Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: pSer65 -4EBP1 is abundantly expressed in ccRCC tumor and co-localizes with tumor necrosis factor receptor (TNFR) 2. Representative confocal images of single-immunofluorescence staining of ccRCC tumor grades (G1 to G4) and corresponding nontumor tissue (NK). A: Top panels: Staining for total 4EBP1 (t4EBP1). A marked signal was detected in NK, confined to normal tubular cells (t), some isolated cells within glomeruli (Glom), and the interstitium ( red arrowheads ). A similar pattern and intensity were found in tumor cells in all ccRCC grades. Bottom panels: Staining for pSer65 -4EBP1. Signal was undetectable in NK and infrequently present in G1 ccRCC tumor cells but accumulated with increasing intensity in ccRCC according to malignant grade (G2 to G4). Signal was mainly cytoplasmic but also found infrequently in the nucleus of some tumor cells. Inset: low-power image; arrowhead indicates the panel labeled super-res shows a high-power image of 4E-BP1 staining in the nuclei taken by super-resolution microscopy. B: Signal intensity per cell was quantified as corrected total cell fluorescence (CTCF). Two-way analysis of variance was performed. C: Immunoblot of lysates from three independent ccRCC G4 tumor resections (T) alongside corresponding nontumor kidney (N) from the same tissue, showing an increased abundance of pSer65 -4EBP1 in all three tumors compared with normal tissue. β-Actin and Ponceau S stain show protein loading. Bar graph shows the relative intensity of pSer65 -4EBP1 band normalized to N for each pair. D: Representative confocal images of ccRCC G4 staining showing co-localization of signal for pSer65 -4EBP1 and TNFR2 in tumor cells and in infiltrating cells within the interstitium ( inset ; arrowheads ). Data are expressed as means ± SD ( B ). n = 3 per group with similar results ( A , B , and D ). ∗∗∗ P < 0.001 versus NK and G1; ∗∗∗∗ P < 0.0001 versus NK, G1, and G2. Scale bars: 50 μm ( A and D , main images ); 25 μm ( D , inset ). Original magnification: ×40 ( A ); ×63 ( A , super-res image , and D , main images and inset ). NS, not significant.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Immunofluorescence, Staining, Isolation, Labeling, Super-Resolution Microscopy, Fluorescence, Western Blot

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Activation of tumor necrosis factor receptor (TNFR) 2 by TNFR2-specific mutein (R2TNF) induces expression of pSer65 -4EBP1 and its co-localization with TNFR2 in tumor cells in organ cultures of ccRCC. A: Representative confocal images of combined immunofluorescence in low-grade ccRCC organ culture sections stained for total (t) 4EBP1. A similar signal intensity was found in tumor cells (identified using the tumor marker RCC-MA) in control [untreated (UT)] and R2TNF-treated cultures. B: In comparison, R2TNF induced expression of pSer65 -4EBP1 in tumor cells with a weak infrequent signal in controls. A signal for pSer65 -4EBP1 is also seen in endothelial cells in blood vessels (Bv; white arrows ), negative for RCC-MA. Numbers indicate percentage co-localization in tumor cells. C: pSer65 -4EBP1 and TNFR2 co-localization in tumor cells, in vascular endothelial cells (Bv; white arrow s ), and in infiltrating cells ( yellow arrowheads ). Inset : Cosignal in a bilobed tumor cell in another area on the same section. Data are given as means ± SD ( A and B ). n = 3 per group with similar results ( A and B ). Scale bars: 50 μm ( A – C, main image s ); 10 μm ( C , inset ). Original magnification: ×40 ( A – C , main images ); ×63 ( C , inset ).

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Activation Assay, Expressing, Immunofluorescence, Organ Culture, Staining, Marker, Control, Comparison

