Journal: The European journal of neuroscience

Article Title: Chronic fluoxetine induces region-specific changes in translation factor eIF4E and eEF2 activity in the rat brain.

doi: 10.1111/j.1460-9568.2006.04817.x

Figure Lengend Snippet: Fig. 1. Chronic fluoxetine treatment leads to enhanced phosphorylation of eukaryotic initiation factor 4E (eIF4E) in the dentate gyrus. (A) and (B) Group mean + SEM changes in immunoreactivity levels in Western blots performed in homogenates from prefrontal cortex (PFC), hippocampus (HPC) and dentate gyrus (DG) following acute (single i.p. injection; n ¼ 5) or chronic (daily injection for 21 days; n ¼ 8) injection of fluoxetine (FLX) or saline (SAL) control. Changes in eIF4E phosphorylation were normalized to total eIF4E levels. Optical density values are expressed as the ratio treated ⁄ control. *Significantly different from saline-treated control (Student’s t-test, P < 0.05). (C) Two representative Western blots from adjacent lanes are shown. b-actin served as a loading control and levels were unchanged across samples.

Article Snippet: Sections were preincubated in 0.3% H2O2 for 15 min to reduce endogenous peroxidase activity, before being incubated in primary antibody recognizing phospho-Ser209 eIF4E [rabbit polyclonal, Cell Signalling; 1 : 25 dilution in 0.05 m Trisbuffered saline (TBS), 0.25% Triton, pH 7.6] for 48 h at 4 C. Subsequently, sections were washed with TBS and incubated at room temperature with biotinylated donkey anti-rabbit IgG (Amersham Biosciences; 1 : 200 dilution) followed by ABC complex (Vector ABC kit, Vector Laboratories, Burlingame, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Injection, Saline, Control

Journal: The European journal of neuroscience

Article Title: Chronic fluoxetine induces region-specific changes in translation factor eIF4E and eEF2 activity in the rat brain.

doi: 10.1111/j.1460-9568.2006.04817.x

Figure Lengend Snippet: Fig. 3. Immunohistochemical localization and enhanced staining of phospho-eukaryotic initiation factor 4E (eIF4E) in the dentate gyrus following chronic fluoxetine treatment. Upper panels show staining in coronal hippocampal sections in rats receiving chronic treatment with fluoxetine or saline control. White boxes show region from the dentate gyrus magnified in the lower panels. Increased staining in fluoxetine-treated rats was detected among cells (white arrow) within the subgranular zone and hilar region of the dentate gyrus. A band of intense staining (straddled by white bars) corresponding to the inner molecular layer of the dentate gyrus was also evident in control and fluoxetine-treated rats. Similar results were obtained in multiple sections across five animals in each group.

Article Snippet: Sections were preincubated in 0.3% H2O2 for 15 min to reduce endogenous peroxidase activity, before being incubated in primary antibody recognizing phospho-Ser209 eIF4E [rabbit polyclonal, Cell Signalling; 1 : 25 dilution in 0.05 m Trisbuffered saline (TBS), 0.25% Triton, pH 7.6] for 48 h at 4 C. Subsequently, sections were washed with TBS and incubated at room temperature with biotinylated donkey anti-rabbit IgG (Amersham Biosciences; 1 : 200 dilution) followed by ABC complex (Vector ABC kit, Vector Laboratories, Burlingame, CA, USA).

Techniques: Immunohistochemical staining, Staining, Saline, Control

Journal: Journal of medicinal chemistry

Article Title: Novel C-4 Heteroaryl 13- cis -Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

doi: 10.1021/jm501792c

Figure Lengend Snippet: Effect of compounds 16, 20, and 22 on Mnk1, Mnk2, peIF4E, eIF4E, and cPARP proteins in MCF-7 cells. Equal protein concentrations from MCF-7 (A), MDA-MB-231 (B), and MDA-MB-468 (C) cells treated for 24 h with 16, 20, and 22 at the various concentrations as indicated and 6 (20 μmol/L) were separated by SDS–PAGE and Western blots probed with antibodies to Mnk1, Mnk2, peIF4E, eIF4E, and cPARP. Vehicle treated cells were included as control, and all blots were reprobed for GAPDH for loading control.

