Journal: Cells

Article Title: Contribution of the CK2 Catalytic Isoforms α and α’ to the Glycolytic Phenotype of Tumor Cells

doi: 10.3390/cells10010181

Figure Lengend Snippet: CK2 expression and activity in SK-N-BE and U2OS cells. ( A ) Titration of the antibody reactivity towards CK2 catalytic subunits. The indicated amounts of recombinant CK2 catalytic subunits (myc-α’ or α) were loaded on SDS-PAGE, and either blotted for the WB (western blot) analysis or stained by Colloidal Coomassie Blue; ( B ) CK2 expression and activity in w.t. and KO clones of the cells used for this study. 10 μg proteins from cell lysates were analyzed by WB with the indicated antibodies. The last two right lanes belong to an independent experiment. As a reporter of CK2 endogenous activity, pS129 Akt signal has been quantified, normalized to total Akt signal, and reported in the bar graph as % of w.t. cells. At least three independent experiments were performed; representative western blots are shown, while quantification in the bar graphs reports the mean values ± SEM of all experiments and of the two clones of the same KO. Statistical significance refers to w.t. cells. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001

Article Snippet: The CK2 inhibitor CX-4945 (5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic) was purchased from MedChemExpress and the chemical inducer of hypoxia DMOG (dimethyloxallyl glycine) was from Sigma.

Techniques: Expressing, Activity Assay, Titration, Recombinant, SDS Page, Western Blot, Staining, Clone Assay

Journal: Cells

Article Title: Contribution of the CK2 Catalytic Isoforms α and α’ to the Glycolytic Phenotype of Tumor Cells

doi: 10.3390/cells10010181

Figure Lengend Snippet: Lactate production in response to CK2 knock out or pharmacological inhibition. ( A ) The extracellular lactate secreted in the culture medium in 8 h by the indicated cells was analyzed by means of a luminescence assay. Values for each KO are the means ± SEM obtained with the different clones of the same KO. At least three independent experiments were performed, in duplicate. Significance refers to w.t. cells ( B ) Cells were analyzed for the extracellular lactate secreted in the culture medium in 6 h, in the absence or in the presence of the indicated concentrations of CX-4945. Three independent experiments were performed, in duplicate. Results (means ± SEM) are reported as % compared to the respective w.t. cells, in the absence of CX-4945. Significance refers to untreated control. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.

Article Snippet: The CK2 inhibitor CX-4945 (5-[(3-Chlorophenyl)amino]-benzo[c]-2,6-naphthyridine-8-carboxylic) was purchased from MedChemExpress and the chemical inducer of hypoxia DMOG (dimethyloxallyl glycine) was from Sigma.

Techniques: Knock-Out, Inhibition, Luminescence Assay, Clone Assay, Control

Journal: Science advances

Article Title: Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy.

doi: 10.1126/sciadv.abo1215

Figure Lengend Snippet: Fig. 4. CK2 phosphorylates FAM134C. (A) List of putative residues with CK2 consensus at the C terminus of FAM134C (data obtained from the PhosphositePlus website). (B) U2OS cells expressing doxycycline-inducible HA-tagged FAM134C were subjected to IP. Inputs and immunoprecipitates were analyzed by immunoblotting using the indicated antibodies. (C) Bar graph shows quantification of phosphorylated/total HA-FAM134C ratio relative to (B). Mean ± SEM. N = 3 biological replicates. Student’s paired t test: ***P < 0.0005. CX4945: CK2 inhibitor (4 M; 6 hours) and BafA1 (200 nM; 6 hours). (D) CK2 and subunit interaction with FAM134 proteins based on IP-MS interactome experiment from U2OS FLAG-HA-FAM134A, FLAG-HA-FAM134B, and FLAG-HA-FAM134C doxycycline-inducible cells. NA, not detected. Numeric values represent log2 difference. (E) In vitro CK2 phosphorylation radioactive assay. C-terminal FAM134C WT and phosphomutant (T440A and 3A) peptides and CK2 (control substrate) were incubated with CK2 in the presence of radioactive adenosine triphosphate (ATP). Substrate phosphorylation was detected by autoradiography. Equal amounts of proteins were verified by Coomassie staining. On the bottom, relative quantitation of the bands is performed by analysis with the CyclonePlus Storage Phos- phor System (PerkinElmer); the unit amounts of each peptide are indicated, and activity is reported as % of that measured with 5 units of CK2. Mean ± SEM. N = 3 biolog- ical replicates. One-way ANOVA and Tukey’s multiple comparison test: ****P < 0.0001 and *P < 0.05. (F) Western blot analysis of CK2-dependent phosphorylation of WT- and 3A-HA-FAM134C250–466 peptides transfected in Atg7KO MEFs untreated or treated with the CK2 inhibitor CX4945 (4 M; 6 hours). Vinculin was used as loading control. NT, not transfected.

Article Snippet: CK2 inhibitor CX4945 (Selleckchem, #S2248) was used at 4 M for 6 hours.

Techniques: Expressing, Western Blot, Protein-Protein interactions, In Vitro, Phospho-proteomics, Radioactivity, Control, Incubation, Autoradiography, Staining, Quantitation Assay, Activity Assay, Comparison, Transfection

Journal: Science advances

Article Title: Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy.

doi: 10.1126/sciadv.abo1215

Figure Lengend Snippet: Fig. 5. Regulation of FAM134C by CK2. (A) Representative Western blot analysis of IP experiments of doxycycline-inducible HA-FAM134C treated with CX4945 was indicated (4 M; 4 hours). Vinculin was used as loading control for input. Arrowheads indicate a nonspecific band. On the right, the bar graph shows quantification of immunoprecipitated LC3B relative to untreated samples. Mean ± SEM. N = 3 biological replicates. Student’s paired t test: *P < 0.05. (B) Representative Western blot analysis of HA-FAM134C in U2OS cells overexpressing doxycycline-inducible HA-FAM134C treated with DMSO, Torin1 (250 nM; 6 hours), CK2 inhibitor CX4945 (4 M; 6 hours), or CK2 inhibitor SGC-CK2-1 (50 nM; 6 hours) in the absence or presence of BafA1 (100 nM). -Actin was used as a loading control. Bar graph shows quantification of HA-FAM134C/-actin ratio relative to DMSO-treated samples. Mean ± SEM. N = 3 biological replicates. Sidak’s and Tukey’s multiple comparison test: ****P < 0.0001, ***P < 0.0005, and *P < 0.05. (C) Representative immunofluorescence staining of Myc (green), LAMP1 (red), and nuclei (blue) in U2OS cells transiently transfected with Myc-FAM134C-WT, Myc-FAM134C-3D, and Myc-FAM134C-3A. Cells were pretreated for 2 hours with BafA1 (100 nM), and then DMSO or CK2 inhibitor CX4945 (4 M) was added for 6 hours. Scale bar, 10 m. On the right, bar graph shows quantification of Myc-FAM134C in LAMP1-positive vesicles. Mean ± SEM. N = 3 biological replicates. n = 15 cells per experiment. Two-way ANOVA and Tukey’s multiple comparison test: ****P < 0.0001.

Article Snippet: CK2 inhibitor CX4945 (Selleckchem, #S2248) was used at 4 M for 6 hours.

