Journal: International Journal of Molecular Sciences

Article Title: eIF4E-Dependent Translation Potentially Regulates Apoptosis and BDNF/TrkB Signaling in the Medial Prefrontal Cortex During Morphine-Induced CPP

doi: 10.3390/ijms27115097

Figure Lengend Snippet: 4EGI-1 aggravates morphine-induced apoptotic injury and attenuates BDNF and TrkB expression in N2a cells. ( a ) Schematic illustration of the experimental design. N2a cells were treated with 4EGI-1 (50 μM), morphine (1000 μM), or their combination, followed by apoptosis, cell viability assays and Western blotting. ( b ) Representative DAPI staining images showing that morphine induced apoptotic nuclear changes in N2a cells, which were further aggravated by cotreatment with 4EGI-1. Blue fluorescence indicates DAPI-stained nuclei, and white arrows indicate nuclei with apoptotic morphology. Scale bar = 50 μm. ( c ) Cell viability analysis showing that 4EGI-1 alone did not significantly affect cell viability but further enhanced the morphine-induced reduction in viability. n = 3, * p < 0.05, **** p < 0.0001. ( d ) Representative Western blots of Bax, Bcl-2 and cleaved caspase-3 in N2a cells under the indicated treatments. ( e ) Quantitative analysis showing that morphine increased Bax and cleaved caspase-3 expression and decreased Bcl-2 expression, while 4EGI-1 pretreatment further enhanced Bax and cleaved caspase-3 expression. n = 4, * p < 0.05, ** p < 0.01, ns, not significant. ( f ) Representative Western blots of total TrkB, p-TrkB and BDNF in N2a cells under the indicated treatments. ( g ) Quantification showing that morphine increased total TrkB, p-TrkB and BDNF expression, whereas 4EGI-1 pretreatment attenuated these morphine-induced changes. n = 4, * p < 0.05, ** p < 0.01.

Article Snippet: 4EGI-1 (HY-19831, 10 mM in DMSO, MedChemExpress, Monmouth Junction, NJ, USA) was prepared freshly before each experiment and diluted to a working concentration of 80 μM according to previous studies [ ].

Techniques: Expressing, Western Blot, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: eIF4E-Dependent Translation Potentially Regulates Apoptosis and BDNF/TrkB Signaling in the Medial Prefrontal Cortex During Morphine-Induced CPP

doi: 10.3390/ijms27115097

Figure Lengend Snippet: Injection of 4EGI-1 into the mPFC inhibited morphine-induced conditioned place preference without affecting motor activity. ( a ) Schematic illustration of the experimental design. Mice underwent habituation and pre-test, followed by conditioning and post-test. 4EGI-1 was microinjected into the mPFC before each morphine conditioning session during training. ( b ) Morphine significantly increased CPP score, whereas intra-mPFC administration of 4EGI-1 before morphine conditioning significantly attenuated this effect. n = 8, **** p < 0.0001 compared to the pre-test, ^^^^ p < 0.0001 compared to the Veh + Saline, #### p < 0.0001 compared to the Veh + Morphine. Veh, vehicle. ( c ) Representative heatmaps showing intra-mPFC administration of 4EGI-1 reduced occupancy in the morphine-paired chamber during the post-test. ( d ) Quantification of total distance traveled, mean speed and entries into each chamber during the pre-test and post-test phases, showing no significant differences among groups. n = 8, ns, not significant.

Article Snippet: 4EGI-1 (HY-19831, 10 mM in DMSO, MedChemExpress, Monmouth Junction, NJ, USA) was prepared freshly before each experiment and diluted to a working concentration of 80 μM according to previous studies [ ].

Techniques: Injection, Conditioned Place Preference, Activity Assay, Saline

Journal: International Journal of Molecular Sciences

Article Title: eIF4E-Dependent Translation Potentially Regulates Apoptosis and BDNF/TrkB Signaling in the Medial Prefrontal Cortex During Morphine-Induced CPP

doi: 10.3390/ijms27115097

Figure Lengend Snippet: Injection of 4EGI-1 into the mPFC does not produce detectable changes in anxiety-like behavior or general cognitive performance in mice. ( a ) Left: Representative movement trajectories of OFT; right: quantitative analysis. Compared with the vehicle group, mice in the 4EGI-1 group showed no significant differences in either the distance traveled or the time spent in the center and peripheral zones. n = 12, ns, not significant. ( b ) Left: Representative movement trajectories of EPM; right: quantitative analysis. Compared with the vehicle group, the 4EGI-1 group showed no significant differences in either the time spent in each arm or the number of entries into each arm. n = 12, ns, not significant. ( c ) Left: Representative movement trajectories of YMT; right: quantitative analysis. No significant differences were observed between the 4EGI-1 and vehicle groups in either the time spent in the novel arm or the spontaneous alternation rate. n = 12, ns, not significant. ( d ) Representative exploration trajectories from the NOR test are presented on the left, while the quantitative analysis of the recognition index is shown on the right. No significant differences were observed between the 4EGI-1 and vehicle groups in either test 1 or test 2. n = 12, ns, not significant.

