Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

doi: 10.1210/me.2003-0478

Figure Lengend Snippet: LβT2 cells were serum starved overnight (A–C) and amino acid starved 1 h(B and C) before GnRH treatment at indicated doses and time points. Extracts were subjected to SDS-PAGE followed by Western blotting with the following antibodies. A, Anti-4E-BP1 reveals three electrophoretic forms (γ, β α) with the histogram representing the proportion of inactive γ-isoform relative to total 4E-BP1. B, Antiphospho-eIF4E (Ser 209) and eIF4E; histogram represents ratio of phospho-eIF4E relative to total eIF4E. C, Anti-phospho-eIF4G (Ser 1108); histogram represents values expressed as a percentage of maximal induction. Blots are representative images of each experiment. Histograms represent quantitative chemiluminescent image analysis of at least three independent experiments. The asterisks show significant difference from the control mean (P ≤ 0.05) as determined by ANOVA and post hoc Dunnett’s comparison to control test.

Article Snippet: 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Western Blot, Control, Comparison

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

doi: 10.1210/me.2003-0478

Figure Lengend Snippet: LβT2 cells were serum starved overnight and amino acid starved for 1 h followed with 10 nm GnRH for the times shown. Extracts underwent SDS-PAGE and immunoblotted with the indicated antiserum. A, Three electrophoretic forms (γ, β α) of 4E-BP1; the histogram depicts the proportion of inactive γ-isoform relative to total 4E-BP1. B, Histogram represents proportion of phospho-eIF4E relative to total eIF4E. C, Percent of maximal eIF4G phosphorylation. Blots are representative images. Histograms are the result of quantitative chemiluminescent imaging analysis of at least three separate experiments. The asterisks show significant difference from the control mean (P ≤ 0.05) as determined by ANOVA and post hoc Dunnett’s comparison to control test.

Article Snippet: 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Phospho-proteomics, Imaging, Control, Comparison

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

doi: 10.1210/me.2003-0478

Figure Lengend Snippet: LβT2 cells were serum starved overnight, followed by amino acid starvation for 1 h and subsequently pretreated with 10 µm PD98059, 1.0 µm LY294002, or 10 nm rapamycin for 30 min, after which cells were treated with 10 nm GnRH for 15 min. Extracts were separated by SDS-PAGE and immunoblotted with the indicated antiserum. A, Histogram represents activation of phospho-Mnk. B, Histogram represents ratio of phospho-eIF4E to total eIF4E normalized to control. Blots are representative images and histograms are the result of quantitative chemiluminescent imaging analysis of at least three separate experiments. The asterisks show significant difference from the control mean (P ≤ 0.05), as determined by ANOVA and post hoc Dunnett’s comparison to control test.

Article Snippet: 4E-BP1, phospho-eIF4E, and AKT antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) whereas phospho-eIF4G, -AKT, -Mnk, and -eIF4E antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Activation Assay, Control, Imaging, Comparison

Journal: iScience

Article Title: Alox15 via H 2 O 2 mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation

doi: 10.1016/j.isci.2026.114796

Figure Lengend Snippet: 12/15-LOX is required for TXA2-induced platelet activation and hemostasis (A–C) Eight-weeks-old WT and 12/15-LOX −/− mice were subjected to measurement of body weight (A), tail bleeding time (B), and whole blood clotting time (C) ( n = 10). (D) Platelet-rich plasma (PRP) from WT and 12/15-LOX −/− mice were incubated with and without F 2 -TXA2 (1 μM) for the indicated periods at RT and photographed. The percentage of clot retraction and extruded serum volume were calculated as described in the methods ( n = 3). (E) Wahed platelets were plated onto fibrinogen-coated coverslips and after 1 h stained with phalloidin and DAPI and observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6). The pictures were captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software ZEN 2.6. (F) Washed platelets from WT mice were labeled with calcein acetoxymethyl ester (10 μM) for 30 min and placed onto fibrinogen-coated wells in a 96-well plate. Platelets were then incubated with and without F 2 -TXA2 at the indicated concentrations for 30 min, washed with PBS and the bound platelets were lysed with lysis buffer and the fluorescence intensity was measured at 494 excitation and 517 emission ( n = 3). (G) PRP from WT mice treated with and without F 2 -TXA2 at the indicated concentrations was subjected to aggregation assay in an aggregometer ( n = 3). (H) Washed platelets from WT and 12/15-LOX −/− mice were subjected adhesion assay as shown in panel F ( n = 3). (I) PRP from WT and 12/15-LOX −/− mice with and without the indicated treatments were subjected to aggregation assay in an aggregometer ( n = 3). (J) Washed platelets from WT and 12/15-LOX −/− mice were incubated with and without F 2 -TXA2 for 30 min and plated onto fibrinogen-coated coverslips for 1 h. Platelets were then fixed, permeabilized, and stained with phalloidin to visualize F-actin, and pictures were captured. (K and L) Platelets from WT and 12/15-LOX −/− mice were incubated with and without F 2 -TXA2 (1 μM) for indicated time periods, and RNA and protein extracts were prepared and analyzed by qRT-PCR (K) and western blotting (L) for 12-LOX, 12/15-LOX and β-actin mRNA and protein levels using their specific primers or antibodies, respectively ( n = 3). (M) Platelets from WT and 12/15-LOX −/− mice were treated with and without F 2 -TXA2 for 30 min, and protein extracts were prepared and analyzed by western blotting for the levels of phospho and total eIF4E and 4EBP1 using their specific antibodies ( n = 3). (N) All the conditions were the same as in panel M except that the extracts were immunoprecipitated with anti-4EBP1 antibody, and the immunocomplexes were analyzed by western blotting for eIF4E and normalized for 4EBP1. The input protein was analyzed for β-actin levels. (O and P) Platelets from WT mice were incubated with and without F 2 -TXA2 in the presence and absence of rapamycin (100 nM) or torin1 (100 nM) for 30 min, and protein extracts were analyzed by western blotting for p4EBP1, 4EBP1, 12/15-LOX, and β-actin levels using their specific antibodies ( n = 3). (Q) Platelets from WT mice and 12/15-LOX −/− mice were assessed for 12(S)-HETE levels using a kit from Cayman ( n = 7). (R–W) Platelets from WT mice and 12/15-LOX −/− mice were treated with and without U46619 (1 μM) or ADP (40 μM) for 30 min and 12(S)-HETE levels were measured (R and U) ( n = 7) or subjected to adhesion assay (S and V) ( n = 3) or aggregation assay (T and W) ( n = 3). All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗ p < 0.01 versus WT mice or control; # p < 0.01 versus F 2 -TXA2 or WT + F 2 -TXA2 or U46619. Scale bars: 10 μm in (E) and (J).

Article Snippet: Anti-eIF4E antibody , Santa Cruz Biotechnology , sc-9976.

Techniques: Activation Assay, Coagulation, Clinical Proteomics, Incubation, Staining, Inverted Microscopy, Microscopy, Software, Labeling, Lysis, Fluorescence, Cell Adhesion Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Control

Journal: Scientific Reports

Article Title: The translation factor eIF4E is a key mediator of doxorubicin resistance: insights from a triple-negative breast cancer model

doi: 10.1038/s41598-025-31313-6

Figure Lengend Snippet: In the chemoresistant variant MDA R , eIF4E’s higher expression and activity are registered under high dox concentrations. A ) Characterization of targets eIF4E, eIF4E-p Ser−209, and p-4EBP1/2/3 on MDA N and MDA R cellular variants under increasing dox range (0–1.6.6 µM) treatment. Densitometry determinations of eIF4E ( B ) and eIF4E-p Ser−209 ( C ), respectively. D ) Detection of phosphorylation levels of eIF4E activity regulator 4E-BP1/2/3 under dox range concentration (0–1.6.6 µM); densitometry analysis is shown using GAPDH as the control. Three replicates data were statistically analyzed using one-way ANOVA, and Newman–Keuls’s multiple comparison test * represents a p-value < 0.05, **represents a p-value < 0.01, and *** represents a p-value < 0.001.

Article Snippet: Cells were seeded at a density of 7.5 × 10 4 cells/plates and incubated overnight in a standard growth medium without antibiotics. eIF4E siRNA (h) (sc-35284) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Variant Assay, Expressing, Activity Assay, Phospho-proteomics, Concentration Assay, Control, Comparison

Journal: Scientific Reports

Article Title: The translation factor eIF4E is a key mediator of doxorubicin resistance: insights from a triple-negative breast cancer model

doi: 10.1038/s41598-025-31313-6

Figure Lengend Snippet: eIF4E pathway, a critical factor for chemoresistance. MDA N and MDA R cells were stimulated with dox increasing concentrations (0–1.6.6 µM) at 48 h treatment; ( A ) Western blot analysis of the scaffold protein eIF4G and helicase protein eIF4A and their densitometric analysis ( B ) and ( C ) respectively. ( D )Western-blot of VEGF expression in both variants, GAPDH was used as a loading control. ( E ) Densitometry analysis of VEGF. ( F ) Metalloproteinase-9 activity evaluated by zymography and respective densitometric analysis ( G ); results showed the mean and standard deviation (X ± S.D.) of three biological replicates. To assess this process, MDA N control sample was used as control. ( H ) Representative images of cell invasion experiments in MDA N and MDA R cells under treatment with increasing dox concentrations (0–1.6.6 µM) and concomitant FBS (10%). ( I ) Densitometric analysis of invasion assays in the MDA N and MDA R variant. Results showed three biological replicates’ mean and standard deviation ( n = 3, X ± S.D.). To evaluate this process, DMEM plus 10% fetal bovine serum (FBS) was used as a positive control. Data were statistically analyzed using one-way ANOVA and Newman–Keuls’s multiple comparison test, * represents a p value < 0.05** represents a p value < 0.01.

