Journal: PLoS ONE

Article Title: Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma

doi: 10.1371/journal.pone.0081345

Figure Lengend Snippet: ( A ) Human glioma cell lines LN229 and U251 with or without silencing of eEF-2 kinase expression were plated in 96-well plates (5×10 3 /well) and treated with TMZ (100 μM) at 37°C for 24 h; ( B ) LN229 and U251 cells were plated in 96-well plates (5×10 3 /well) and treated with TMZ (100 μM) in the presence or absence of 0.5μM of NH125 at 37°C for 24 h. Cell viability was measured by CCK-8 assay. ( C ) LN229 and U251 cells were transfected with an eEF-2 kinase-targeted siRNA or a non-targeting control siRNA (NT control). At different time points, expression of eEF-2 kinase was analyzed by Western blot,. β-actin was used as a loading control. ( D ) LN229 and U251 cells were treated with NH125 (0.5μM) or vehicle for various periods of time. At the end of treatment, the level of phospho-eEF2 (Thr56) was examined by western blot. β-actin was used as a loading control. Each bar represents mean ± SD of triplicate determinations; results shown are the representative of three identical experiments; data are expressed as the percentage of the controls. ** p < 0.01.

Article Snippet: Temozolomide and dimethyl sulfoxide (DMSO) were purchased from Sigma (St Louis, MO); 1-Hexadecyl- 2-methyl-3-(phenylmethyl)-1H-imi-dazolium iodide (NH125) was obtained from Tocris Bioscience (St. Louis, MO); the antibodies to phospho-eEF2, eEF-2, casepase-3, PARP, and LC3B, were purchased from Cell Signaling Technology (Danvers, MA); rabbit polyclonal anti-eEF2 kinase antibody was obtained from Novus Biologicals (Littleton, CO); p62 was purchased from Enzo Life Sciences (Plymouth Meeting, PA); β-actin antibody was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA); eEF-2 kinase-siRNA and control siRNA were synthesized by Shanghai Gene-Pharma Co. (Shanghai, China); the Cell Counting Kit-8 (CCK-8) was purchased from DojinDo Molecular Technologies, Inc. (Rockville, MA); the Annexin V-FITC apoptosis detection kit and Matrigel were purchased from BD Biosciences (San Diego, CA); the Pierce BCA Protein Assay Kit was obtained from Thermo Scientific Corp (Hudson, New Hampshire); oligofectamine reagent was purchased from Invitrogen Corp (Carlsbad, CA); other Western blot reagents were obtained from Bio-Rad Laboratories (Hercules, CA).

Techniques: Expressing, CCK-8 Assay, Transfection, Control, Western Blot

Journal: Molecular Cancer

Article Title: PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

doi: 10.1186/1476-4598-13-157

Figure Lengend Snippet: Phospho-proteomic analysis reveals decreased levels of p-eEF2 in SKBR3-L cells. (A) Example of a 3D view of p-eEF2 protein abundance with graphs of protein abundance analyzed by DeCyder software in SKBR3-par and SKBR3-L cells. The solid line represents the average of three replicate measurements (dotted lines) of protein abundance. (B) Schematic depiction of mTOR-mediated activation of eEF2; active mTOR phosphorylates and activates p70S6k, which in turn phosphorylates and deactivates eEF2k thus preventing the phosphorylation of eEF2 resulting in active eEF2. (C) Immunoblot analysis of total and phosphorylated eEF2 (Thr56) in SKBR3-par and SKBR3-L cells following 24 hr lapatinib treatment.

Article Snippet: Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2 tyr1221/1222 , p-EGFR tyr1173 , AKT, p-AKT ser473 , ERK, pERK thr202/tyr204 , eEF2, p-eEF2 thr56 , mTOR, p-mTOR ser2448 , eEF2k, p-eEF2k ser366 (Cell Signaling Technology), p-eEF2k ser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich).

Techniques: Quantitative Proteomics, Software, Activation Assay, Phospho-proteomics, Western Blot

Journal: Molecular Cancer

Article Title: PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

doi: 10.1186/1476-4598-13-157

Figure Lengend Snippet: mTOR and eEF2k mediated regulation of eEF2 phosphorylation. (A) Immunoblot analysis of total and phosphorylated mTOR (Ser2448) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (B) Effect of rapamycin on growth of SKBR3-par and SKBR3-L cells. Error bars represent the mean ± SD (n = 3). (C) Immunoblot analysis of total and phosphorylated eEF2 (Thr56) following 24 hr. treatment with lapatinib and/or rapamycin. (D) Immunoblot analysis of total and phosphorylated eEF2k (Ser366, 359) in SKBR3-par and SKBR3-L cells following 24 hr. lapatinib treatment. (E) Immunoblot examining the effect of NH125 alone and in combination with lapatinib on the phosphorylation of eEF2 (Thr56) in SKBR3-par cells. *denotes p ≤ 0.05.

Article Snippet: Membranes were incubated at 4°C overnight with primary antibody at a 1:1000 dilution in blocking solution (unless otherwise stated) against p-HER2 tyr1221/1222 , p-EGFR tyr1173 , AKT, p-AKT ser473 , ERK, pERK thr202/tyr204 , eEF2, p-eEF2 thr56 , mTOR, p-mTOR ser2448 , eEF2k, p-eEF2k ser366 (Cell Signaling Technology), p-eEF2k ser359 (1:200) (SantaCruzBiotechnology), HER2 (Calbiochem), EGFR (1:250) (Neomarkers), and α-tubulin, anti-mouse and anti-rabbit secondary antibodies (Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot