eif3  (Bethyl)

Journal: Cellular & Molecular Biology Letters

Article Title: The promoting roles of GLP1R and GIPR in stemness maintenance and multiple lineage-specific differentiation of PDLSCs

doi: 10.1186/s11658-026-00867-2

Figure Lengend Snippet: Translation shut off by upregulated IFIT proteins. a Heatmap showing the differentially expressed genes (|log 2 FC|> 1, P < 0.05) compared between GLP1R knockdown, GLP1RA, 2GRA, and NC among the multiple lineage differentiation system of PDLSCs. b–e Dumbbell plots illustrating the changes in gene expression of IFIT1 , IFIT2 , IFIT3 , IRF9 , IFI44 , IFI6 , IFIH1 , OAS1 , OASL , RSAD2 , and BST2 between GLP1R knockdown, GLP1RA, 2GRA, and NC in osteogenesis ( b ), adipogenesis ( c ), chondrogenesis ( d ), and neurogenesis ( e ) from RNA sequencing data. f Western blot assay showing the expression of classic markers of osteogenesis COL1A1, BGLAP, and RUNX2 in PDLSCs for osteogenic differentiation system treated with GLP1R knockdown, GLP1RA, and 2GRA. g Western blot assay showing the expression of classic markers of adipogenesis CEBPA, FABP4, and PPARG in PDLSCs for adipogenic differentiation system treated with GLP1R knockdown, GLP1RA, and 2GRA. h Western blot assay show the expression of classic markers of chondrogenesis ACAN, COL2A1, and SOX9 in PDLSCs for chondrogenic differentiation system treated with GLP1R knockdown, GLP1RA, and 2GRA. i Western blot assay show the expression of classic markers of neurogenesis MAP2, NES, and TUBB3 in PDLSCs for adipogenic differentiation system treated with GLP1R knockdown, GLP1RA, and 2GRA. j Western blot assay showing the interaction of IFIT1 with IFIT2, IFIT3, eIF3C, and eIF3E within the immunoprecipitation of IFIT1 from PDLSCs under undifferentiated, osteogenic, GLP1R knockdown, GLP1RA, and 2GRA treated conditions. k Western blot assay show the interaction of eIF3C with IFIT1, IFIT2, IFIT3, eIF3E, and RPS3 within the immunoprecipitation of eIF3C from PDLSCs under undifferentiated, osteogenic, GLP1R knockdown, GLP1RA, and 2GRA treated conditions. Red boxes highlight the robust protein interaction in undifferentiated PDLSCs and osteogenic PDLSCs with GLP1R knockdown. KD, knockdown; NC, PDLSCs for regular differentiation accordingly; Un, undifferentiated PDLSCs; OS, osteogenesis; 2GRA, GLP1R/GIPR agonist-1

Article Snippet: The information and the concentration of antibodies used in this study were listed as follows: Phospho-(Ser/Thr) PKA substrate antibody (1: 500, cat. no. 9621, CST, USA), GLP1R (1: 2,000, cat. no. 26196-1-AP, Proteintech, China), GIPR (1: 2,000, cat. no. 28322-1-AP, Proteintech), STRO-1 (1: 250, cat. no. 39-8401, Thermo Fisher Scientific), CD146 (1: 2,000, cat. no. 65181-1-Ig, Proteintech), Vimentin (1: 2,000, cat. no. 10366-1-AP, Proteintech), CREB1 (1: 2,000, cat. no. 67927-1-Ig, Proteintech), p-CREB1 (Ser133) (1: 2,000, cat. no. 28792-1-AP, Proteintech), ERK1/2 (1: 2,000, cat. no. 66192-1-Ig, Proteintech), p-ERK1/2 (Thr202/Tyr204) (1: 2,000, cat. no. 28733-1-AP, Proteintech), β-catenin (1: 2,000, cat. no. 51067-2-AP, Proteintech), p-β-catenin (Ser33) (1: 2,000, cat. no. 80067-1-RR, Proteintech), STAT3 (1: 2,000, cat. no. 10253-2-AP, Proteintech), p-STAT3 (Ser727) (1: 2,000, cat. no. 80199-2-RR, Proteintech), Ki-67 (1: 2,000, cat. no. 84192-4-RR, Proteintech), PCNA (1: 2,000, cat. no. 10205-2-AP, Proteintech), CCND3 (1: 2,000, cat. no. 26755-1-AP, Proteintech), β-actin (1: 2,000, cat. no. 66009-1- Ig, Proteintech), COL1A1 (1: 2,000, cat. no. 67288-1-Ig, Proteintech), OCN (1: 2,000, cat. no. 20277-1-AP, Proteintech), RUNX2 (1: 2,000, cat. no. 20700-1-AP, Proteintech), C/EBPα (1: 2,000, cat. no. 29388-1-AP, Proteintech), FABP4 (1: 2,000, cat. no. 12802-1-AP, Proteintech), PPARγ (1: 2,000, cat. no. 16643-1-AP, Proteintech), ACAN (1: 2,000, cat. no. 68350-1-Ig, Proteintech), COL2A1 (1: 2,000, cat. no. 28459-1-AP, Proteintech), SOX9 (1: 2,000, cat. no. 67439-1-Ig, Proteintech), MAP2 (1: 2,000, cat. no. 17490-1-AP, Proteintech), Nestin (1: 2,000, cat. no. 19483-1-AP, Proteintech), TUBB3 (1: 2,000, cat. no. 66375-1-Ig, Proteintech), IFIT1 (1: 2,000, cat. no. ab305301, Abcam), IFIT2 (1: 2,000, cat. no. 12604-1-AP, Proteintech), IFIT3 (1: 2,000, cat. no. 15201-1-AP, Proteintech), eIF3C (1: 2,000, cat. no. 12733-1-AP, Proteintech), eIF3E (1: 2,000, cat. no. 10899-1-AP, Proteintech), RPS3 (1: 2,000, cat. no. 66046-1-Ig, Proteintech), SPP1 (1: 2,000, cat. no. 22952-1-AP, Proteintech), FGF18 (1: 2,000, cat. no. 11495-1-AP, Proteintech), MMP14 (1: 2,000, cat. no. 14552-1-AP, Proteintech), BMP2 (1: 2,000, cat. no. 66383-1-Ig, Proteintech), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1: 5,000, cat. no. SA00001-2, Proteintech), and HRP-conjugated goat anti-mouse IgG (1: 5,000, cat. no. SA00001-1, Proteintech).

Techniques: Knockdown, Gene Expression, RNA Sequencing, Western Blot, Expressing, Immunoprecipitation

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