Journal: Nature communications

Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function.

doi: 10.1038/s41467-024-54356-1

Figure Lengend Snippet: Fig. 4 | Compound 4 inhibits eIF4G:eIF4E binding and cap-dependent transla- tion in cell lysate assays. a Lysates from SW620 cells were incubated with 1–100 µM compound 4 or 5 or positive control peptide (RIIY) for 30 min. Endo- genous eIF4E was immunoprecipitated and immunoblotted for eIF4G, 4E-BP1 and eIF4E. Quantitation of 4E-BP1 (b) or eIF4G (c) with endogenous eIF4E in SW620 and HeLa cell lysates, determined by the electro-chemiluminescent binding assay fol- lowing incubation for 30 min with DMSO vehicle (Cont), 100 µM compound 4 or 100 µM RIIY peptide. Complexes were immobilised by an eIF4E antibody and captured eIF4E, eIF4G and 4E-BP1 detected by their respective secondary anti- bodies. Values represent ratios of 4E-BP1:eIF4E or eIF4G:eIF4E electro- chemiluminescence relative to DMSO control (n = 2 biological replicates). d Electro-chemiluminescent assay for binding of eIF4G or 4E-BP1 with eIF4E in SW620 (n = 2 biological replicates), or (e) in HeLa lysates (n = 3 biological replicates, mean ± SD) following incubation for 30 min with compound 4 or 5. Results are expressed as luminescence signals relative to DMSO control. f Quantification of eIF4E:eIF4G interaction in H1299 cells by electro-chemiluminescent assay. Cell lysates treated with RIIY 4E-BP1 derived peptide or RIIG negative control peptide at 0.1–100 µM for 30 min (n = 2 biological replicates). g Quantification of the endo- genous eIF4E:eIF4G interaction in H1299 cell lysates at 0.1–100 µM (for 6 h) of compound 4 or 5, as measured by electro-chemiluminescent assay (mean ± SD from n = 3 biological replicates). h HeLa cell lysates for in vitro translation were incubated for 30 min with 1, 10, 100 µM of compound 4 or 5. Results are expressed as firefly or renilla luminescence signal normalized to DMSO control and expressed as % (mean ± SD from n = 3 biological replicates). Significance was determined using two-sided unpaired t-test comparing compound 4 to compound 5 at each concentration. Statistically significant p-values (p < 0.05) are shown on the plot and source data is located in the Source Data file.

Article Snippet: Blots were probed at 4 °C overnight for eIF4E (Cell Signaling, Cat#2067, RRID:AB_2097675), MCL1 (Cell Signaling, cat#39224, RRID:AB_2799149), monoclonal ANTI-FLAG® M2 (Sigma-Aldrich, cat#F1804, RRID:AB_262044), and vinculin (Cell Signaling, cat#13901, RRID:AB_2728768).

Techniques: Binding Assay, Incubation, Positive Control, Immunoprecipitation, Quantitation Assay, Control, Derivative Assay, Negative Control, In Vitro, Concentration Assay

Journal: bioRxiv

Article Title: DDX3 regulates the cap-independent translation of the Japanese encephalitis virus via its interactions with PABP1 and the untranslated regions of the viral genome

doi: 10.1101/2024.10.15.618401

Figure Lengend Snippet: (A-D) BHK-21 cells were respectively transfected with 100 pmol of a mixture of eIF4E-specific siRNAs (Table supplement 3) or treated with 20 μM 4E2RCat for 12 hours, and then infected with JEV at an MOI of 0.1. At different time points of post-infection, the cells were labeled with puromycin for 30 min and harvested to analyze puromycin incorporation (A and C). The viral titers in culture supernatants of BHK-21 cells treated with eIF4E-specific siRNAs (B) or 4E2RCat (D) were measured by TCID 50 assay (n=3). (E) Schematic diagram of the virus rescue using JEV RNA transcripts modified with three different 5′ termini: m7G(5′)ppp(5′)A, G(5′)ppp(5′)A and ppp(5′)A. (F) Cytopathic effects and immunofluorescence assay of cells transfected with full-length viral RNA transcripts with different 5′ termini. (G) The viral titers in culture supernatants of cells transfected with 2μg RNA transcripts at 24, 36 and 48hpt (n=3). *, p<0.05; **, p<0.01; ns, no statistical difference. Data are presented as mean ± standard deviation (SD) of three independent experiments and tested by Student’s t -test (B, D, and G).

Article Snippet: Mouse anti-GST (AE001), rabbit anti-Myc (AE009), rabbit anti-eIF4E (A2162), rabbit anti-Phospho-eIF4E (AP0747), rabbit anti-Phospho-eIF2α (A0764), rabbit anti-eIF4A (A24293), rabbit anti-eIF2A (A9709), rabbit anti-eIF3A(A0573), rabbit anti-eIF1A (A20954) antibodies were purchased from ABclonal Technology.

Techniques: Transfection, Infection, Labeling, Virus, Modification, Immunofluorescence, Standard Deviation

Journal: bioRxiv

Article Title: DDX3 regulates the cap-independent translation of the Japanese encephalitis virus via its interactions with PABP1 and the untranslated regions of the viral genome

doi: 10.1101/2024.10.15.618401

Figure Lengend Snippet: (A and B) The firefly luciferase activity assay of monocistronic RNA reporters with 5’terminal modified by either m 7 G(5′)ppp(5′)A or ppp(5′) in BHK-21 cells treated with eIF4E-specific siRNAs or 4E2RCat (n=4). (C-F) BHK-21 cells were respectively treated with eIF4E-specific siRNAs (100pmol) or 4E2RCat (20μM) and then transfected with JEV genomic RNA of WT or ΔDB2 mutant with a type I cap structure. At the indicated time points of post-transfection, NS1′ protein was detected by immunoblotting (C and E) and viral titers in culture supernatants were measured by TCID 50 assays (D and F) (n=3). (G-J) BHK-21 cells were respectively treated with eIF4E-specific siRNAs or 4E2RCat and then infected with rGI and rGI-ΔDB2 at an MOI of 0.05. At the indicated time points of post-infection, viral titers in culture supernatants were measured by TCID 50 assays (G and I) (n=3), and NS1′ protein was detected by immunoblotting (H and J). (K and L) The survival rate of mice (n=10) intraperitoneally mock-infected or infected with the indicated JEV at doses of 10 3 and 10 5 TCID 50 . The significant differences were determined by the Kaplan-Meier analysis. (M) Histopathological analysis of brain lesions of the dead mice. *, p<0.05; **, p<0.01; ***, p<0.001. Data are presented as mean ± SD and tested by one-way ANOVA (A-B, D, F, G and I).

Article Snippet: Mouse anti-GST (AE001), rabbit anti-Myc (AE009), rabbit anti-eIF4E (A2162), rabbit anti-Phospho-eIF4E (AP0747), rabbit anti-Phospho-eIF2α (A0764), rabbit anti-eIF4A (A24293), rabbit anti-eIF2A (A9709), rabbit anti-eIF3A(A0573), rabbit anti-eIF1A (A20954) antibodies were purchased from ABclonal Technology.

Techniques: Luciferase, Activity Assay, Modification, Transfection, Mutagenesis, Western Blot, Infection