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Tumor necrosis factor receptor (TNFR) 2–specific mutein (R2TNF) induces co-localization of pSer65 -4EBP1 and TNFR2 in mitochondria (m). Representative panels of high-power confocal images of combined immunofluorescence (IF). A and B: pSer65 -4EBP1 and heat shock protein (HSP) 60 (mitochondrial matrix protein; A ) or TNFR2 and HSP60 ( B ) in low-grade ccRCC organ culture sections. Images show R2TNF-induced co-localization of the two proteins in mitochondria in tumor cells ( arrowheads ). C and D: Super-resolution images ( super-res) of tumor cells show mitochondrial co-localization of the two proteins ( white arrowheads ; C ), confirmed by triple IF ( D ), which shows pSer65 -4EBP1/TNFR2/HSP60 + ( arrows ) within a tumor cell ( white dotted lines ). E: Representative immunogold electron micrographs show presence of pSer65 -4EBP1 and TNFR2 (15-nm colloidal gold) in mitochondria, labeled with HSP60 (5-nm colloidal gold) on R2TNF-treated low-grade ccRCC organ sections ( white arrows ). F: The close proximity of pSer65 -4EBP1 ( red arrows ) and TNFR2 ( blue arrows ) in mitochondria and the presence of TNFR2 on the cell surface compared with untreated (UT) cultures, which show a rare signal. n = 3 per group with similar results ( A – F ). Scale bars: 25 μm ( A and B ); 75 μm ( C and D ); 200 nm ( E and F, left and right panels ); 500 nm ( F , middle panel ). Original magnifications: ×40 ( A and B ); ×63 ( C and D ). N, nucleus.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Immunofluorescence, Organ Culture, Labeling

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Tumor necrosis factor receptor (TNFR) 2–specific mutein (R2TNF) induces co-localization of pSer65 -4EBP1 and TNFR2 with cytochrome c oxidase (Cox) subunits. A: Representative confocal images of combined immunofluorescence of pSer65 -4EBP1 or TNFR2 with Cox1, Cox4, or Cox5b in low-grade ccRCC organ culture sections show R2TNF-induced cosignal of Ser65 -4EBP1 or TNFR2 and Cox1, Cox4, and Cox5. B: High-power images show R2TNF-induced cosignal of Ser65 -4EBP1 or TNFR2 and Cox1. C: Quantification of signal intensity of R2TNF-induced cosignal compared with untreated (UT) presented as corrected total cell fluorescence (CTCF). Two-way analysis of variance was performed. Data are given as means ± SD ( C ). n = 3 per group with similar results ( C ). ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 versus controls. Scale bars: 50 μm ( A ); 25 μm ( B , top panels ); 10 μm ( B , bottom panels ). Original magnification: ×40 ( A ); ×63 ( B ).

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Immunofluorescence, Organ Culture, Fluorescence

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: Tumor necrosis factor receptor 2–specific mutein (R2TNF)–induced cell proliferation and pSer65 -4EBP1 expression in tumor cells are reduced by mammalian target of rapamycin (mTOR) inhibitors. A: Representative confocal images of combined immunofluorescence for pSer65 -4EBP1 (green) and proliferative marker phosphorylated-histone S10 (pH3 S10 ; red) in low-grade ccRCC organ culture either left untreated (UT) or treated with R2TNF for 3 hours at 37°C show a significant increase in proliferative tumor cells (pH3 S10+ ) also positive for pSer65 -4EBP1 in R2TNF-treated cultures compared with UT. B: Similar cultures pretreated with mTOR inhibitors Torin 2, Ku63794, or rapamycin for 1 hour before R2TNF addition show a significant reduction in pSer65 -4EBP1 + /pH3 S10+ tumor cells, more pronounced in cultures pretreated with Torin 2 and Ku63794 than rapamycin. C: Quantification of tumor cell proliferation as a proliferative index (percentage positive of total cells) in UT or R2TNF-treated organ cultures of ccRCC (RCCoC) versus staining in normal tubular cells in organ cultures of nontumor tissue (NKoC) and in the absence or presence of the mTOR inhibitors. D: Quantification of double-stained pSer65 -4EBP1 + /pH3 S10 positive tumor cells in UT and R2TNF-treated cultures ± mTOR inhibitors. Two-way analysis of variance was performed. Data are given as means ± SD ( C and D ). n = 3 per group with similar results ( C and D ). ∗ P < 0.05, ∗∗∗ P < 0.001 versus R2TNF; ∗∗∗∗ P < 0.0001 versus UT. Scale bars: 50 μm ( A ); 75 μm ( B ). Original magnifications: ×2.3 ( B ); ×40 ( C ). NS, not significant.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Expressing, Immunofluorescence, Marker, Organ Culture, Staining

Journal: The American Journal of Pathology

Article Title: Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation

doi: 10.1016/j.ajpath.2024.02.019

Figure Lengend Snippet: A model of tumor necrosis factor receptor (TNFR) 2–driven cell cycle entry in tumor cells in ccRCC. Selective ligation of TNFR2 by TNFR2-specific mutein (R2TNF) induces an increased expression of TNFR2 and phosphorylation of 4EBP1 at serine 65 ( pSer65 -4EBP1) and facilitates co-localization of the two proteins in cytoplasm and mitochondria alongside increased expression of nuclear- and mitochondrial-encoded cytochrome c oxidase (COX) subunits. pSer65 -4EBP1 is also detected in the nucleus in some tumor cells. Mammalian target of rapamycin (mTOR) inhibitors cause a significant reduction in R2TNF-mediated cell cycle activation and pSer65 -4EBP1 expression, accompanied by an increased cell death with the relative potency of Torin 2 > Ku63794 > rapamycin. mTORC, mTOR complex.

Article Snippet: Rabbit anti–phosphorylated 4EBP1 S65 , AP0032 , ABclonal (Woburn, MA) , IF.

Techniques: Ligation, Expressing, Phospho-proteomics, Activation Assay

Journal: Cancer Cell International

Article Title: HBx mutant-regulated RPL13AP25 mediates suboptimal virological response to entecavir and HCC progression

doi: 10.1186/s12935-025-03873-0

Figure Lengend Snippet: Hyperphosphorylation of eIF4EBP1 is enhanced by overexpression of HBx-H94Y/K130M and RPL13AP25. ( A ) Western blot used to show the expression of proteins, as indicated, in HepG2.2.15 cells were transfected with vector control (vc) as indicated, HBx-wt or HBx-H94Y/K130M. Phosphorylated eIF4EBP1 at residues T37/46, S65 and T70, total eIF4EBP1, and HBx (detected using Flag antibody) are shown. GAPDH served as loading control. * P < 0.05, ** P < 0.01. ( B ) Target genes in HepG2.2.15 and J7 cells were infected with HBV and overexpressed RPL13AP25 were determined using western blot analysis. Phosphorylated eIF4EBP1 (T37/46, S65, T70) and total eIF4EBP1 were shown. GAPDH served as a loading control. * P < 0.05, ** P < 0.01. ( C ) Target genes in RPL13AP25-depleted HepG2.2.15 and J7/HBV cells were detected using western blot analysis. NC denotes the negative control siRNA. GAPDH served as loading control. * P < 0.05, ** P < 0.01. ( D ) Immunohistochemical staining of phosphorylated eIF4EBP1 (S65 and T70) and H&E staining of tumor sections were shown. Results show that tumors overexpressing RPL13AP25 exhibit increased phosphorylated eIF4EBP1. Scale bar, 50 μm. * P < 0.05. ( E ) The expression levels of RPL13AP25 and phosphorylation of eIF4EBP1 in HBV-HCC patients with HBx-wt ( n = 3) and HBx-H94Y/K130M ( n = 6) were determined using qRT-PCR and western blot analysis, respectively. 18 S rRNA and GAPDH were used as loading control. * P < 0.05. ( F ) The migration and invasion assay in eIF4E-silenced cell lines was analyzed by Transwell assay. Migratory cells were assessed using crystal violet staining, quantified by ImageJ software. Scale bar, 100 μm. ** P < 0.01

Article Snippet: In this study, the following antibodies were used: p-eIF4EBP1 (T37/46) (Abclonal, AP1381), p-eIF4EBP1 (S65) (Abclonal, AP0032), p-eIF4EBP1 (T70) (Abclonal, AP0031), eIF4EBP1 (Abclonal, A1248), Flag (Merck, Rockville, MD, USA, F1804) and GAPDH (Merck, G8795).

Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Infection, Negative Control, Immunohistochemical staining, Staining, Phospho-proteomics, Quantitative RT-PCR, Migration, Invasion Assay, Transwell Assay, Software

Journal: Frontiers in Neuroscience

Article Title: 4E-BP1 expression in embryonic postmitotic neurons mitigates mTORC1-induced cortical malformations and behavioral seizure severity but does not prevent epilepsy in mice

doi: 10.3389/fnins.2023.1257056

Figure Lengend Snippet: DNA plasmid information.

Article Snippet: pCAG-4E-BP1 CA (4E-BP1 F113A ) , 3.0 , Addgene #81122; the F113A mutation resists phosphorylation by mTORC1 and constitutively binds to eIF4E ( ; ) .

Techniques: Plasmid Preparation, Concentration Assay, Mutagenesis, Phospho-proteomics, Generated

Journal: Frontiers in Physiology

Article Title: A Systems Biology Approach to Deciphering the Etiology of Steatosis Employing Patient-Derived Dermal Fibroblasts and iPS Cells

doi: 10.3389/fphys.2012.00339

Figure Lengend Snippet: List of the pre-validated antibodies used in reverse phase protein array (RPPA) .

Article Snippet: 4EBP1 , , NB200157 , Novus Biologicals.

Techniques: Protein Array, Phospho-proteomics