Article Snippet: 4EGI-1 (HY-19831, 10 mM in DMSO, MedChemExpress, Monmouth Junction, NJ, USA) was prepared freshly before each experiment and diluted to a working concentration of 80 μM according to previous studies [ ].

Techniques: Injection

Journal: Chemico-biological interactions

Article Title: Hexavalent chromium promotes malignant transformation via enhanced translation of SUV39H1

doi: 10.1016/j.cbi.2025.111869

Figure Lengend Snippet: (A and B) Cr(VI)-transformed BEAS-2BR cells were treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO control for 24 h. Cell lysates were subjected to immunoblotting for the indicated proteins (A) or immunoprecipitated with anti-eIF4E antibodies followed by immunoblotting (B). (C) Cap-dependent translation activity was measured in Cr(VI)-transformed BEAS-2BR cells treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO for 24 h. Results are expressed as percentage change relative to DMSO control. (D) Representative images of anchorage-dependent colony formation in Cr(VI)-transformed BEAS-2BR cells treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO. (E and F) Representative images (E) and quantification (F) of anchorage-independent colony formation in soft agar by Cr(VI)-transformed BEAS-2BR cells treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO control. Scale bar, 200 μm. (G and H) Representative images (G) and quantification (H) of sphere formation in suspension culture by Cr(VI)-transformed BEAS-2BR cells treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO. Results are expressed as percentage change relative to DMSO control. Scale bar, 400 μm. All graphical data are presented as mean ± SD (n = 3 replicates) from one representative of three (C) or two (F and H) independent experiments. ***p < 0.001, by Student’s t test.

Article Snippet: Rapamycin and INK128 were purchased from Selleckchem and MedChemExpress, respectively, and potassium dichromate and 4EGI-1 were obtained from Sigma.

Techniques: Inhibition, Transformation Assay, Control, Western Blot, Immunoprecipitation, Activity Assay, Suspension

Journal: Chemico-biological interactions

Article Title: Hexavalent chromium promotes malignant transformation via enhanced translation of SUV39H1

doi: 10.1016/j.cbi.2025.111869

Figure Lengend Snippet: (A and B) Cr(VI)-transformed BEAS-2BR cells were pretreated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO control for 30 min, followed by exposure to 2.5 μM Cr(VI) (K 2 Cr 2 O 7 ) for 12 h. One set of cells was fixed immediately for immunofluorescence staining of γH2AX (red) and DAPI (blue) (“Rest 0 h”), while the other set was washed with PBS and incubated for an additional 36 h in Cr(VI)-free medium with continued inhibitor treatment before staining (“Rest 36 h”). Quantification shows the percentage of γH2AX-positive nuclei relative to total DAPI-stained nuclei per field (B). Scale bar, 50 μm. (C-F) BEAS-2BR cells stably expressing raptor shRNA_1, raptor shRNA_2, or control shRNA (C and D), as well as cells stably expressing HA-4E-BP1-WT, HA-4E-BP1–4A, or vector control (E and F), were chronically exposed to 0.125 μM K 2 Cr 2 O 7 for 25 weeks, then treated with 2.5 μM K 2 Cr 2 O 7 for 12 h. One set of cells was fixed immediately for immunofluorescence staining of γH2AX (red) and DAPI (blue) (“Rest 0 h”), while the other set was washed with PBS and incubated for an additional 36 h in Cr(VI)-free medium before staining (“Rest 36 h”). Quantification shows the percentage of γH2AX-positive nuclei (D and F). Scale bars, 50 μm. All graphical data are presented as mean ± SD (n = 3 replicates) from one representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t test; ns, not significant, by one-way ANOVA.

Article Snippet: Rapamycin and INK128 were purchased from Selleckchem and MedChemExpress, respectively, and potassium dichromate and 4EGI-1 were obtained from Sigma.

