Journal: International Journal of Oncology

Article Title: Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells

doi: 10.3892/ijo.2012.1635

Figure Lengend Snippet: Downregulation of protein kinase CK2 affects the mTOR and ERK1/2 signaling pathways. Cell lysates (30 μ g) from cells treated as indicated in the figure were analyzed by western blot analysis using antibodies against the indicated proteins or their phosphorylated form. Anti-β-actin was applied to confirm equal protein loading. At least 4 separate experiments were performed obtaining similar results. Data from one representative experiment are shown.

Article Snippet: The primary antibodies employed in this study were: mouse monoclonal anti-CK2β and -CK2α (both from Calbiochem); rabbit polyclonal anti-CK2α′ obtained by immunizing rabbits with a specific peptide (SQPCADNAVLSSGTAAR) deriving from human CK2α′; mouse monoclonal anti-β-actin (Sigma); mouse monoclonal anti-mTOR and -AKT1 (both from BD Biosciences), rabbit monoclonal anti-Raptor and -ERK1/2 (p-T202/Y204); rabbit polyclonal anti-mTOR (p-S2481) and -ERK1/2 (all from Cell Signaling Technology, Beverly, MA, USA); mouse monoclonal anti-AKT (p-S473) and -p70 S6 kinase (p-T389) (both from Cell Signaling Technology) and rabbit polyclonal anti-p70 S6 kinase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot

Journal: International Journal of Oncology

Article Title: Downregulation of protein kinase CK2 induces autophagic cell death through modulation of the mTOR and MAPK signaling pathways in human glioblastoma cells

doi: 10.3892/ijo.2012.1635

Figure Lengend Snippet: Model of the proposed influence of CK2 on autophagy induction. The model suggests the placement and function of CK2 with respect to the major signaling cascades regulating autophagy. CK2 has been reported to be a master regulator of cellular functions in virtue of its ability to play a ‘lateral means’ of pathways intervention . Here, we show that effective CK2-mediated autophagy induction is achieved by the simultaneous targeting of the ERK1/2-and mTOR signaling pathways. Additional details are reported in the text.

Article Snippet: The primary antibodies employed in this study were: mouse monoclonal anti-CK2β and -CK2α (both from Calbiochem); rabbit polyclonal anti-CK2α′ obtained by immunizing rabbits with a specific peptide (SQPCADNAVLSSGTAAR) deriving from human CK2α′; mouse monoclonal anti-β-actin (Sigma); mouse monoclonal anti-mTOR and -AKT1 (both from BD Biosciences), rabbit monoclonal anti-Raptor and -ERK1/2 (p-T202/Y204); rabbit polyclonal anti-mTOR (p-S2481) and -ERK1/2 (all from Cell Signaling Technology, Beverly, MA, USA); mouse monoclonal anti-AKT (p-S473) and -p70 S6 kinase (p-T389) (both from Cell Signaling Technology) and rabbit polyclonal anti-p70 S6 kinase (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hepatitis C virus-induced MDSCs Suppress NK Cell Interferon-gamma Production by Altering Cellular Metabolism via Arginase-1

doi: 10.4049/jimmunol.1501881

Figure Lengend Snippet: NK cells were co-cultured with mock- or HCV-conditioned CD33+ cells separated by a 0.45 μm transwell insert. Following 2 days in culture, NK cells were recovered and phosphorylation of mTOR was assessed by flow cytometry. (A) Cells were gated on singlets, forward and side scatter, mTOR+ cells, and phospho-mTOR+. mTOR and phospho-mTOR positive cells were gated based on FMOs. MFI of phospho-mTOR expression was quantified from the experiment. (B) Activation of the mTOR substrate, 4EBP1, was assessed measuring the phosphorylation of 4EBP1 at residue T69 by flow cytometry. As above, cells were gated on singlets, forward and side scatter, and phosphor-4EBP1+ cells based on FMOs. The MFI of p-4EBP1 of the p-4EBP1+ cells was computed. (C) NK cells were grown in complete media with and without the mTOR inhibitor rapamycin or L-arginine deficient media for 2 days. IFN-γ in the supernatant was measured by ELISA. Results are representative or mean of 8-10 independent experiments. *p<0.01, **p<0.01, ns denotes not significant, (A, B) Wilcoxon signed rank test. **p<0.01, ***p<0.001, (C) Kruskal-Wallis test with Dunn’s post-test.

Article Snippet: The recovered cells were fixed in Cytofix (BD Biosciences), permeabilized using BD Phosflow Perm Buffer (III), and stained with rat anti-mTOR (R&D systems) and mouse anti-phospho-mTOR (BD Phosflow™), or mouse anti-phospho-4EBP1 (pT69) (BD Phosflow™).

Techniques: Cell Culture, Flow Cytometry, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hepatitis C virus-induced MDSCs Suppress NK Cell Interferon-gamma Production by Altering Cellular Metabolism via Arginase-1

doi: 10.4049/jimmunol.1501881

Figure Lengend Snippet: PBMCs were obtained from chronic 7 HCV patients and 7 healthy donors and then frozen in 90% FBS/10% DMSO. NK cells were magnetically sorted from thawed PBMCs and cultured for 2 days with IL-2/IL-12/IL-18. (A) The cell media was collected, spun to remove cell bodies, and IFN-γ in the culture media was determined by ELISA. (B, C) More cells were recovered by detachin treatment, combined with the existing cell pellet, and then phosphorylation levels of mTOR and 4EBP1 were assessed by flow cytometry. (B) Cells were gated on singlets, forward and side scatter, mTOR+ cells. MFIs of phospho-mTOR and total mTOR expression were quantified from the experiment. mTOR and phosphor-mTOR positive cells were gated based on FMOs as shown in Fig. 6. (C) The level of phosphorylated 4EBP1 (pT69) was assessed. As above, cells were gated on singlets, forward and side scatter, and phosphor-4EBP1+ cells based on FMOs. The MFI of p-4EBP1 of the p-4EBP1+ cells was computed. ***p<0.001, ns denotes not significant, (A-C) Mann-Whitney Test.

Article Snippet: The recovered cells were fixed in Cytofix (BD Biosciences), permeabilized using BD Phosflow Perm Buffer (III), and stained with rat anti-mTOR (R&D systems) and mouse anti-phospho-mTOR (BD Phosflow™), or mouse anti-phospho-4EBP1 (pT69) (BD Phosflow™).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, MANN-WHITNEY