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: iScience

Article Title: Vinorelbine causes a neuropathic pain-like state in mice via STING and MNK1 signaling associated with type I interferon induction

doi: 10.1016/j.isci.2024.108808

Figure Lengend Snippet: Vinorelbine increases p -eIF4E in WT DRG neurons and sciatic nerve but not in Sting Gt/Gt and MNK1 KO mice (A) Representative confocal micrographs showing p -eIF4E (magenta) and peripherin (green) immunofluorescence in WT mouse DRG neurons at day 3 post-second administration of vinorelbine (10 mg/kg, i.v.) or vehicle (3% DMSO i.v.). DAPI (cyan) stains nuclei in the tissue. Respective bottom rows show zoomed-in images of a few neurons inside the white dashed-lined box. Scale bar – 10 μm. (B) Analysis of immunoreactivity of p -eIF4E across all neuronal sizes in vinorelbine- and vehicle-administered WT mice. The mean intensity values are plotted as a function of different neuronal sizes. (C) Mean intensity value of p -eIF4E in peripherin-positive neurons in vinorelbine- and vehicle-administered WT mice. Data are presented as mean ± SEM. Section thickness – 20 μm. Scale bar – 50 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (n = 3 per group) as determined by two-way ANOVA followed by Bonferroni’s multiple comparisons test in B. Data are presented as mean ± SEM ∗∗∗∗p < 0.0001 as determined by t test in C.

Article Snippet: Sections were washed in 0.1 M PB pH 7.4 and incubated for 1 h at room temperature with the corresponding secondary antibodies against p -IRF3 and p -eIF4E (0.2 μg/mL, Cat. #111-605-144, RRID: AB_2338078 , Jackson ImmunoResearch Laboratories Inc.) and peripherin (0.2 μg/mL, Cat. #A11039, RRID: AB_2534096 , Fisher Scientific).

Techniques: Immunofluorescence

Journal: iScience

Article Title: Vinorelbine causes a neuropathic pain-like state in mice via STING and MNK1 signaling associated with type I interferon induction

doi: 10.1016/j.isci.2024.108808

Figure Lengend Snippet: Vinorelbine enhances p -eIF4E in WT DRG and sciatic nerve but not in Sting Gt/Gt and MNK1 KO mice (A‒C) Western blot analysis of p -eIF4E in DRGs of WT (n = 8–10 per group), Sting Gt/Gt (n = 6 per group), and MNK1 KO (n = 10–12 per group) vinorelbine- or vehicle-administered mice. (D‒F) Western blot analysis of p -eIF4E in sciatic nerve of WT (n = 10 per group), Sting Gt/Gt (n = 3–4 per group), and MNK1 KO (n = 8–10 per group) vinorelbine- or vehicle-administered mice. (G) Representative western blot images showing p -eIF4E bands in DRGs of WT, Sting Gt/Gt , and MNK1 KO vinorelbine- or vehicle-administered mice. (H) Representative western blot images showing p -eIF4E mean intensity levels in sciatic nerve of WT, Sting Gt/Gt , and MNK1 KO vinorelbine- or vehicle-administered mice. Data are presented as mean ± SEM ∗p < 0.05 as determined by t test in D.

Article Snippet: Sections were washed in 0.1 M PB pH 7.4 and incubated for 1 h at room temperature with the corresponding secondary antibodies against p -IRF3 and p -eIF4E (0.2 μg/mL, Cat. #111-605-144, RRID: AB_2338078 , Jackson ImmunoResearch Laboratories Inc.) and peripherin (0.2 μg/mL, Cat. #A11039, RRID: AB_2534096 , Fisher Scientific).

Techniques: Western Blot

Journal: iScience

Article Title: Vinorelbine causes a neuropathic pain-like state in mice via STING and MNK1 signaling associated with type I interferon induction

doi: 10.1016/j.isci.2024.108808

Figure Lengend Snippet:

Article Snippet: Sections were washed in 0.1 M PB pH 7.4 and incubated for 1 h at room temperature with the corresponding secondary antibodies against p -IRF3 and p -eIF4E (0.2 μg/mL, Cat. #111-605-144, RRID: AB_2338078 , Jackson ImmunoResearch Laboratories Inc.) and peripherin (0.2 μg/mL, Cat. #A11039, RRID: AB_2534096 , Fisher Scientific).

Techniques: Recombinant, Software