Journal: The American journal of pathology

Article Title: CK2α/CSNK2A1 Phosphorylates SIRT6 and Is Involved in the Progression of Breast Carcinoma and Predicts Shorter Survival of Diagnosed Patients.

doi: 10.1016/j.ajpath.2016.08.007

Figure Lengend Snippet: Figure 2 Inhibition of CSNK2A1 decreased the proliferation and invasiveness of breast cancer cells. A and B: Treatment with the CSNK2 inhibitors, CX4945 (A) and emodin (B), significantly inhibited the growth of both MCF7 and T47D cells in a dose- and time-dependent manner. C: The knockdown of CSNK2A1 inhibited the proliferation of both MCF7 and T47D cells as indicated by MTT and colony-forming assays. D: The sub-G1 and G0/G1 population increased with the knockdown of CSNK2A1 in flow cytometric cell cycle analysis. E and F: The knockdown of CSNK2A1 significantly reduced cell migration (E) and invasion (F) in both MCF7 and T47D cell lines. *P < 0.05, **P < 0.01, and ***P < 0.001. CSNK2A1, casein kinase 2 a1.

Article Snippet: Two CSNK2 inhibitors, CX4945 (Santa Cruz Biotechnology, Santa Cruz, CA) and emodin (Sigma-Aldrich, St. Louis, MO), were used.

Techniques: Inhibition, Knockdown, Cell Cycle Assay, Migration

Journal: Cells

Article Title: Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

doi: 10.3390/cells9051138

Figure Lengend Snippet: CK2 controls membrane expression of TMEM16A in CFBE airway epithelial cells. ( A ) Expression of double-tagged (eGFP and extracellular HA-tag) TMEM16A in CFBE airway epithelial cells. Membrane localized TMEM16A (Alexa647 positivity) was detected by an extracellular anti-HA-Alexa647-conjugated antibody. ( B , C ) RT-PCR and densitometric analysis indicating successful knockdown of CK2α’, #significant inhibition (unpaired t -test; p = 0.01). ( D , E ) Immunocytochemistry of TMEM16A expressed endogenously in CFBE cells. Membrane expression was reduced by knockdown of CK2α’, #significant inhibition (unpaired t -test; p = 0.000000002). Mean ± SEM. In parentheses are numbers of experiments.

Article Snippet: The CK2 inhibitors CX-4945 (silmitasertib) and TBB (4,5,6,7-Tetrabromobenzotriazole) were purchased from Cayman Chemicals and Sigma, respectively.

Techniques: Membrane, Expressing, Reverse Transcription Polymerase Chain Reaction, Knockdown, Inhibition, Immunocytochemistry

Journal: Cells

Article Title: Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

doi: 10.3390/cells9051138

Figure Lengend Snippet: Inhibitors of CK2 inhibit TMEM16A in CFBE airway epithelial cells. ( A – F ) Whole cell current overlay recorded in patch clamp experiments and current/voltage relationships. ATP (100 µM) activated TMEM16A whole cell Cl − currents that were strongly inhibited by the CK2-inhibitors TBB (10 µM; #significant inhibition, unpaired t -test; p = 0.01, ( A , B )) and CX4945 (20 µM; #significant inhibition, unpaired t -test; p = 0.02; ( C , D )), and siRNA-knockdown of CK2α’ (#significant inhibition, unpaired t -test; p = 0.0001; ( E , F )). ( G , H ) Plasma membrane (PM) expression of endogenous TMEM16A in CFBE cells and inhibition of PM expression by the CK2-inhibitor CX4945 (#significant inhibition, unpaired t -test; p = 0.000000000007). Mean ± SEM #significant inhibition ( p < 0.05; unpaired t -test). In parentheses are numbers of experiments.

Article Snippet: The CK2 inhibitors CX-4945 (silmitasertib) and TBB (4,5,6,7-Tetrabromobenzotriazole) were purchased from Cayman Chemicals and Sigma, respectively.

Techniques: Patch Clamp, Inhibition, Knockdown, Clinical Proteomics, Membrane, Expressing

Journal: Cells

Article Title: Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

doi: 10.3390/cells9051138

Figure Lengend Snippet: Role of CK2 for plasma membrane expression of TMEM16A in Cal33 head and neck cancer cells. ( A , B ) RT-PCR and densitometric analysis indicating successful knockdown of CK2α’ by siRNA for CK2α’ in Cal33 head and neck cancer cells (#significant inhibition, unpaired t -test; p = 0.01). Knockdown of CK2α’ did not inhibit transcription of TMEM16A. ( C , D ) Western blot analysis indicating successful knockdown of CK2α’ but unaffected expression of TMEM16A. ( E , F ) Plasma membrane (PM) expression of TMEM16A expressed endogenously in Cal33 cells and inhibition of PM expression by knockdown of CK2α’ (#significant inhibition, unpaired t -test; p = 0.00000002). ( G ) Current/voltage relationships of ATP-activated TMEM16A whole cells currents, indicating inhibition of TMEM16A by knockdown of CK2α’ (#significant inhibition, unpaired t -test; p = 0.01). Mean ± SEM. In parentheses are numbers of experiments.

