α eif3e  (Bethyl)

Journal: Cell reports

Article Title: eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules

doi: 10.1016/j.celrep.2025.116643

Figure Lengend Snippet: (A) Diagram of a simplified eIF3 complex with only eIF3d and eIF3e labeled for clarity. (B) Outline of the experimental design of Ribo-seq and RNA-seq. (C) siRNA knockdown (KD) of eIF3d and eIF3e in MCF7-SIX1 cells, followed by western blot analysis. (D) Venn diagram of the overlapping and distinct differentially translated mRNAs after eIF3d and eIF3e KD. p values were calculated with Fisher’s exact test. (E) Heatmap of specific mRNAs that are shared or specific to eIF3d vs. eIF3e. The values are Z scores of the TE. (F) Gene set enrichment analysis for the hypoxia hallmark dataset when eIF3e and eIF3d are knocked down.

Article Snippet: Western blots were performed on the resulting supernatants using the following antibodies: α-GAPDH (mouse, GeneTex GT239), α-eIF3a (rabbit, Bethyl A302–002A), α-eIF3b (rabbit, Bethyl A301–760A), α-eIF3c (rabbit, Bethyl A300377A), α-eIF3d (rabbit, Bethyl A301–758A), α-eIF3e (rabbit, Bethyl A302–984A), α-eIF3k (rabbit, Bethyl A301–762A), and α-eIF3l (rabbit, Bethyl A304–754A).

Techniques: Labeling, RNA Sequencing, Knockdown, Western Blot

Journal: Cell reports

Article Title: eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules

doi: 10.1016/j.celrep.2025.116643

Figure Lengend Snippet: (A and B) Western blot analysis of HIF1α after eIF3d and eIF3e knockdown (KD) in normoxic and hypoxic (4 h at 1% O2) conditions in (A) MCF7-SIX1 and (B) MDA-MB-231 cells. (C and D) Log 2 fold changes of RPFs ( y axis) and RNA levels ( x axis) comparing siCtrl cells in normoxia vs. hypoxia in (C) MCF7-SIX1 and (D) MDA-MB-231 cells. Only mRNAs with a statistically significant difference (adjusted p value [adj p ] < 0.05) are shown. (E–H) Comparison of TE changes from normoxia to hypoxia in (E) MCF7-SIX1 and (F) MDA-MB-231 siCtrl cells ( x axis) vs. si3e cells ( y axis) or (G and H) si3d cells ( y axis). Only mRNAs with a statistically significant TE difference (adj p < 0.05) are shown. (I and J) Venn diagram showing the overlap of mRNAs with statistically significant TE changes in MCF7-SIX1 and MDA-MB-231 for (I) eIF3e KD or (J) eIF3d KD. (K and L) Western blot analysis of high-confidence targets in (K) MCF7-SIX1 and (L) MDA-MB-231 cells.

Article Snippet: Western blots were performed on the resulting supernatants using the following antibodies: α-GAPDH (mouse, GeneTex GT239), α-eIF3a (rabbit, Bethyl A302–002A), α-eIF3b (rabbit, Bethyl A301–760A), α-eIF3c (rabbit, Bethyl A300377A), α-eIF3d (rabbit, Bethyl A301–758A), α-eIF3e (rabbit, Bethyl A302–984A), α-eIF3k (rabbit, Bethyl A301–762A), and α-eIF3l (rabbit, Bethyl A304–754A).

Techniques: Western Blot, Knockdown, Comparison

Journal: Cell reports

Article Title: eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules

doi: 10.1016/j.celrep.2025.116643

Figure Lengend Snippet: (A) Representative images of 231HFM cells grown in tumorspheres embedded in Matrigel and collagen I ± eIF3d and eIF3e KD. (B) Fluorescence ratio of GFP:dsRed over time ± eIF3d and eIF3e KD. (C) Invasive area of the 231 tumorspheres over time ± eIF3d and eIF3e KD. For (B) and (C), statistical significance was calculated using a longitudinal mixed-effects model in which si3e and si3d were both compared to siCtrl. Data are represented as the mean ± SEM from 6–9 replicates per time point and condition. (D) Density and contour plot of the normalized invasive area (x axis) and the GFP:dsRed ratio ( y axis) for siCtrl (left), si3e (middle), and si3d (right). The Spearman correlation coefficient and p value are denoted for each.

