Journal: Cancer biology & therapy

Article Title: Inhibition of cytosolic phospholipase A2 alpha increases chemosensitivity in cervical carcinoma through suppressing β-catenin signaling.

doi: 10.1080/15384047.2019.1579961

Figure Lengend Snippet: Figure 7. cPLA2α inhibition suppresses Akt/eIF4E/β-catenin signaling in cervical cells. cPLA2α inhibition by inhibitors (a) or siRNA (b) significantly decreases cPLA2α enzyme activity in cervical cancer cells. cPLA2α inhibition by inhibitors (c) or siRNA (d) decreases βAkt (Ser473), p-eIF4E (Ser209), and β-catenin in CaLo cells. (e) Pyrrophenone and RSC-3388 at 100 nM significantly inhibit TOPflash activation. CaLo cells transfected with TOPflash plasmid were treated as indicated. The graph represents mean ± s.e.m. of TOPflash signal relative to control. cPLA2α inhibitors (f) and siRNA (g) significantly decrease the transcript level of C-MYC, BCL9, and CYCLIN D genes in CaLo cells. *p < 0.05 compared to control or siRNA con (scrambled siRNA).

Article Snippet: Antibodies for cPLAα, p-Akt, Akt, p-eIF4E, eIF4E, β-catenin, and β-actin were from Santa Cruz, US; cPLAβ antibody was from Biorbyt, US; and cPLAδ antibody was from St John’s Laboratory, US.

Techniques: Inhibition, Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Control

Journal: Cancer gene therapy

Article Title: Targeting and killing of prostate cancer cells using lentiviral constructs containing a sequence recognized by translation factor eIF4E and a prostate-specific promoter.

doi: 10.1038/sj.cgt.7700885

Figure Lengend Snippet: Figure 3 Western analysis of eIF4E level in cancer and noncancer cell lines. Protein extracts from cancer cells (LNCaP, MCF-7, PC- 3M, and DU145) and noncancer cells (BPH-1, Plat-E, Huvec-c, and 267-B1) were probed with antibodies to eIF4E and b-actin in a Western blot. Density scans (means7s.e.m., n ¼ 3) of eIF4E/b-actin are shown in the bottom panel.

Article Snippet: The membranes were blocked with 5% skim milk in trisbuffered saline (TBS) containing 0.05% tween-20 and probed with 1:1000 rabbit polyclonal anti-eIF4E antibody (Cell Signalling), 1:500 anti-HTK (a gift from Dr William C Summers, Yale University), or 1:500 anti-b-actin antibody (Sigma), followed by 1:2000 polyclonal goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad).

Techniques: Western Blot

Journal: Cancer research

Article Title: Translation initiation factor eIF4E is a target for tumor cell radiosensitization

doi: 10.1158/0008-5472.CAN-12-0329

Figure Lengend Snippet: Effect of eIF4E knockdown on clonogenic cell survival. Cultures were transfected with siRNA specific to eIF4E (eIF4E KD) or non-targeted siRNA (Scramble). A) Representative immunoblots from each cell line showing extent of eIF4E protein reduction 72h after transfection. B) 72h post-transfection cells were plated at specified densities and colony-forming efficiency was determined 10–14 days later. Surviving fractions for eIF4E KD cells were calculated after normalizing to the surviving fraction obtained for cells receiving the scrambled siRNA. Values shown represent the means ± SE for 3–4 independent experiments. *p < 0.04 according to Student’s t test (all tumor cell lines compared to HMEC).

Article Snippet: RIP-Chip and Microarray Analysis: The RIP-Chip kit and anti-eIF4E antibody were obtained from MBL International (Woburn, Ma); the procedure was performed in biological triplicate according to manufacturer’s protocol.

Techniques: Transfection, Western Blot

Journal: Cancer research

Article Title: Translation initiation factor eIF4E is a target for tumor cell radiosensitization

doi: 10.1158/0008-5472.CAN-12-0329

Figure Lengend Snippet: The effects of eIF4E knockdown on cellular radiosensitivity. A) MDA-MB-231, B) A549, C) DU145, D) MRC9, and E) HMEC cells were transfected with non-targeted siRNA (Scramble) or siRNA specific for eIF4E (eIF4E KD). 72h post-transfection cells were plated, allowed to attach for 6h, and irradiated. Colony-forming efficiency was determined 10–14 days later and survival curves were generated after normalizing for cell killing from siRNA alone. DEFs were calculated at a surviving fraction of 0.1. Values shown represent the mean ± SE for 3–4 independent experiments. * p < 0.05; ** p < 0.1 according to Student’s t test.

Article Snippet: RIP-Chip and Microarray Analysis: The RIP-Chip kit and anti-eIF4E antibody were obtained from MBL International (Woburn, Ma); the procedure was performed in biological triplicate according to manufacturer’s protocol.

