Journal: eLife

Article Title: Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

doi: 10.7554/eLife.19850

Figure Lengend Snippet: ( A ) Ratio of F- to G-actin in MCF-7 cells during CaAR as determined by fractionation and Western blotting (mean ± SD, n = 5). ( B ) Lifeact-GFP intensity at the cortex of MCF-7 cells after stimulation by laser ablation. ( C ) Levels of CTGF and NR4A3 transcripts in MCF-7 cells at the indicated times after addition of 1 µM ionomycin (at t = 0) as determined by quantitative RT-PCR (mean ± SD, n = 2, normalized against untreated control cells at each time point). ( D ) MCF-7 cells were pretreated with the indicated drugs (ROCK inhibitor Y27632, CaM-kinase inhibitors KN62 and KN93, calcineurin inhibitor cyclosporin A (CsA), actin inhibitors latrunculin A (LatA) and cytochalasin D (CytoD); concentrations are indicated in µM) for 10 min before addition of 1.5 µM ionomycin, and levels of CTGF transcripts were determined. Values are mean ± SD, n = 3. ( E ) Lifeact-GFP expressing MCF-7 cells were pretreated with the indicated inhibitors (CsA: 1 µM, Y27632: 10 µM, C3: 2.5 µg/ml) for at least 2 hr before the addition of 1 µM ionomycin to induce CaAR. Upper right: MCF-7 cells expressing Lifeact-GFP stimulated by the addition of 40% FBS and then monitored by fluorescence microscopy. Times in sec. Scale bars 10 µm. DOI: http://dx.doi.org/10.7554/eLife.19850.023

Article Snippet: Cells were treated with 50 μM blebbistatin (Sigma-Aldrich, St Louis, MO USA) to inhibit myosin II ATPase activity, 400 nM latrunculin A (Enzo Life Sciences) to sequester actin monomers, 1 μM thapsigargin to inhibit Ca 2+ uptake into the ER, 1 μM cytochalasin D (Sigma) to depolymerize actin, 10–50 μM cyclosporin A (Sigma) to inhibit calcineurin, 1–10 μM KN93 or KN62 (Tocris) to inhibit Ca 2+ /calmodulin-dependent protein kinase II, 10–20 μM Y27632 (Sigma) to inhibit ROCK, 2.5 μg/μl C3 transferase (Cytoskeleton) to inhibit Rho1, 1 μM nocodazole (Sigma) to depolymerize microtubules, 10–20 μM SMIFH2 (Sigma) to inhibit formins and 50–100 μM CK666 or CK869 (Sigma) to inhibit the ARP2/3 complex.

Techniques: Fractionation, Western Blot, Quantitative RT-PCR, Control, Expressing, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: Ca 2+ /calmodulin-dependent protein kinase IIγ enhances stem-like traits and tumorigenicity of lung cancer cells

doi:

Figure Lengend Snippet: A. Relative gene expression of OCT4, NANOG, MYC, and KLF4 in ZRLC-1 or HCC827 cells treated with DMSO, KN92, or KN93. Data are expressed as mean ± SEM of n = 3 independent cell dishes per condition. * p < 0.05, ** p < 0.01, *** p < 0.001 versus cells treated with KN92. B. Detection of Oct4, c-Myc, pCaMKIIγ, and GAPDH protein by western blots in ZRLC-1 or HCC827 cells treated with DMSO, KN92, or KN93. C. Quantitative analysis of oncosphere formation by 3000 ZRLC-1 or HCC827 cells treated with DMSO, KN92, or KN93. Data are expressed as mean ± SEM of n = 3 independent cell dishes per condition. * p < 0.05 versus cells treated with KN92. D. ZRLC-1 or HCC827 cells pre-treated with KN92 or KN93 were separately injected subcutaneously into nude mice. Data are expressed as mean ± SEM of n = 5 mice per group. * p < 0.05, ** p < 0.01. NS: no significance. Tumor incidence is displayed on the graph.

Article Snippet: KN93 (Cat. No. 422711), KN92 (Cat. No. 422709), Akt Inhibitor IV (Cat. No. 124011), β-catenin/Tcf Inhibitor V (Cat. No. 219334), and Wnt Pathway inhibitor XI (Cat. No. 681674) were purchased from Merck Millipore.