Article Snippet: Cells were seeded at a density of 7.5 × 10 4 cells/plates and incubated overnight in a standard growth medium without antibiotics. eIF4E siRNA (h) (sc-35284) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Control, Activity Assay, Zymography, Standard Deviation, Variant Assay, Positive Control, Comparison

Journal: Scientific Reports

Article Title: The translation factor eIF4E is a key mediator of doxorubicin resistance: insights from a triple-negative breast cancer model

doi: 10.1038/s41598-025-31313-6

Figure Lengend Snippet: Doxorubicin chemoresistance is associated with the ABCB1 transporter. ( A ) Expression levels of ABCC1 in MDA N and MDA R cells under treatment with doxorubicin concentration range (0–1.6.6 µM) through qPCR determination; ( B ) under the same conditions, characterization of ABCB1 levels. In both cases, GAPDH was used as a reference gene, Results showed three biological replicates’ mean and standard deviation ( n = 3, X ± S.D.). Molecular docking assay of ABCB1 (PDB: 7A69) under dox interaction, an increment in the interaction region is shown ( C ), and prediction of ABCB1 residues determinants of doxorubicin interaction obtained by MOE program ( D ). (E) Overlay of the ligand (doxorubicin) in its initial (green) and final (purple) conformations within the transmembrane binding pocket after a 10 ns molecular dynamics simulation. (F) RMSD of the ligand over the simulation time (10 ns), calculated with respect to the initial docking pose. ( G ) Overall survival plot for low and high expression of the ABCB1 gene in BC patients from GEPIA. ( H ) The overall survival plot for low and high expression of eIF4E in breast cancer patients was obtained from the GEPIA database ( http://gepia2.cancer-pku.cn/#index ). Data were statistically analyzed using one-way ANOVA and Newman–Keuls’s multiple comparison test **represents a p value < 0.01 and *** p-value < 0.001.

Article Snippet: Cells were seeded at a density of 7.5 × 10 4 cells/plates and incubated overnight in a standard growth medium without antibiotics. eIF4E siRNA (h) (sc-35284) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Concentration Assay, Standard Deviation, Docking Assay, Binding Assay, Comparison

Journal: Scientific Reports

Article Title: The translation factor eIF4E is a key mediator of doxorubicin resistance: insights from a triple-negative breast cancer model

doi: 10.1038/s41598-025-31313-6

Figure Lengend Snippet: Nrf2 is associated with the eIF4E expression. ( A ) WB evaluated Nrf2 protein expression levels in MDA N and MDA R variants under increasing dox concentrations (0–1.6.6 µM); ( B ) Densitometry analysis of Nrf2. ( C) WB of control eIF4E expression using siRNA on MDA N cells with respective densitometric analysis ( D ). ( E ) MMP-9 activity was evaluated by zymography after treatment with siRNA eIF4E and densitometric analysis ( F ). ( G ) WB evaluated eIF4E, p-eIF4E, and Nrf2 expression under eIF4E siRNA and concomitant treatment with dox (1.6 µM). Densitometric analysis of eIF4E ( H ) and p-eIF4E ( I ). Results showed three biological replicates mean and standard deviation ( n = 3, X ± S.D.). Data were statistically analyzed using one-way ANOVA and Newman–Keuls’s multiple comparison test, *represents a p -value < 0.05, and **represents a p value < 0.01 concerning the control. GAPDH was used as a loading control for WB.