Techniques: Transfection, Irradiation, Generated

Journal: Cancer research

Article Title: Translation initiation factor eIF4E is a target for tumor cell radiosensitization

doi: 10.1158/0008-5472.CAN-12-0329

Figure Lengend Snippet: Mechanism of radiosensitization by eIF4E knockdown. In the following experiments MDA-MB-231 cells were transfected with siRNA specific to eIF4E (eIF4E KD) or non-targeted siRNA (Scramble). All experiments were carried out 72 hours post-transfection. A) Cell cycle phase distribution was determined. Values represent the mean of three independent experiments. B) Cells were irradiated with 2 or 4Gy and collected at the specified time; γH2AX foci were counted in at least 50 cells per condition. Values shown represent the means ± SE for 3 independent experiments, *p < 0.04 according to Student’s t test (eIF4E KD compared to scramble). C) Cells were irradiated (2 Gy) and collected at the specified time points. Cells were classified as being in mitotic catastrophe by the presence of nuclear fragmentation, which was defined as a single cell containing two or more distinct nuclear lobes. At least 50 cells per condition were scored. Values represent the mean ± SE for 3 independent experiments. *p< 0.04

Article Snippet: RIP-Chip and Microarray Analysis: The RIP-Chip kit and anti-eIF4E antibody were obtained from MBL International (Woburn, Ma); the procedure was performed in biological triplicate according to manufacturer’s protocol.

Techniques: Transfection, Irradiation

Journal: Cancer research

Article Title: Translation initiation factor eIF4E is a target for tumor cell radiosensitization

doi: 10.1158/0008-5472.CAN-12-0329

Figure Lengend Snippet: The effect of radiation on eIF4E activation. A) MDA-MB-231 cells were irradiated (2 Gy) and collected at the specified times and subjected to immunoblot analysis. Actin was used as a loading control. B) m7-GTP affinity chromatography was performed on MDA-MB-231 cells that were irradiated and collected 1h after 2 Gy, and compared to unirradiated counterparts. m7-GTP bound and unbound proteins (flow through) were resolved via SDS-PAGE followed by immunoblot analysis. eIF4E was used as a loading control. Blots are representative of two independent experiments.

Article Snippet: RIP-Chip and Microarray Analysis: The RIP-Chip kit and anti-eIF4E antibody were obtained from MBL International (Woburn, Ma); the procedure was performed in biological triplicate according to manufacturer’s protocol.

Techniques: Activation Assay, Irradiation, Western Blot, Affinity Chromatography, SDS Page

Journal: Cancer research

Article Title: Translation initiation factor eIF4E is a target for tumor cell radiosensitization

doi: 10.1158/0008-5472.CAN-12-0329

Figure Lengend Snippet: Rip Chip analysis of the effects of radiation on eIF4E mRNA clients. MDA-MB-231 cells were irradiated (2 Gy) and collected 6 hours later. eIF4E was immunoprecipitated, RNA bound to eIF4E was isolated and subjected to microarray analysis and mRNAs whose binding to eIF4E after irradiation were classified using IPA. A) The top ten biological functions (containing 100 or more genes) of the mRNAs whose binding to eIF4E was increased by radiation. B) The biological functions of the mRNAs (with greater than 10 genes) within the DNA Replication, Recombination, and Repair category are further delineated. C) Network 4 and D) Network 6 are shown with dark red indicating not bound to bound and lighter red indicating fold increase ≥ 1.5. E) Immunoblot analysis of DNA Damage response related proteins predicted by RIP-Chip analysis to be induced by radiation. MDA-MB-231 cells were radiated (6 Gy) and collected at the specified times. Actin was used as a loading control. Blots are representative of two independent experiments.

Article Snippet: RIP-Chip and Microarray Analysis: The RIP-Chip kit and anti-eIF4E antibody were obtained from MBL International (Woburn, Ma); the procedure was performed in biological triplicate according to manufacturer’s protocol.

Techniques: Irradiation, Immunoprecipitation, Isolation, Microarray, Binding Assay, Western Blot

Journal: Cancer research

Article Title: Translation initiation factor eIF4E is a target for tumor cell radiosensitization

doi: 10.1158/0008-5472.CAN-12-0329

Figure Lengend Snippet: Functions associated with the top ten networks for genes who were increasingly bound to eIF4E after radiation (2Gy 6h) in MDA-MB-231 cells.

Article Snippet: RIP-Chip and Microarray Analysis: The RIP-Chip kit and anti-eIF4E antibody were obtained from MBL International (Woburn, Ma); the procedure was performed in biological triplicate according to manufacturer’s protocol.

Techniques: Expressing, Cell Function Assay, Modification