Article Snippet: Western blots were performed on the resulting supernatants using the following antibodies: α-GAPDH (mouse, GeneTex GT239), α-eIF3a (rabbit, Bethyl A302–002A), α-eIF3b (rabbit, Bethyl A301–760A), α-eIF3c (rabbit, Bethyl A300377A), α-eIF3d (rabbit, Bethyl A301–758A), α-eIF3e (rabbit, Bethyl A302–984A), α-eIF3k (rabbit, Bethyl A301–762A), and α-eIF3l (rabbit, Bethyl A304–754A).

Techniques: Fluorescence

Journal: Cell reports

Article Title: eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules

doi: 10.1016/j.celrep.2025.116643

Figure Lengend Snippet: (A and B) Overall survival rates of METABRIC patients ( n = 1980) with breast tumor (A) eIF3e gains/amplifications or (B) eIF3d gains/amplifications compared to no copy-number alterations (CNAs) in the METABRIC dataset. (C and D) Overall survival of patients in the METABRIC datasets stratified by (C) eIF3e and (D) eIF3d RNA-seq signatures using the intersection of targets in both MCF7-SIX1 and MDA-MB-231 cells. For clarity, only the first and fourth quartiles are shown. (E and F) Overall survival rates of patients in the METABRIC dataset stratified by (E) the combination of hypoxia signature and eIF3e or (F) eIF3d enrichment/depletion. The p values and hazard ratios were calculated using a Cox proportional hazards regression where each group was compared to the control group (e.g., no CNAs for eIF3e, heterozygous (het) CNA loss for eIF3d, and bottom 25% for signatures).

Article Snippet: Western blots were performed on the resulting supernatants using the following antibodies: α-GAPDH (mouse, GeneTex GT239), α-eIF3a (rabbit, Bethyl A302–002A), α-eIF3b (rabbit, Bethyl A301–760A), α-eIF3c (rabbit, Bethyl A300377A), α-eIF3d (rabbit, Bethyl A301–758A), α-eIF3e (rabbit, Bethyl A302–984A), α-eIF3k (rabbit, Bethyl A301–762A), and α-eIF3l (rabbit, Bethyl A304–754A).

Techniques: RNA Sequencing, Control

Journal: Cell reports

Article Title: eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules

doi: 10.1016/j.celrep.2025.116643

Figure Lengend Snippet: (A) Volcano plot of the isothermal shift assay (ITSA) with log2 fold change ( x axis) and −log10 p value ( y axis). (B) Cellular thermal shift assay (CETSA) followed by western blot showing protein levels of eIF3e and multiple other eIF3 subunits in addition to the loading control of GAPDH. (C and D) Dose response of 8430 for an isothermal dose-response fingerprint (ITDRF) assay. (E and F) Dose response of 209 for an ITDRF assay. For (D) and (F), data are represented as the mean ± SD from 3 replicates.

Article Snippet: Western blots were performed on the resulting supernatants using the following antibodies: α-GAPDH (mouse, GeneTex GT239), α-eIF3a (rabbit, Bethyl A302–002A), α-eIF3b (rabbit, Bethyl A301–760A), α-eIF3c (rabbit, Bethyl A300377A), α-eIF3d (rabbit, Bethyl A301–758A), α-eIF3e (rabbit, Bethyl A302–984A), α-eIF3k (rabbit, Bethyl A301–762A), and α-eIF3l (rabbit, Bethyl A304–754A).

Techniques: Shift Assay, Thermal Shift Assay, Western Blot, Control

Journal: Cell reports

Article Title: eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules

doi: 10.1016/j.celrep.2025.116643

Figure Lengend Snippet: (A and B) Western blot analysis showing HIF1α after 8430 and 209 treatment (24 h pre-treatment) in normoxic and hypoxic (4 h at 1%O2) conditions for (A) MCF7-SIX1 and (B) HEK293T cells. (C) Log 2 fold change of RPFs ( y axis) and RNA levels ( x axis) comparing DMSO treatment in normoxia vs. hypoxia in MCF7-SIX1 cells. Each dot represents a mRNA transcript. (D) Comparison of significant TE changes from normoxia to hypoxia in DMSO-treated cells ( x axis) to TE changes from normoxia to hypoxia after treatment with a209 ( y axis). (E) Barplot of the number of differentially translated mRNAs with 209 treatment in normoxia and hypoxia. (F and G) Barplot of the odds ratio for the overlap of mRNA with TE changes in a209 treatment compared to those with TE changes in eIF3e or eIF3d KD in (F) normoxia and (G) hypoxia. p values were calculated using Fisher’s exact test.