Article Snippet: The CK2 inhibitors CX-4945 (silmitasertib) and TBB (4,5,6,7-Tetrabromobenzotriazole) were purchased from Cayman Chemicals and Sigma, respectively.

Techniques: Clinical Proteomics, Membrane, Expressing, Reverse Transcription Polymerase Chain Reaction, Knockdown, Inhibition, Western Blot

Journal: Cells

Article Title: Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

doi: 10.3390/cells9051138

Figure Lengend Snippet: Inhibition of proliferation by knockdown of CK2α’ and TMEM16A. ( A ) Cell proliferation assessed in MTT assays and shown as absorbance. Both siRNA-knockdown of CK2α’ and TMEM16A inhibited cell proliferation (#significant inhibition, unpaired t -tests; p = 0.0001). Simultaneous knockdown of CK2α’ and TMEM16A had a more pronounced inhibitory effect on cell proliferation (#significant inhibition, unpaired t -test; p = 0.0015). ( B ) Inhibition of cell proliferation by the CK2-inhibitor CX4945 (20 µM) and additional inhibitory effect of TMEM16A-knockdown (#significant inhibition, unpaired t -test; p = 0.0001). Mean ± SEM. In parentheses are numbers of experiments.

Article Snippet: The CK2 inhibitors CX-4945 (silmitasertib) and TBB (4,5,6,7-Tetrabromobenzotriazole) were purchased from Cayman Chemicals and Sigma, respectively.

Techniques: Inhibition, Knockdown

Journal: Cells

Article Title: Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

doi: 10.3390/cells9051138

Figure Lengend Snippet: Blockers of CK2 and TMEM16A inhibit proliferation of Cal33 and BHY head and neck cancer cells. ( A ) Blocking CK2 by CX4945 (20 µM) and blocking TMEM16A by niclosamide (0.5 µM) inhibited proliferation of Cal33 cells. Simultaneous application of both blockers had an additive effect (#significant inhibition, unpaired t -tests; p = 0.0001). ( B ) Enhanced cell proliferation of BHY cells induced by the TMEM16A-activator, Eact. Blocking CK2 by CX4945 (20 µM) and blocking TMEM16A by niclosamide (0.5 µM) inhibited proliferation of BHY cells. Simultaneous application of both blockers had an additive effect (#significant inhibition, unpaired t -tests; p = 0.000015). Mean ± SEM. In parentheses are numbers of experiments.

Article Snippet: The CK2 inhibitors CX-4945 (silmitasertib) and TBB (4,5,6,7-Tetrabromobenzotriazole) were purchased from Cayman Chemicals and Sigma, respectively.

Techniques: Blocking Assay, Inhibition

Journal: Cells

Article Title: Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

doi: 10.3390/cells9051138

Figure Lengend Snippet: Blockers of CK2 and TMEM16A inhibit receptor-mediated Ca 2+ signaling. ( A , B ) Original recordings and summaries for basal and ATP-induced intracellular Ca 2+ concentrations in Cal33 cells. Increase of intracellular Ca 2+ by 10 and 100 µM ATP, respectively. Both CX4945 (20 µM; #significant inhibition, ANOVA; p = 0.0004) and niclosamide (1 µM; #significant inhibition, ANOVA; p = 0.0002) largely reduced ATP-induced Ca 2+ increase. Mean ± SEM. In parentheses are numbers of experiments.

Article Snippet: The CK2 inhibitors CX-4945 (silmitasertib) and TBB (4,5,6,7-Tetrabromobenzotriazole) were purchased from Cayman Chemicals and Sigma, respectively.

Techniques: Inhibition

Journal: Scientific Reports

Article Title: Phosphorylation of p23-1 cochaperone by protein kinase CK2 affects root development in Arabidopsis

doi: 10.1038/s41598-019-46327-0

Figure Lengend Snippet: p23-1 phosphorylation by CK2-like activity. ( A ) Representative autoradiography (left) and Coomassie staining (right) after radioactive phosphorylation of increasing concentrations of recombinant p23-1 and p23-2, as indicated, by 10 μg of Arabidopsis total protein extract and separation via SDS-PAGE. The arrows indicate the migrations of each p23 isoform. Equally labeled radioactive bands in all lanes are due to autophosphorylation of the proteins present in the extract (see lane 1, where no p23 was present). The migration of the most abundant protein rubisco (~55 kDa) is also indicated. ( B ) Kinetics showing phosphorylation by CK2 with increasing concentrations of p23-1 and p23-2. The calculated kinetics values are shown in the box. Vmax is reported as pmol/min/mg, Km as μM. Quantification was performed by excising bands from the gel, as shown in panel A, and scintillator counting. Values are the means ± SD of independent experiments. ( C ) p23-1 (0.1 μg) was phosphorylated by 10 μg of Arabidopsis total protein extract in the presence (as indicated) of 2 μM TBB (TBB), 10 nM CX4945 (CX), or 1 μM Staurosporin (ST), or DMSO solvent as the control (CTR). Representative autoradiography is shown after protein separation by SDS-PAGE. The arrow indicates the migration of p23-1. Mw markers migrations are also shown on the left.