Article Snippet: Cells were seeded at a density of 7.5 × 10 4 cells/plates and incubated overnight in a standard growth medium without antibiotics. eIF4E siRNA (h) (sc-35284) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Control, Activity Assay, Zymography, Standard Deviation, Comparison

Journal: Scientific Reports

Article Title: The translation factor eIF4E is a key mediator of doxorubicin resistance: insights from a triple-negative breast cancer model

doi: 10.1038/s41598-025-31313-6

Figure Lengend Snippet: The eIF4E/ABCB1 axis in chemoresistance, the role of the 4E1RCat molecule. ( A ) Expression levels of ABCB1 in MDA N and MDA R cells under treatment with dox (1.6 µM) and 4E1RCat (3 µM) for 48 h through qPCR determination, GAPDH was used as a reference gene. ( B ) p-eIF4E expresión levels evaluated by WB under dox (1.6 µM) and 4E1RCat (3 µM) concomitant treatment, MDA N and MDA R variants were treated for 48 h. ( C ) Densitometry analysis of p-eIF4E; Results showed three biological replicates’ mean and standard deviation ( n = 3, X ± S.D.) and expressed as % of control; data were statistically analyzed using one-way ANOVA and Newman–Keuls’s multiple comparison test ** represents a p -value < 0.01 concerning the control. GAPDH was used as a control. ( D , E ) Molecular docking assay of eIF4E (PDB used 4UED) under 4E1RCat and concomitant interaction of 4E1RCat and 4EBP-1. Cyan: eIF4E, Green: Tryptophan, Red: 4E Binding Protein − 1.

Article Snippet: Cells were seeded at a density of 7.5 × 10 4 cells/plates and incubated overnight in a standard growth medium without antibiotics. eIF4E siRNA (h) (sc-35284) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Standard Deviation, Control, Comparison, Docking Assay, Binding Assay

Journal: Scientific Reports

Article Title: The translation factor eIF4E is a key mediator of doxorubicin resistance: insights from a triple-negative breast cancer model

doi: 10.1038/s41598-025-31313-6

Figure Lengend Snippet: Integrative model of the eIF4E–Nrf2–ABCB1 axis in doxorubicin resistance. Schematic summary of the proposed mechanism by which sustained eIF4E activation in MDA R cells promotes the selective translation of Nrf2 and ABCB1, enhancing antioxidant response and drug efflux. The convergence of translational control and stress-adaptive signaling reduces intracellular doxorubicin accumulation, thereby reinforcing the chemoresistant phenotype in triple-negative breast cancer cells.

Article Snippet: Cells were seeded at a density of 7.5 × 10 4 cells/plates and incubated overnight in a standard growth medium without antibiotics. eIF4E siRNA (h) (sc-35284) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Metabolic synthetic lethality by targeting NOP56 and mTOR in KRAS -mutant lung cancer

doi: 10.1186/s13046-022-02240-5

Figure Lengend Snippet: Synthetic lethality by targeting NOP56 and mTOR in KRAS -mutant lung cancer cells. A , Immunoblots of H358 cells expressing scramble control or NOP56 -specific shRNAs after treated with rapamycin (1 μM) for 24 h. B-G H358 cells expressing scramble control or sh NOP56 -specific shRNAs were transfected with control siRNAs or the indicated siRNAs specifically targeting S6, eIF4E, alone and in combination. The cells were then subjected to immunoblots (B, D, F) and viability assay (C, E, G) 72 h post-transfection. Data are presented as mean ± SD ( n = 3). H , H358 cells expressing scrambled control or NOP56 -specific shRNAs were transfected with IRE1α- specific or control siRNAs for 48 h, followed by treatment with rapamycin (1 μM) for 24 h before immunoblotting. I , H358 cells expressing control or NOP56- specific shRNAs were transfected with IRE1α- specific or control siRNAs for 24 h, followed by treatment with rapamycin (5 μM) for 72 h before apoptosis assay. Data are presented as mean ± SD ( n = 3). * p < 0.05, *** P < 0.001 and **** P < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. J , H358 cells expressing control or NOP56- specific shRNAs were preincubated overnight with vehicle (DMSO) or the JNK inhibitor SP600125, followed by treatment with rapamycin for 72 h before apoptosis assay. Data are presented as mean ± SD ( n = 3). ** p < 0.01, *** P < 0.001 and ns P >0.05 by two-way ANOVA with Tukey’s multiple comparisons test. K , Proposed model of cellular gauge for IRE1α-regulated UPR. In KRAS -mutant cancer cells, intact NOP56 keeps ROS in check so that IRE1α-regulated UPR is minimal (basal level; left). Intermediate levels of IRE1α-regulated UPR ensue from NOP56 depletion, which activates p38-AKT/mTOR and promotes cell survival (middle). At “dangerous” level of ROS, IRE1α-regulated UPR initiates JNK-dependent apoptosis (right)

Article Snippet: NOP56 (CAT#: SR307156), EIF4E (CAT#: SR320018), RPS6 (CAT#: SR304160) , RAPTOR (CAT#: SR324724) , and RICTOR (CAT#: SR326062) were knocked down by specific pooled siRNA duplexes purchased from OriGene Technologies, with control siRNA Duplex as a negative control.

Techniques: Mutagenesis, Western Blot, Expressing, Control, Transfection, Viability Assay, Apoptosis Assay