Article Snippet: Western blots were performed on the resulting supernatants using the following antibodies: α-GAPDH (mouse, GeneTex GT239), α-eIF3a (rabbit, Bethyl A302–002A), α-eIF3b (rabbit, Bethyl A301–760A), α-eIF3c (rabbit, Bethyl A300377A), α-eIF3d (rabbit, Bethyl A301–758A), α-eIF3e (rabbit, Bethyl A302–984A), α-eIF3k (rabbit, Bethyl A301–762A), and α-eIF3l (rabbit, Bethyl A304–754A).

Techniques: Western Blot, Comparison

Journal: PLoS Pathogens

Article Title: Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

doi: 10.1371/journal.ppat.1005982

Figure Lengend Snippet: (A) Immune fluorescence images of mock-treated an MERS-CoV infected Vero cells. Cells were infected with an MOI of 1 and fixed using 3% paraformaldehyde in PBS at 10h or 24h post infection. Cells were stained for dsRNA, and stress granule markers eIF3 and G3BP2. (B) Immune fluorescence images of cells treated with arsenic acid (0.5 mM for 60 min) or transfected with poly(I:C) and stained for eIF3, G3BP1 and G3BP2.

Article Snippet: Cells were permeabilized with PBS + 0.2% Triton X-100, washed trice with blocking buffer (PBS + 2% bovine serum albumin [BSA] + 50mM NH 4 Cl), and incubated with blocking buffer for 1 h. Cell monolayers were incubated for 1 h with primary antibody mouse-α-G3BP1 (BD, 1:1,000), rabbit-α-TIA1 (Santa-Cruz, 1:50), mouse-α-dsRNA (J2, English&Scientific Consulting, 1:1,000), goat-α-eIF3 (Santa-Cruz, 1:100), rabbit-α-G3BP2 (Bethyl Laboratory, 1:200; or Assay Biotech, 1:500), or rabbit-α-MERS-CoV (1:500: raised against the MERS-CoV M carboxyl terminal peptide CRYKAGNYRSPPITADIELALLRA), and then for 30 min with secondary antibody donkey-α-mouse-Cy3 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Alexa488 (Jackson ImmunoResearch, 1:1000), bovine-α-goat-Alexa647 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Cy5 (Jackson ImmunoResearch, 1:200), donkey-α-mouse-Alexa 488 (Invitrogen, 1:200) or donkey-α-goat-Alexa 594 (Invitrogen, 1:200) and Hoechst-33258 (1:2,000) diluted in blocking buffer.

Techniques: Fluorescence, Infection, Staining, Transfection

Journal: PLoS Pathogens

Article Title: Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

doi: 10.1371/journal.ppat.1005982

Figure Lengend Snippet: (A, B) Immune fluorescence images of HeLa-wt cells (A) and HeLa-PKR KO cells (B) transfected with the indicated pEGFP-expression plasmids. Next day, SG formation was triggered using arsenic acid (0.5 mM for 30 min). Cells were fixed and stained for eIF3 (shown in red) or G3BP2 (shown in cyan). EGFP expression is shown in green. (C, D) Quantification of SG-positive HeLa-wt cells (C) and HeLa-PKR KO cells (D) treated with Pateamine A (100 nM for 2h), arsenic acid (0.5 mM for 30 min), or heat shock (50°C for 30 min). SG-positive cells were quantified from three randomly selected images. Shown are means with standard deviations, which were analyzed using an unpaired t-test. (*, p<0.05; ns, not significant).