Article Snippet: Western blotting was done as mentioned previously, 8 , 13b using the following antibodies: AR, Bad, Bax, breast cancer l-2, eIF4E, Mnk1, Mnk-2, N-cadherin, PARP, peIF4E ser209 purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: SDS Page, Western Blot

Journal: Journal of medicinal chemistry

Article Title: Novel C-4 Heteroaryl 13- cis -Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

doi: 10.1021/jm501792c

Figure Lengend Snippet: Compounds 20 and 22 suppress breast xenograft tumor growth in vivo. (A) Nude female mice bearing MDA-MB-231 xenograft tumors (n = 6/group) were treated with 20 or 22, administered ip 20 mg kg−1 day−1, 5 days per week for 28 days. %T/C values are indicated to the right of each growth curve, and the error bars are the SEM. Compounds 20 and 22 treatments, each significantly suppressed tumor growth (for 20, (*) p = 0.0001 and for 22, (**) p = 0.0001). (B) Representative photos of the tumor-bearing mice and excised tumors in the control and the two treatment groups at termination of experiment (day 28). (C) Body weight changes of the mice during the course of treatments. Animals were monitored for changes in body weight as a surrogate marker for toxicity in control and treatment groups. (D) Effect of compounds on the expression of proteins modulated by Mnk/eIF4E signaling. Total cell lysates in mice treated with vehicle, compounds 20 and 22, were prepared separately using RIPA buffer. Total protein (50 μg/well) from pooled samples (n = 6) was run on 10% SDS–PAGE and probed with antibodies for Mnk1, Mnk2, p-eIF4E, eIF4E, cyclin D1, breast cancer l-2, Bax, Bad, and GAPDH.

Article Snippet: Western blotting was done as mentioned previously, 8 , 13b using the following antibodies: AR, Bad, Bax, breast cancer l-2, eIF4E, Mnk1, Mnk-2, N-cadherin, PARP, peIF4E ser209 purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: In Vivo, Marker, Expressing, SDS Page

Journal: Journal of medicinal chemistry

Article Title: Novel C-4 Heteroaryl 13- cis -Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

doi: 10.1021/jm501792c

Figure Lengend Snippet: Compound 20 suppresses prostate xenograft tumor growth in vivo. (A) Castrated SCID male mice bearing CWR22Rv1 xenograft tumors (n = 6/group) were treated with 20, administered ip 20 mg kg−1 day−1, 5 days per week for 28 days. %T/C values are indicated to the right of each growth curve, and the error bars are the SEM. Compound 20 treatment significantly suppressed tumor growth ((*) p = 0.0001). (B) Body weight changes of the mice during the course of treatments. Animals were monitored for changes in body weight as a surrogate marker for toxicity in control and treatment groups. (C) Effect of compounds on the expression of proteins modulated by AR and Mnk/eIF4E signaling. Total cell lysates in mice treated with vehicle, 20, and 22 were prepared separately using RIPA buffer. Total protein (50 μg/well) from pooled samples (n = 6) was run on 10% SDS–PAGE and probed with antibodies for fAR, AR-V7, Mnk1, Mnk2, p-eIF4E, eIF4E, cyclin D1, breast cancer l-2, Bad, and GAPDH.

