating the distribution of gene targets, which are either mutually or exclusively regulated by eIF4E1 and eIF4E3. (b,c) Heatmap depicting 50 top gene targets regulated by eIF4E1 (b) and eIF4E3 (c). Values depicted are Z-ratios of expression fold change in the translatome (TL) after eliminating changes in transcriptome (TR) in comparison with vector expressing HLY-1 cells that is, TL-TR data sets. Z-ratio values are as depicted in the colour scale shown. (d) Principal component analysis showing data distribution of each data subset that is, eIF4E1-translatome (E1TL, blue), eIF4E3-translatome (E3TL, green), empty vector-translatome (EVTL, brown), eIF4E1-transcriptome (E1TR, red), eIF4E3-transcriptome (E3TR, purple) and empty vector-transcriptome (EVTR, turquoise). (e) IPA core molecular network analysis of significantly upregulated genes in eIF4E1 translatome exhibiting molecular network interactions that focused on NF-?B complex as a major node. (f) IPA core analysis of significantly upregulated genes in eIF4E3 translatome displayed molecular network interactions that showed several diffused nodes leading to downregulation of DICER1 and upregulation of n-Myc. Details of all the bioinformatics analyses performed and the statistical tests used are described in the methods section of Microarray analysis. (g) Western blot showing the protein expression of DICER1, n-Myc and c-Myc in vector control, eIF4E1 and eIF4E3 expressing HLY-1 cells. (h) Western blot showing three NF-?B targets, YY1, BTK and CDK6 in HLY-1 cells either overexpressing eIF4E1 or treated with eIF4E1 shRNA to knockdown eIF4E1 expression. Full immunoblots are shown in Supplementary Fig. 10.

Fig 4: Suppression of cellular translation is attributable to HIV-1-dependent dephosphorylation of eIF4E and 4E-BP1.(A) HEK 293 cells were transfected with HIV-1NL4-3 or ?VifVprX (?VV) for 24 or 48 hour, as indicated. Equivalent whole-cell extracts were immunoblotted with antiserum against phospho-eIF2a Ser51, total eIF2a, phospho-eIF4E Ser209 or total eIF4E, respectively. (B) CEMx174 cells were infected with HIV-1 or ?VifVprX (?VV) and evaluated at 12 hour intervals. Equivalent whole-cell extracts were immunoblotted with the indicated antiserum.

Fig 5: Analysis of 4E-BP2 immunoprecipitates.(A) Samples of cerebral cortex, C, or CA1 from control (SHC3d) and ischemic animals with long-term reperfusion (R3d), were immunoprecipitated with anti-4E-BP2 antibody and analyzed by western blotting with: anti-eIF4E antibody and anti-mouse IRDye 680LT-conjugated as secondary antibody (in red); and anti-4E-BP2 antibody and anti-rabbit IRDye 800CW-conjugated (in green). Bar graph shows the quantification of eIF4E in the immunoprecipitates; no significant differences in the 4E-BP2 levels were found (p = 0.520). (B) 4E-BP2 immunoprecipitates of cerebral cortex from R3d and SHC3d control, were subjected to 2-DGE and western blotting (wb) with anti-4E-BP2 antibody (in grey); or were labeled with Cy3- and Cy5-dye, subjected to 2-DGE and after analyzed with an fluorescence imager (Cy images) and by western blotting with anti-4E-BP2 antibody as in (A) (wb, in green). The right numbers represent the apparent MW in kDa from protein markers.