Article Snippet: Cells were permeabilized with PBS + 0.2% Triton X-100, washed trice with blocking buffer (PBS + 2% bovine serum albumin [BSA] + 50mM NH 4 Cl), and incubated with blocking buffer for 1 h. Cell monolayers were incubated for 1 h with primary antibody mouse-α-G3BP1 (BD, 1:1,000), rabbit-α-TIA1 (Santa-Cruz, 1:50), mouse-α-dsRNA (J2, English&Scientific Consulting, 1:1,000), goat-α-eIF3 (Santa-Cruz, 1:100), rabbit-α-G3BP2 (Bethyl Laboratory, 1:200; or Assay Biotech, 1:500), or rabbit-α-MERS-CoV (1:500: raised against the MERS-CoV M carboxyl terminal peptide CRYKAGNYRSPPITADIELALLRA), and then for 30 min with secondary antibody donkey-α-mouse-Cy3 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Alexa488 (Jackson ImmunoResearch, 1:1000), bovine-α-goat-Alexa647 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Cy5 (Jackson ImmunoResearch, 1:200), donkey-α-mouse-Alexa 488 (Invitrogen, 1:200) or donkey-α-goat-Alexa 594 (Invitrogen, 1:200) and Hoechst-33258 (1:2,000) diluted in blocking buffer.

Techniques: Fluorescence, Transfection, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

doi: 10.1371/journal.ppat.1005982

Figure Lengend Snippet: (A) Immune fluorescence images of HeLa-wt cells that were mock-treated or infected with wt mengovirus or the indicated recombinant mengoviruses (MOI = 10). Cells were fixed at 6h post infection and stained for dsRNA (shown in green), eIF3 (shown in red), and G3BP1 (shown in cyan). Nuclei were stained using Hoechst-33258 (shown in blue). (B) SG-positive cells were quantified from three randomly selected images. Shown are means with standard deviations, analyzed using an unpaired t-test (***, p<0.001; ns, not significant). (C) Western blot analysis of PKR and phospho-PKR in cells infected with indicated viruses. Capsid staining was used as a control for virus replication efficiency and tubulin staining was used as loading control.

Article Snippet: Cells were permeabilized with PBS + 0.2% Triton X-100, washed trice with blocking buffer (PBS + 2% bovine serum albumin [BSA] + 50mM NH 4 Cl), and incubated with blocking buffer for 1 h. Cell monolayers were incubated for 1 h with primary antibody mouse-α-G3BP1 (BD, 1:1,000), rabbit-α-TIA1 (Santa-Cruz, 1:50), mouse-α-dsRNA (J2, English&Scientific Consulting, 1:1,000), goat-α-eIF3 (Santa-Cruz, 1:100), rabbit-α-G3BP2 (Bethyl Laboratory, 1:200; or Assay Biotech, 1:500), or rabbit-α-MERS-CoV (1:500: raised against the MERS-CoV M carboxyl terminal peptide CRYKAGNYRSPPITADIELALLRA), and then for 30 min with secondary antibody donkey-α-mouse-Cy3 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Alexa488 (Jackson ImmunoResearch, 1:1000), bovine-α-goat-Alexa647 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Cy5 (Jackson ImmunoResearch, 1:200), donkey-α-mouse-Alexa 488 (Invitrogen, 1:200) or donkey-α-goat-Alexa 594 (Invitrogen, 1:200) and Hoechst-33258 (1:2,000) diluted in blocking buffer.

Techniques: Fluorescence, Infection, Recombinant, Staining, Western Blot

Journal: PLoS Pathogens

Article Title: Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

doi: 10.1371/journal.ppat.1005982

Figure Lengend Snippet: (A) Immune fluorescence images of HeLa-wt and HeLa-PKR KO cells infected with EMCV-L-Zn (MOI = 10). Cells were fixed at 6h post infection and SG formation was visualized using antibodies directed against G3BP1 (shown in green) and eIF3 (shown in red). Nuclei were stained using Hoechst-33258 (shown in blue). (B) In parallel with A , RNA was isolated at 8h post infection and relative IFNβ mRNA levels were quantified by RT-PCR. Means and standard deviations of triplicate measurements are shown. (C) Virus production after wt and recombinant mengovirus infection (MOI = 0.01) in HeLa and HeLa-PKR KO cells. Supernatant was collected 24h post infection and virus progeny was titrated by end-point dilution with 3-fold dilution steps. (D) Schematic representation of the virus competition assay. Briefly, two viruses are mixed 1:1 and used to infection HeLa-wt or HeLa-PKR KO cells. Progeny virus was collected 48h post infection and viral RNA was isolated. RT-PCR was used to amplify the MERS-CoV 4a insert, which was analyzed using agarose gel electrophoresis. (E, F, G) Agarose gel analysis of the 4a insert region from virus competition assays with the indicated viruses. To distinguish between wild-type and mutant 4a genes, 4a-wt specific HindIII digestion was used.