Techniques: Western Blot, Control, Immunoprecipitation, Comparison, Immunofluorescence, Staining, Transfection

Journal: Science advances

Article Title: Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy.

doi: 10.1126/sciadv.abo1215

Figure Lengend Snippet: Fig. 6. CK2-dependent phosphorylation of FAM134C is modulated by mTORC1. (A) Western blot analysis of Atg7KO MEF cells transfected with WT- or 3A-HA- FAM134C250–466 peptides and treated with Torin1 (1 M; 6 hours). (B) Western blot analysis of U2OS cells transfected with GFP-CK2 plasmid treated with DMSO, Torin1 (1 M), or HBSS (4 hours). Vinculin (A) or -actin (B) was used as loading controls. Quantification (A and B) of phosphorylated/total protein ratio. N = 6 (A) and N = 4 (B) biological replicates. Mean ± SEM. Two-way ANOVA and Sidak’s multiple comparison test: **P < 0.005 and ***P < 0.0005. (C) Phosphorylation analysis of CK2 in U2OS cells. N = 3 biological replicates. Student’s paired t test: *P < 0.05. (D and E) Radioactive phosphorylation assays using (D) mTOR (200 ng; 20 min) and CK2 [203–215] peptide at the indicated concentrations and (E) mTOR (200 ng) and CK2 [203–215] peptide (1.2 mM) for the indicated times. Mean ± SEM. N = 2. (F) Western blot analysis of MEF cells treated with DMSO or Torin1 (1 M; 6 hours). N = 3 biological replicates. H3 histone was used as loading control. Bar graphs show quantifications (mean ± SEM) of phosphorylated AKT and CDC37/total protein ratio. Student’s paired t test: *P < 0.05 and **P < 0.005 (G) Western blot analysis of WT, tamoxifen-inducible Raptor KO (Rptor-indKO), or Rictor KO (Rictor-indKO) MEFs. WT MEFs were treated with CX4945 (4 M; 6 hours) as control. Asterisk (*) denotes bands changing intensity. N = 3 biological replicates. Bar graphs show quantifications (mean ± SEM) of phosphorylated AKT and CDC37/total protein ratio. Two-way ANOVA and Sidak’s multiple comparison test: *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Article Snippet: CK2 inhibitor CX4945 (Selleckchem, #S2248) was used at 4 M for 6 hours.

Techniques: Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Comparison, Control

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 overexpression in AML cells causes IKAROS phosphorylation which is reversed by CK2 inhibitor, CX-4945. ( A ) Baseline protein levels of CK2α, CK2α’, CK2β, IKAROS and BCL-XL in the myeloid leukemia cell panel [U937, THP-1, K562, and primary AML cells (labelled AML-1) were measured by western blot and compared to CD34+ Hematopoietic Stem Cells (HSC). Multiple IKAROS bands represent Ikaros isoforms 1 and 2. ( B ) qRT-PCR showing mRNA level of CK2α and BCL-XL in various AML cells compared to CD34+ HSC. **** ( p < 0.0001). ( C ) Radio-blot showing increased Phosphorylated IKAROS (p-IKAROS) in AML cells compared to HSC. ( D ) Radio-immunoblot showing dose-dependent decrease in Phospho-IKAROS level following CX-4945 treatment. U937 and THP-1 were treated with 10 µM and AML-1 was treated with 5μM (IC50–3.791 μM) CX-4945 for 48 h. IC50 values of CX-4945 treated cells are shown in . ( E ) Western blot showing decrease in amount of phosphorylated CK2 substrates with molecular masses of approximately 175, 120, 80, 70 and 56 kDa as indicated by arrows (left panel) and western blot showing phosphorylation extent of specific CK2 substrate, AKT1 at Ser129 (right panel).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Over Expression, Phospho-proteomics, Western Blot, Quantitative RT-PCR

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 inhibition suppresses BCL-XL expression in AML cells and xenograft model. ( A ) U937 and AML-1 were treated with 5 and 10 µM concentration of CX-4945 for 48 h before RNA and protein extraction. mRNA level of BCL-XL was measured by qRT-PCR. ( B ) U937 and K562 cells were treated with 5 and 10 µM of CX-4945 for 48 hours and protein was extracted. BCL-XL protein level was measured by western blot. ( C ) Downregulation of CK2α in U937 was achieved using shRNA. Validation of CK2α protein knockdown in U937-shCK2α cell line is shown in . qRT-PCR shows mRNA level of CK2α and BCL-XL in CK2α shRNA treated U937 cells. ( D ) Overexpression of CK2α was achieved by retroviral transduction of U937 cells. Validation of CK2α protein overexpression is shown in . qRT-PCR showed increased BCL-XL mRNA level in CK2α overexpressed U937 (left panel) and THP-1 cells (right panel). ( E ) WST assay showing increased cell viability in CK2α overexpressing U937 and THP-1 cells. U937 cells transduced with retroviral vector CK2α-gfp-luc were transplanted into NRG-S mice. 25,000 cells were injected via the tail vein. Bioluminescence imaging using IVIS 100 was obtained weekly following transplant. ( F ) Bioluminescence signal in xenograft mice engrafting U937-gfp-luc cell or control (U937-ctl-gfp-luc) shown at week 2 and 3 post transplantation. ( G ) Kaplan Maier plot showing survival probability in U937-CK2-gfp-luc cells vs control. P -value summaries are as follows: p > 0.05 (ns-not significant); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Inhibition, Expressing, Concentration Assay, Protein Extraction, Quantitative RT-PCR, Western Blot, shRNA, Biomarker Discovery, Knockdown, Over Expression, Retroviral, Transduction, WST Assay, Plasmid Preparation, Injection, Imaging, Control, Transplantation Assay

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CX-4945 treatment shows therapeutic efficacy in AML Patient Derived Xenograft (PDX). ( A ) Schema showing AML PDX generation and treatment. AML-1 PDX was developed by injecting NRG-S mice with one million cells per mouse via the tail vein. Treatment was started when 2–5% human CD45+ cells were detected in peripheral blood (PB). Mice received 100 mg/kg of CX-4945 via gavage twice daily for 21 days. ( B ) Percent AML (human CD45+, CD13+,CD33+) in bone marrow (BM) and spleen of treated and untreated AML-1 PDX mice by flow cytometry. ( C ) Kaplan Maier plot showing survival probability of treated and untreated AML-1 PDX mice. Cell line derived xenograft (CDX) mouse models was developed using luciferase, and green fluro-protein (gfp) labeled U937 cells (U937-gfp-luc) or THP-1 cells (THP1-gfp-luc) transplanted into immunocompromised NRG-S mice as described in methods. Mice were treated with vehicle or CX-4945 at a dose of 100 mg/kg twice daily via gavage for up to 7 days. ( D ) Engraftment was monitored using bioluminescence imaging shown as the mean of the total flux in photons/second of mice in each group. ( E ) Following treatment, mice were sacrificed, and bone marrow mononuclear cells were collected. Human CD45+ cells in bone marrow were measured using flow cytometry. Flow cytometry using conjugated BCL-XL antibody was used to quantify intracellular BCL-XL protein level in FACS enriched human CD45+cells in the bone marrow of treated and untreated mice. shows histogram of BCL-XL and CK2α protein level in vehicle and CX-4945 treated U937 and THP-1 CDX. ( F ) Mean fluorescence intensity(MFI) is graphed, showing decreased BCL-XL protein level. P -value summaries are as follows: p > 0.05 (ns-not significant); p ≤ 0.05 (*); p < 0.01 (**); p < 0.001 (***).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Drug discovery, Derivative Assay, Flow Cytometry, Luciferase, Labeling, Imaging, Fluorescence

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 inhibitor increases IKAROS DNA-binding to BCL-XL . Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) and analysis of genome-wide occupancy of IKAROS was performed on U937 following the CX-4945 treatment at 10 µM concentration for 72 h. A change in IKAROS binding to promoter regions of the BCL-XL gene was analyzed following the CX-4945 treatment. ( A ) A chIP-seq signal map for IKAROS binding to the BCL-XL/BCL- 2L1 promoter region in U937-untreated labeled as WT (wild-type) (top panel) and CX-4945 treated U937 (bottom panel). Y-axis represents the log-2-fold change enrichment of IKAROS binding (** p < 0.01). ( B ) U937 and primary AML cells (AML-1) cells were treated with 10 and 5 μM of CX-4945, respectively (based on the IC50 value) for 48 and 72 h. IKAROS binding to the BCL-XL promoter region was confirmed using the qChIP assay in WT and CX-4945 treated cells. Binding at 72 h was not significantly increased compared to the 48 h treatment (not shown in the graph). Results are the mean +/– SD of three independent experiments. P-value summaries are as follows: p > 0.05 (ns-non significant); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Next-Generation Sequencing, ChIP-sequencing, Genome Wide, Concentration Assay, Labeling