Article Snippet: The CK2 inhibitor CX4945 (5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic) was purchased from Glixx Laboratories.

Techniques: Phospho-proteomics, Activity Assay, Autoradiography, Staining, Recombinant, SDS Page, Labeling, Migration, Solvent, Control

Journal: Scientific Reports

Article Title: Phosphorylation of p23-1 cochaperone by protein kinase CK2 affects root development in Arabidopsis

doi: 10.1038/s41598-019-46327-0

Figure Lengend Snippet: p23-1 phosphorylation by a monomeric 40-kDa CK2. ( A ) p23-1 (0.1 μg, lanes 1 and 2) was incubated with recombinant human monomeric CK2 ( α , 15 ng) or tetrameric CK2 ( α 2 β 2 , 5 ng); β-casein (1 μg, lanes 3 and 4) was used to ensure that the amount of each CK2 isoform chosen had the same catalytic activity towards a model substrate. After radioactive phosphorylation (10 min at 30 °C), samples were resolved by SDS-PAGE. A representative autoradiograph is shown. ( B ) Representative autoradiography of an in-gel kinase assay: 10 or 20 μg protein from Arabidopsis total extract (in duplicate, as indicated) was resolved by SDS-PAGE in which p23-1 (10 μg/ml) was included in the gel. In lane 1, human recombinant CK2 α (hsCK2 α 50 ng, Mw 40 kDa) was loaded as a positive control. After electrophoresis and protein renaturation, the gel was incubated with a radioactive phosphorylation mixture and analyzed by autoradiography. The migration of Mw markers is shown on the left.

Article Snippet: The CK2 inhibitor CX4945 (5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic) was purchased from Glixx Laboratories.

Techniques: Phospho-proteomics, Incubation, Recombinant, Activity Assay, SDS Page, Autoradiography, Kinase Assay, Positive Control, Electrophoresis, Migration

Journal: Scientific Reports

Article Title: Phosphorylation of p23-1 cochaperone by protein kinase CK2 affects root development in Arabidopsis

doi: 10.1038/s41598-019-46327-0

Figure Lengend Snippet: p23-1 phosphorylation activity of Arabidopsis mutant lines. ( A ) The specific CK2 peptide substrate CK2-tide was incubated with 10 μg total extract proteins from wild type (wt) or the CK2 αC mutant ( α 3), CK2 αAαB mutant ( α A α B) or of CK2 αAαBαC (triple) mutant lines in the presence of a radioactive phosphorylation mixture. Blank controls were performed in the presence of 10 nM CX4945. Each phosphorylation activity is reported after subtraction of the relative blank control. ( B ) p23-1 wt or the S201A mutant (0.1 μg; lane #6) was incubated with 10 μg total extract proteins from wild type (wt), CK2 αC mutant ( α C), CK2 αAαB mutant ( α A α B) or the CK2 αAαBαC (triple) mutant, as indicated. CX4945 (10 nM) was present where indicated. A representative autoradiograph after protein separation by SDS-PAGE is shown. The migration of Mw markers is shown on the left. ( C ) Quantification of p23-1 radioactivity is shown, obtained by Cyclone Plus Storage Phosphor System (PerkinElmer) analysis from the gel of panel B.

Article Snippet: The CK2 inhibitor CX4945 (5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic) was purchased from Glixx Laboratories.

Techniques: Phospho-proteomics, Activity Assay, Mutagenesis, Incubation, Control, Autoradiography, SDS Page, Migration, Radioactivity

Journal: Scientific Reports

Article Title: Phosphorylation of p23-1 cochaperone by protein kinase CK2 affects root development in Arabidopsis

doi: 10.1038/s41598-019-46327-0

Figure Lengend Snippet: Arabidopsis CK2 nuclear catalytic isoform expression. ( A ) Expression levels of CK2 αA , CK2 αB or CK2 αC assayed by quantitative Real-Time PCR of the 3′-region of the mRNA. Values are reported as the percentage of the Actin 2 ( ACT2 ) expression level in the shoot (white bars) or root (black bars) of 10-day-old Arabidopsis seedlings. ( B ) Expression levels of CK2 αA , CK2 αB or CK2 αC assayed by quantitative Real-Time PCR on the 5′-region of the mRNA. Values are reported as the percentage of the Actin 2 ( ACT2 ) expression level in the shoot (white bars) or root (black bars) of 10-day-old Arabidopsis seedlings.

Article Snippet: The CK2 inhibitor CX4945 (5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic) was purchased from Glixx Laboratories.

Techniques: Expressing, Real-time Polymerase Chain Reaction