Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in cancer cell survival under chronic extracellular acidosis. A549 (A) or HCT116 (B) cells were cultured in pH 6.7 (6.7EXT) or pH 7.4 (7.4EXT) buffered medium for about 3 months. Cells were then cultured in pH 6.7 or 7.4-buffered media for 48 h with/without 1 mM IPTG and/or 30 μM A484954, before lysis and Western blot analysis. A549 (C) or HCT116 (D, G) cells were also subjected to flow cytometry to determine subG1 population. (E) HCT116 cells were cultured at pH 7.4 in the presence or absence of 1 mM IPTG and 30 μM A484954 for 48 h, and G1 cell size was then determined by flow cytometry. Results are shown as forward scatter-height (FSC-H). (F) HCT116 cells were cultured as described for panel E and then subjected to flow cytometry analysis; the percentage of sub-G1 population is shown. (H) HCT116 cells cultured at pH 6.7 (6.7EXT) or 7.4 (7.4EXT) for approximately 3 months were transfected with GFP-spectrin alone or GFP-spectrin plus FLAG-tagged eEF2K as indicated. Cells were lysed 8 h after transfection, and eEF2K levels were analyzed by Western blotting. Data are expressed as mean ± SE from 3 independent experiments. P values were obtained by two-way ANOVA. *, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001. For panels A and B, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Cell Culture, Lysis, Western Blot, Flow Cytometry, Transfection

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Acidosis activates eEF2K in cancer cells. (A) Validation of the P-eEF2 Thr56 antibody for immunohistochemistry. eEF2K WT and knockout (KO) MEFs were stained with P-eEF2 Thr56. Scale bar, 50 μm. (B) Representative sequential sections of human lung cancer to support expression of NHE-1, GLUT-1, and P-eEF2 Thr56. Nuclei were counterstained with hematoxylin. Ten lung cancer carcinoma samples were stained in total, 7 of which showed strong areas of GLUT-1 expression; of these, 6 codisplayed NHE-1 and P-eEF2 Thr56. Scale bar, 100 μm. A549 (C) or HCT116 (D) cells were cultured in medium buffered at pH 6.4, 6.8, or 7.4 for the indicated times. IPTG (1 μM) was added to A549 (E) or HCT116 (F) cells 5 days before the experiment. Cells were then incubated in medium buffered at different pH values for 48 h with/without 1 mM IPTG, 30 μM A484954, or 25 μM etoposide, followed by lysis and Western blot analysis. (G) A549 cells were treated as described for panel E, and Western blot analysis was then performed to study the phosphorylation of p70S6K1 at Thr389 and S6 at Ser240/Ser244. For panels C to G, data shown are representative of three independent experiments.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Biomarker Discovery, Immunohistochemistry, Knock-Out, Staining, Expressing, Cell Culture, Incubation, Lysis, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: Activation of eEF2K is important in cancer cell survival under acute acidic conditions. A549 (A) or HCT116 (B) cells were treated as described for Fig. 9E and ​andF,F, respectively, and then subjected to cell ATP assay using the CellTitre-Glo kit. A549 (C) or HCT116 (E) cells were treated as described for panels A and B, respectively, and then subjected to cytotoxicity assay using CellTox Green kit. A549 (D) or HCT116 (F) cells were cultured as described for panels A and B, respectively, and then subjected to flow cytometry analysis. The percentage of sub-G1 population is presented. For panels A, B, C, and E, results are expressed as means ± SE from 3 independent experiments in duplicate. For panel D, results are means ± SE from 3 independent experiments. For panel E, results are means ± SE from 2 independent experiments and a third one in duplicate. P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001).

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Activation Assay, ATP Assay, Cytotoxicity Assay, CellTox Assay, Cell Culture, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

doi: 10.1128/MCB.00012-15

Figure Lengend Snippet: The role of eEF2K in protein synthesis under acidosis. (A) IPTG (1 μM) was added to A549 cells 5 days before experiment. A549 cells were then transferred to media at different pH values for 1 h with/without 1 μM IPTG. Rates of protein synthesis were determined, and results are presented as counts per minute (cpm) per microgram protein and expressed as means ± SE from 4 independent experiments. (B) Quantification of IPTG-treated cells as described for panel A expressed as a percentage of control (no IPTG treatment). For panels A and B, P values were obtained either by two-way ANOVA (*, 0.01 ≤ P < 0.05; **, 0.01 < P ≤ 0.001; ***, P < 0.001) or by one-way ANOVA followed by Dunnett's test (@, 0.01 ≤ P < 0.05; #, 0.01 < P ≤ 0.001; $, P < 0.001). (C) A549 cells were incubated at pH 6.7 or 7.4 for 1 h. Lysates were fractionated on sucrose density gradients. Positions of the 40S, 60S, and 80S ribosomal particles and polysome fractions are shown. Absorbance values (254 nm) are in arbitrary units and on the same scale for each panel. Representative data from 4 independent experiments are shown.

Article Snippet: HCT116 and A549 cells expressing inducible short hairpin RNA (shRNA) against eEF2K were generously provided by Janssen Pharmaceutica.

Techniques: Control, Incubation