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 and IKAROS regulate sensitivity towards daunorubicin. CK2 overexpressing U937 ( A , B ) and THP-1 ( C , D ) were treated with 10 nM or 50 nM of daunorubicin for 48 hand stained with 7-AAD and Annexin V for flow cytometry to determine apoptosis. Flow plots ( A , C ) and percent apoptotic cells (early + late apoptosis) are shown in ( B , D ). Flow plots showing representative results from three replicates. The percentage of cells in the right upper and lower quadrant of each flow chart represents the percentage of late and early apoptotic cells, respectively. Q1-dead, Q2-late apoptosis, Q3-early apoptosis, Q4-live. ( E ) Cytotoxicity and drug response measured by MTT assay after treating CK2 overexpressing U937 and THP-1 cells and respective controls with various concentrations of daunorubicin for 48 h. p > 0.05 (ns); p < 0.05 (*); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Staining, Flow Cytometry, MTT Assay

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: CK2 inhibition potentiates daunorubicin-induced apoptosis in AML cells. Cells were treated with 10 µM of CX-4945 or a combination of 10 µM CX-4945 with 10 nM of daunorubicin for up to 48 h. Cells were stained with 7-AAD and Annexin V for flow cytometry to assess apoptosis. ( A ) Flow plots showing representative results from three replicate experiments. The percentage of cells in the right upper and lower quadrant of each flow chart represents the percentage of late and early apoptotic cells, respectively, in U937 (top row), THP-1 (middle row), and AML-1 cells (bottom row) Q1: Dead, Q2: Late apoptosis, Q3: Early apoptosis, Q4: Live. Graphed in ( B ) are the mean +/−SD of triplicates from two independent experiments showing the percent of apoptosis cells following the drug treatment, as indicated above. ( C ) CK2α silencing in U937 cells achieved using ShCK2α and sorted for GFP after 24 h. Sorted cells were treated with 10 nM of DNR for 24 h before staining for Annexin V and 7AAD to assess apoptosis. The graph shows the combined percent apoptotic cells (early + late) in each group with and without the DNR treatment. ( D ) Cytotoxic drug response measured by the MTT assay after treating CK2α ShRNA treated U937 cells and the respective controls with various daunorubicin concentrations for 24 h. ( E ) Cells were pretreated as above for 48 h and were plated in a Methocult medium. Colonies were counted under an inverted light microscope. Colonies that contained around 50 cells or more were counted for analysis. Graphed in E is the number of colonies after 14 days as the mean of three replicates +/− SD of two independent experiments. ( F ) The qRT-PCR showing decreases in the mRNA level in U937 cells treated with daunorubicin alone (10 nM) or a combination of CX-4945 and daunorubicin. p -value summaries are as follows: p > 0.05 (ns); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques: Inhibition, Staining, Flow Cytometry, MTT Assay, shRNA, Light Microscopy, Quantitative RT-PCR

Journal: Cancers

Article Title: Mechanistic Basis for In Vivo Therapeutic Efficacy of CK2 Inhibitor CX-4945 in Acute Myeloid Leukemia

doi: 10.3390/cancers13051127

Figure Lengend Snippet: Model illustration of the regulation of apoptosis in AML by CK2 and IKAROS via repression of BCL-XL.

Article Snippet: The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences.