Techniques: Gene Expression, Western Blot, Injection

Journal: Oncotarget

Article Title: Ca 2+ /calmodulin-dependent protein kinase IIγ enhances stem-like traits and tumorigenicity of lung cancer cells

doi:

Figure Lengend Snippet: A. Detection of Akt and β-catenin signals in H1299 parental or oncosphere cells by western blots. B. Detection of Akt and β-catenin signals in H1299 cells tranduced with control or CaMKIIγ vector by western blots. C. Detection of Akt and β-catenin signals in ZRLC-1 cells treated with DMSO, KN92, or KN93 by western blots. D. Detection of Oct4 and c-Myc in CaMKIIγ-overexpressed H1299 cells treated with Akt or β-catenin inhibitor by western blots. E. Lysates of CaMKIIγ-overexpressed A549 and H1299 cells were incubated with CaMKIIγ or Akt antibody for immunoprecipitation and the immune complex was then purified, separated by SDS-PAGE, and immunoblotted with Akt or CaMKIIγ antibody. F. Dual luciferase reporter assay of Wnt pathway (TOP, FOP, and the ratio of TOP/FOP) in control or CaMKIIγ-overexpressed H1299 cells. Data are expressed as mean ± SEM of n = 4 independent cell dishes per condition. ** p < 0.01. G. Quantitative western blot analysis of oncosphere formation by control or CaMKIIγ-overexpressed A549 or H1299 cells treated with Akt or β-catenin inhibitor. Data are expressed as mean ± SEM of n = 4 independent cell dishes per condition. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KN93 (Cat. No. 422711), KN92 (Cat. No. 422709), Akt Inhibitor IV (Cat. No. 124011), β-catenin/Tcf Inhibitor V (Cat. No. 219334), and Wnt Pathway inhibitor XI (Cat. No. 681674) were purchased from Merck Millipore.

Techniques: Western Blot, Control, Plasmid Preparation, Incubation, Immunoprecipitation, Purification, SDS Page, Luciferase, Reporter Assay

Journal: Oncotarget

Article Title: Ca 2+ /calmodulin-dependent protein kinase IIγ enhances stem-like traits and tumorigenicity of lung cancer cells

doi:

Figure Lengend Snippet: A. Detection of acetylation and methylation of histone in control or CaMKIIγ-overexpressed H1299 cells by western blots. B. Detection of acetylated histone3 (H3Ac) in ZRLC-1 cells treated with DMSO, KN92, or KN93 by western blots. C. Detection of H3Ac, Oct4, and c-Myc in KN93-treated ZRLC-1 cells cultured with TSA by western blots. D. Detection of H3Ac in control or CaMKIIγ-overexpressed H1299 cells treated with Akt inhibitor by western blots. E. CHIP-enriched DNA from control or CaMKIIγ-overexpressed H1299 cells treated with Akt inhibitor using anti-H3Ac antibody is amplified by real-time PCR, depending on primers designed according to the transcription regulatory region of OCT4. Data are expressed as enrichment assessed relative to the input DNA ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. F. Detection of histone acetylation transferases in control or CaMKIIγ-overexpressed H1299 cells by western blots. G. Detection of histone acetylation transferases in H1299 parental or oncosphere cells by western blots. (H) CHIP-enriched DNA from control or CaMKIIγ-overexpressed H1299 cells treated with Akt inhibitor using anti-PCAF antibody is amplified by real-time PCR, depending on primers designed according to the transcription regulatory region of OCT4. Data are expressed as enrichment assessed relative to the input DNA ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: KN93 (Cat. No. 422711), KN92 (Cat. No. 422709), Akt Inhibitor IV (Cat. No. 124011), β-catenin/Tcf Inhibitor V (Cat. No. 219334), and Wnt Pathway inhibitor XI (Cat. No. 681674) were purchased from Merck Millipore.

Techniques: Methylation, Control, Western Blot, Cell Culture, Amplification, Real-time Polymerase Chain Reaction