Article Snippet: Western blotting was done as mentioned previously, 8 , 13b using the following antibodies: AR, Bad, Bax, breast cancer l-2, eIF4E, Mnk1, Mnk-2, N-cadherin, PARP, peIF4E ser209 purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: In Vivo, Marker, Expressing, SDS Page

Journal: Carcinogenesis

Article Title: Microvesicles derived from normal and multiple myeloma bone marrow mesenchymal stem cells differentially modulate myeloma cells' phenotype and translation initiation.

doi: 10.1093/carcin/bgx045

Figure Lengend Snippet: Figure 4. TI and autophagy are essential for MM cell lines’ phenotype. U266 and RPMI 8226 MM cell lines were incubated with MM-MVs (50 μg/ml) and with eIF4E/eIF4G complex inhibitor for 3 days. Then, the MM cells were assayed for cell count and migration (transwell assay) and immunoblotted for PCNA, and Cyclin D1. Analysis of our observations (A) and representative immunoblots (B) are presented. Tubulin served as loading control. Results are expressed as percent (mean ± SE, n ≥ 4) of respec- tive protein expression in control cells not treated with MVs (dotted line). Next, U266 and MM1S cell lines were co-treated with MM-MSCs MVs and autophagy inhibitor (3MA) for 3 days then assayed for dead cell count (trypan blue, C), and apoptosis (Annexin/7AAD analysis and representative dot plots, C). Representative immunoblot of LC3-II in 3MA treated U266 is presented (D). Asterisks depict statistical significance (*P < 0.05, **P < 0.01).

Article Snippet: The following proteins were detected using rabbit/mouse anti-human: peIF4E (Ser209)/total eIF4E, peIF4GI(Ser1108)/total eIF4GI, p4EBP(Ser65)/total 4EBP, pmTOR(Ser2448)/ total mTOR, pMNK(Thr197/Thr202)/total MNK/pERK1/2 and pJNK (Cell Signaling Technology, Danvers, MA, USA); SMAD5 (Epitomics, Burlingame, CA); c-Myc, HIF1α, NFkB, PCNA (Santa-Cruz, CA); tubulin, LC3-II (Sigma).

Techniques: Incubation, Cell Counting, Migration, Transwell Assay, Western Blot, Control, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Dual targeting of AKT and mammalian target of rapamycin: A potential therapeutic approach for malignant peripheral nerve sheath tumor

doi: 10.1158/1535-7163.mct-08-1008

Figure Lengend Snippet: Figure 1. MPNST exhibit dereg- ulation of the mTOR and AKT path- ways. A, expression of activated mTOR downstream effectors 4EBP1 (top) and S6RP (bottom) in MPNST was assessed via immuno- histochemistry. A tissue microarray consisting of human NF1-associated plexiform neurofibromas and NF1 associated as well as sporadic MPNST was used for immuno- histochemistry. A significantly higher proportion of MPNST cells expressed p4EBP1 and pS6RP compared with neurofibromas (rep- resentative tissue microarray spots are shown; original pictures were captured at ×200). Similarly, stain- ing intensity for both markers was significantly higher in the malignant tumors compared with the benign lesions. Results of Wilcoxon rank- sum tests: blue line, median of the data; bars, 25% and 75% quar- tiles; dots, mean outliers. The two lines are whisker lines, depicting the largest or smallest values that are not outliers. Statistical P values are shown. B, pAKT was evaluated as above; a significantly higher pro- portion of MPNST cells were found to exhibit a stronger pAKT expres- sion level. Furthermore, metastatic lesions expressed higher levels of pAKT compared with localized (pri- mary and recurrent) tumors.

Article Snippet: AKT, pAKT (Ser473), mTOR, pmTOR (Ser2448), S6K1, pS6K1 (Thr398), S6 ribosomal protein (S6RP), pS6RP (Ser235/ Ser236), 4EBP1, p4EBP1 (Thr70), eIF4E, peIF4E (Ser209), pGSK-3α/β (Ser21/Ser9), GSK-3α/β, pMDM2 (Ser166), LC3B, PTEN, and cyclin D1 were all purchased from Cell Signaling; c-Met, MDM2, BCN1, EGFR, β-actin, and antimouse and anti-rabbit second antibody were acquired from Santa Cruz Biotechnology.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining, Whisker Assay