Techniques:

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

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: ( A ) Schematic of the domain structures of NIX, BNIP3, FUNDC1, and BCL2L13. LC3-interacting motif (LIR), Minimal essential region (MER), Bcl-2 Homology domain (BH), transmembrane domain (TMD). ( B ) Representative SDS-PAGE gels of NIX(1-182aa)-GST, BNIP3(1-158aa)-GST, FUNDC1(1-50aa)-GST, and BCL2L13(1-465aa)-GST. Arrows indicate the predicted molecular weight. ( C-H ) Microscopy-based bead assay of agarose beads coated with the indicated GST-tagged cargo receptors and incubated with (C) GFP-tagged FIP200-CTR (residues 1429-1591), (D) GFP-tagged full-length FIP200, (E) FIP200-CTR and kinases TBK1, MBP-ULK1, CK2, or Src (Y530F; constitutively active mutant), (F) full-length FIP200 and kinases TBK1, MBP-ULK1, CK2, or Src (Y530F; constitutively active mutant), (G) FIP200-CTR and Lambda Protein Phosphatase, (H) full-length FIP200 and Lambda Protein Phosphatase. Samples were analyzed by confocal imaging and one of three representative experiments is shown.

Article Snippet: The following chemicals were used in this study: Rapalog A/C hetero-dimerizer (635057, Takara), Bafilomycin A1 (sc-201550, Santa Cruz Biotech), TBK1 inhibitor GSK8612 (S8872, Selleck Chemicals), ULK1/2 inhibitor (MRT68921, BLDpharm), Vps34-IN1 inhibitor (APE-B6179, ApexBio), CK2 kinase inhibitor (CX4945, Selleckchem), Deferiprone (379409, Sigma Aldrich), oligomycin A (A5588, ApexBio), Antimycin A1 (A8674, Sigma-Aldrich), Q-VD-OPh (A1901, ApexBio), and DMSO (D2438, Sigma).

Techniques: SDS Page, Molecular Weight, Microscopy, Incubation, Mutagenesis, Imaging

Journal: bioRxiv

Article Title: Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

doi: 10.1101/2024.08.28.609967

Figure Lengend Snippet: (A) Representative SDS-PAGE gels of purified Src (Y530F), CK2 complex, TBK1, and MBP-ULK1. Arrows indicate the predicted molecular weight. ( B-C ) Measurement of kinase activity using a plate-reader based read-out. Kinases were incubated with or without a substrate peptide or kinase inhibitor. Kinase activity was compared between our purified CK2 complex (home-made) and commercially available CK2, or between wild-type (WT) and Y530F mutant Src. ( D ) Measurement of kinase activity by mixing recombinantly purified mCherry-OPTN and TBK1 for the indicated time and western blot analysis using antibodies for phosphorylated OPTN (S177) as a read out for TBK1 activity. ( E ) As in D, but after mixing recombinantly purified MBP-ULK1 and the PI3KC3-C1 complex (composed of ATG14, Beclin-1, Vps15, Vps34) for the indicated time and using antibodies for phosphorylated Beclin-1 (Ser30) as a read out for ULK1 activity. One-way ANOVA with Dunnett’s multiple comparisons test (B, C). **P<0.005, ****P<0.0001. ns, not significant.

Article Snippet: The following chemicals were used in this study: Rapalog A/C hetero-dimerizer (635057, Takara), Bafilomycin A1 (sc-201550, Santa Cruz Biotech), TBK1 inhibitor GSK8612 (S8872, Selleck Chemicals), ULK1/2 inhibitor (MRT68921, BLDpharm), Vps34-IN1 inhibitor (APE-B6179, ApexBio), CK2 kinase inhibitor (CX4945, Selleckchem), Deferiprone (379409, Sigma Aldrich), oligomycin A (A5588, ApexBio), Antimycin A1 (A8674, Sigma-Aldrich), Q-VD-OPh (A1901, ApexBio), and DMSO (D2438, Sigma).

Techniques: SDS Page, Purification, Molecular Weight, Activity Assay, Incubation, Mutagenesis, Western Blot