Article Snippet: Cells were permeabilized with PBS + 0.2% Triton X-100, washed trice with blocking buffer (PBS + 2% bovine serum albumin [BSA] + 50mM NH 4 Cl), and incubated with blocking buffer for 1 h. Cell monolayers were incubated for 1 h with primary antibody mouse-α-G3BP1 (BD, 1:1,000), rabbit-α-TIA1 (Santa-Cruz, 1:50), mouse-α-dsRNA (J2, English&Scientific Consulting, 1:1,000), goat-α-eIF3 (Santa-Cruz, 1:100), rabbit-α-G3BP2 (Bethyl Laboratory, 1:200; or Assay Biotech, 1:500), or rabbit-α-MERS-CoV (1:500: raised against the MERS-CoV M carboxyl terminal peptide CRYKAGNYRSPPITADIELALLRA), and then for 30 min with secondary antibody donkey-α-mouse-Cy3 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Alexa488 (Jackson ImmunoResearch, 1:1000), bovine-α-goat-Alexa647 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Cy5 (Jackson ImmunoResearch, 1:200), donkey-α-mouse-Alexa 488 (Invitrogen, 1:200) or donkey-α-goat-Alexa 594 (Invitrogen, 1:200) and Hoechst-33258 (1:2,000) diluted in blocking buffer.

Techniques: Fluorescence, Infection, Staining, Isolation, Reverse Transcription Polymerase Chain Reaction, Recombinant, Competitive Binding Assay, Agarose Gel Electrophoresis, Mutagenesis

Journal: PLoS Pathogens

Article Title: Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

doi: 10.1371/journal.ppat.1005982

Figure Lengend Snippet: (A, B) Vero cells were infected (MOI = 1) with MERS-CoV wt or MERS-CoVΔORF4. At 16h p.i., cells were (A) mock treated, or (B) treated with 0.5 mM arsenic acid for 1h. Subsequently, MERS-CoV infection and SG formation were visualized by IFA using antibodies directed against MERS-CoV M, G3BP1, and eIF3, respectively. (C) Huh7 cells were transfected with poly(I:C), or infected (MOI = 1) with the indicated viruses. RT-qPCR was used to quantify relative IFNβ mRNA levels at the indicated time points. Shown are means and standard deviations of the relative IFNβ mRNA levels compared to mock treated cells.

Article Snippet: Cells were permeabilized with PBS + 0.2% Triton X-100, washed trice with blocking buffer (PBS + 2% bovine serum albumin [BSA] + 50mM NH 4 Cl), and incubated with blocking buffer for 1 h. Cell monolayers were incubated for 1 h with primary antibody mouse-α-G3BP1 (BD, 1:1,000), rabbit-α-TIA1 (Santa-Cruz, 1:50), mouse-α-dsRNA (J2, English&Scientific Consulting, 1:1,000), goat-α-eIF3 (Santa-Cruz, 1:100), rabbit-α-G3BP2 (Bethyl Laboratory, 1:200; or Assay Biotech, 1:500), or rabbit-α-MERS-CoV (1:500: raised against the MERS-CoV M carboxyl terminal peptide CRYKAGNYRSPPITADIELALLRA), and then for 30 min with secondary antibody donkey-α-mouse-Cy3 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Alexa488 (Jackson ImmunoResearch, 1:1000), bovine-α-goat-Alexa647 (Jackson ImmunoResearch, 1:1000), donkey-α-rabbit-Cy5 (Jackson ImmunoResearch, 1:200), donkey-α-mouse-Alexa 488 (Invitrogen, 1:200) or donkey-α-goat-Alexa 594 (Invitrogen, 1:200) and Hoechst-33258 (1:2,000) diluted in blocking buffer.

Techniques: Infection, Transfection, Quantitative RT-PCR