Journal: Molecular Cancer Therapeutics

Article Title: Dual targeting of AKT and mammalian target of rapamycin: A potential therapeutic approach for malignant peripheral nerve sheath tumor

doi: 10.1158/1535-7163.mct-08-1008

Figure Lengend Snippet: Figure 3. Rapamycin abrogates MPNST cell growth but induces the activation of AKT and eIF4E. A, MTS assays showing a rapamycin (48 h) dose- dependent decrease in MPNST cell growth. B, rapamycin (4 h) blocks the activation of the mTOR downstream targets S6K1 and 4EBP1 (Western blot). C, rapamycin at doses indicated induces activation of AKT in both cell lines tested (Western blot). D, rapamycin (4 h) induces the phosphorylation of eIF4E in MPNST cells. This activation is blocked when combining rapamycin with a PI3K inhibitor (LY294002; Western blot).

Article Snippet: AKT, pAKT (Ser473), mTOR, pmTOR (Ser2448), S6K1, pS6K1 (Thr398), S6 ribosomal protein (S6RP), pS6RP (Ser235/ Ser236), 4EBP1, p4EBP1 (Thr70), eIF4E, peIF4E (Ser209), pGSK-3α/β (Ser21/Ser9), GSK-3α/β, pMDM2 (Ser166), LC3B, PTEN, and cyclin D1 were all purchased from Cell Signaling; c-Met, MDM2, BCN1, EGFR, β-actin, and antimouse and anti-rabbit second antibody were acquired from Santa Cruz Biotechnology.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Journal of medicinal chemistry

Article Title: Novel C-4 Heteroaryl 13- cis -Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

doi: 10.1021/jm501792c

Figure Lengend Snippet: Compounds 20 and 22 suppress breast xenograft tumor growth in vivo. (A) Nude female mice bearing MDA-MB-231 xenograft tumors (n = 6/group) were treated with 20 or 22, administered ip 20 mg kg−1 day−1, 5 days per week for 28 days. %T/C values are indicated to the right of each growth curve, and the error bars are the SEM. Compounds 20 and 22 treatments, each significantly suppressed tumor growth (for 20, (*) p = 0.0001 and for 22, (**) p = 0.0001). (B) Representative photos of the tumor-bearing mice and excised tumors in the control and the two treatment groups at termination of experiment (day 28). (C) Body weight changes of the mice during the course of treatments. Animals were monitored for changes in body weight as a surrogate marker for toxicity in control and treatment groups. (D) Effect of compounds on the expression of proteins modulated by Mnk/eIF4E signaling. Total cell lysates in mice treated with vehicle, compounds 20 and 22, were prepared separately using RIPA buffer. Total protein (50 μg/well) from pooled samples (n = 6) was run on 10% SDS–PAGE and probed with antibodies for Mnk1, Mnk2, p-eIF4E, eIF4E, cyclin D1, breast cancer l-2, Bax, Bad, and GAPDH.

Article Snippet: Western blotting was done as mentioned previously, 8 , 13b using the following antibodies: AR, Bad, Bax, breast cancer l-2, eIF4E, Mnk1, Mnk-2, N-cadherin, PARP, peIF4E ser209 purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: In Vivo, Marker, Expressing, SDS Page

Journal: Journal of medicinal chemistry

Article Title: Novel C-4 Heteroaryl 13- cis -Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

doi: 10.1021/jm501792c

Figure Lengend Snippet: Effects of compounds 16, 20, and 22 on the expression of fAR, Mnk, peIF4E, cyclin D1, and cleaved PARP in LNCaP cells. Equal protein concentrations from LNCaP cells treated with 16, 20, and 22 at different concentrations (0.6–20 μM) for 24 h were separated by SDS–PAGE and Western blots probed with antibodies to fAR, Mnk1/2, peIF4E, cyclin D1, and cleaved PARP. Vehicle treated cells were included as a control, and all blots were reprobed for β-actin for loading control.

Article Snippet: Western blotting was done as mentioned previously, 8 , 13b using the following antibodies: AR, Bad, Bax, breast cancer l-2, eIF4E, Mnk1, Mnk-2, N-cadherin, PARP, peIF4E ser209 purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Expressing, SDS Page, Western Blot