Techniques: Inhibition, Transformation Assay, Control, Immunofluorescence, Staining, Incubation, Stable Transfection, Expressing, shRNA, Plasmid Preparation

Journal: Chemico-biological interactions

Article Title: Hexavalent chromium promotes malignant transformation via enhanced translation of SUV39H1

doi: 10.1016/j.cbi.2025.111869

Figure Lengend Snippet: (A) Quantitative PCR analysis of SUV39H1 mRNA levels in Cr(VI)-transformed BEAS-2BR cells relative to passage-matched control cells. (B) Quantitative PCR analysis of SUV39H1 mRNA levels in monosomal and polysomal fractions as indicated in Fig. 1B . (C) Cap-dependent SUV39H1 translation activity was measured in Cr(VI)-transformed and control BEAS-2BR cells. Results are expressed as fold change relative to control cells. (D-F) Cr(VI)-transformed BEAS-2BR cells were treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO control for 24 h, followed by cap-dependent SUV39H1 translation assay (D), immunoblotting for the indicated proteins (E), and qPCR analysis of SUV39H1 mRNA levels (F). Results are expressed as percentage (D) or fold (F) change relative to DMSO control. (G-J) Cr(VI)-transformed BEAS-2BR cells stably expressing raptor shRNA_1, raptor shRNA_2, or control shRNA (G and H), as well as cells stably expressing HA-4E-BP1-WT, HA-4E-BP1–4A, or vector control (I and J), were subjected to cap-dependent SUV39H1 translation assay (G and I) or immunoblotting for the indicated proteins (H and J). Results are expressed as percentage change relative to control shRNA (G) or vector control (I). (K) Immunoblot analysis of Cr(VI)-transformed BEAS-2BR cells stably expressing 4E-BP1 shRNA_1, 4E-BP1 shRNA_2 (two distinct targeting sequences) or control shRNA for the indicated proteins. (L and M) Cr(VI)-transformed BEAS-2BR cells stably expressing 4E-BP1 shRNA_1, 4E-BP1 shRNA_2, or control shRNA were treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO control for 24 h, followed by cap-dependent SUV39H1 translation assay (L) and immunoblotting for the indicated proteins (M). Results are expressed as percentage change relative to control shRNA cells treated with DMSO. All graphical data are presented as mean ± SD (n = 3 replicates) from one representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t test; ns, not significant, by Student’s t test or one-way ANOVA as appropriate.

Article Snippet: Rapamycin and INK128 were purchased from Selleckchem and MedChemExpress, respectively, and potassium dichromate and 4EGI-1 were obtained from Sigma.

Techniques: Real-time Polymerase Chain Reaction, Transformation Assay, Control, Activity Assay, Western Blot, Stable Transfection, Expressing, shRNA, Plasmid Preparation

Journal: Chemico-biological interactions

Article Title: Hexavalent chromium promotes malignant transformation via enhanced translation of SUV39H1

doi: 10.1016/j.cbi.2025.111869

Figure Lengend Snippet: (A) Immunoblot analysis of Cr(VI)-transformed BEAS-2BR cells stably expressing SUV39H1-Myc or vector control. (B and C) Representative images (B) and quantification (C) of sphere formation in suspension culture by Cr(VI)-transformed BEAS-2BR cells stably expressing SUV39H1 or vector control, treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1 or DMSO control. Results are expressed as the percentage of sphere formation relative to vector control cells treated with DMSO. Scale bar, 400 μm. (D and E) Cr(VI)-transformed BEAS-2BR cells stably expressing SUV39H1 or vector control were pre-treated with 50 nM rapamycin, 100 nM INK128, 25 μM 4EGI-1, or DMSO for 30 min, followed by exposure to 2.5 μM Cr(VI) (K 2 Cr 2 O 7 ) for 12 h. Cells were fixed either immediately (“Rest 0 h”) or after a 36-h recovery in Cr(VI)-free medium with continued inhibitor treatment (“Rest 36 h”) and stained for γH2AX (red) and DAPI (blue) (D). Quantification shows the percentage of γH2AX-positive nuclei relative to the total DAPI-stained nuclei per field (E). Scale bar, 50 μm. All graphical data are presented as mean ± SD (n = 3 replicates) from one representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, by Student’s t test; ns, not significant, by one-way ANOVA.

Article Snippet: Rapamycin and INK128 were purchased from Selleckchem and MedChemExpress, respectively, and potassium dichromate and 4EGI-1 were obtained from Sigma.

Techniques: Expressing, Inhibition, Western Blot, Transformation Assay, Stable Transfection, Plasmid Preparation, Control, Suspension, Staining