Journal: Frontiers in Pharmacology

Article Title: Identification of potential diagnostic targets and therapeutic strategies for anoikis-related biomarkers in lung squamous cell carcinoma using machine learning and computational virtual screening

doi: 10.3389/fphar.2025.1500968

Figure Lengend Snippet: Molecular docking and molecular dynamics simulation. (A) The docking result of the CSNK2A1-Dihydroergotamine complex. (B–F) The curve of CSNK2A1 protein and Dihydroergotamine complex: RMSD, RMSF, Rg, Hydrogen bond analysis, and SASA. Curves for the CSNK2A1 protein and the Dihydroergotamine complex. (G) Comparison of conformation of the complex at five different molecular dynamics simulation time points. (H) Free energy distribution. (I) Average binding free energy. (J) Contributions of amino acid residues involved in binding.

Article Snippet: The membrane was blocked with 5% skimmed milk for 1 h at room temperature and then incubated overnight at 4°C with rabbit anti-CSNK2A1 antibody (10992-1-AP, Proteintech, China) and rabbit anti-GAPDH antibody (60004-1-Ig, Proteintech, China).

Techniques: Comparison, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Identification of potential diagnostic targets and therapeutic strategies for anoikis-related biomarkers in lung squamous cell carcinoma using machine learning and computational virtual screening

doi: 10.3389/fphar.2025.1500968

Figure Lengend Snippet: Effect of Dihydroergotamine on NCI-H2170 cells viability, CSNK2A1 expression, migration, invasion. (A) Inhibition of NCI-H2170 cells viability by Dihydroergotamine. (B) Changes in CSNK2A1 expression in NCI-H2170 cells after Dihydroergotamine treatment. (C) Effect of 40 μM Dihydroergotamine on the migration of NCI-H2170 cells. (D) Effect of 40 μM Dihydroergotamine on the invasion of NCI-H2170 cells.

Article Snippet: The membrane was blocked with 5% skimmed milk for 1 h at room temperature and then incubated overnight at 4°C with rabbit anti-CSNK2A1 antibody (10992-1-AP, Proteintech, China) and rabbit anti-GAPDH antibody (60004-1-Ig, Proteintech, China).

Techniques: Expressing, Migration, Inhibition

Journal: iScience

Article Title: MOTS-c modulates skeletal muscle function by directly binding and activating CK2

doi: 10.1016/j.isci.2024.111212

Figure Lengend Snippet: Endogenous and exogenous MOTS-c regulates CK2 activity in the skeletal muscle (A–C) CK2 activity assessed by detecting endogenous proteins containing a pS/pTDXE motif and MOTS-c expression levels in gastrocnemius muscle from young control (2 months) and aged (22 months) mice ( n = 4 per group). (D–F) Effect of 4 weeks of voluntary wheel running exercise on CK2 activity and MOTS-c expression levels in gastrocnemius muscle from young mice ( n = 4 per group). (G and H) Effect of 8 weeks of MOTS-c administration (5 mg/kg/day) on CK2 activity in quadriceps muscle from high-fat-diet (HFD)-fed mice ( n = 4 per group). (I) Summary of MOTS-c and CK2 activity in the skeletal muscle. Data are represented as mean ± SEM for (B, C, E, F, and H). ∗∗ p < 0.01.

Article Snippet: First, 70 nM of CK2α (GenScript) or CK2β (IBL) was flowed over to check the bingeing between MOTS-c and CK2α or CK2β.

Techniques: Activity Assay, Expressing, Control

Journal: iScience

Article Title: MOTS-c modulates skeletal muscle function by directly binding and activating CK2

doi: 10.1016/j.isci.2024.111212

Figure Lengend Snippet: MOTS-c directly binds and activates CK2 in cell-free systems (A–C) Dot blot assays with (A) CK2 complex (contains both CK2α and CK2β subunits) immobilized, MOTS-c flowed over the membrane, and detected by MOTS-c antibody. (B–C) MOTS-c immobilized, CK2 complex flowed over the membrane, and detected by CK2α (B) and CK2β (C) antibodies. (D) CK2α or CK2β immobilized, MOTS-c flowed over the membrane, and detected by MOTS-c antibody. FO, flow over; IB, immunoblotting. (E): Surface plasmon resonance (Biacore assay) of MOTS-c (10 μg/mL) and CK2α (2.5 nM, 5.0 nM, 10 nM, and 20 nM). MOTS-c was immobilized on the sensor chip and CK2α flowed over the sensor chip. K D , dissociation constant. (F) Molecular docking simulation of the binding between MOTS-c and CK2α by using AlphaFold2. (G) CK2 activity assessed by kinase activity assay with/without MOTS-c in cell-free condition. A different dose of MOTS-c (0–100 μM) was used for the assay. Data are represented as mean ± SEM for (G). ∗ p < 0.05, ∗∗ p < 0.01 versus CK2 without MOTS-c group.

Article Snippet: First, 70 nM of CK2α (GenScript) or CK2β (IBL) was flowed over to check the bingeing between MOTS-c and CK2α or CK2β.

Techniques: Dot Blot, Membrane, Western Blot, SPR Assay, Binding Assay, Activity Assay, Kinase Assay

Journal: iScience

Article Title: MOTS-c modulates skeletal muscle function by directly binding and activating CK2

doi: 10.1016/j.isci.2024.111212

Figure Lengend Snippet: MOTS-c modulates CK2 activity in a tissue-specific manner (A) Experimental design of a single MOTS-c administration (7.5 mg/kg) experiment in young mice ( n = 5 per time point). (B) Plasma MOTS-c levels after MOTS-c administration. ∗: p < 0.05 versus time 0. (C) Quantification of CK2 activity assessed by western blotting in each tissue shown in (E–F). (E–F) CK2 activity assessed by western blotting with p-CK2 substrate antibody in gastrocnemius muscle (D), epididymal fat (E), and liver (F) after MOTS-c administration. ∗ p < 0.05, ∗∗ p < 0.01 versus time 0 in same tissue. (G–I) MOTS-c detection following CK2α immunoprecipitation (IP) in gastrocnemius muscle (G), epididymal fat (H), and liver (I) 30 min after MOTS-c administration (7.5 mg/kg). The "-" indicates tissues from non-MOTS-c administered mice, while the "+" indicates tissues from MOTS-c administered mice. (J–K) CK2 activity after 10 min MOTS-c treatment (10 μM) in differentiated skeletal muscle (J) and adipocyte (K). Data are represented as mean ± SEM for (B, C, J, and K).

Article Snippet: First, 70 nM of CK2α (GenScript) or CK2β (IBL) was flowed over to check the bingeing between MOTS-c and CK2α or CK2β.

Techniques: Activity Assay, Western Blot, Immunoprecipitation

Journal: iScience

Article Title: MOTS-c modulates skeletal muscle function by directly binding and activating CK2

doi: 10.1016/j.isci.2024.111212

Figure Lengend Snippet: MOTS-c modulates the CK2 interactome in a tissue-specific manner (A) Experimental design for the interactome analyses in young mice ( n = 3 per condition). Proteome analysis was performed following CK2α immunoprecipitation in gastrocnemius muscle and epididymal fat with/without MOTS-c administration. (B) Principal components (PCs) of control and MOTS-c-treated mouse gastrocnemius muscle and epididymal fat. (C) Venn diagram of interacting proteins in each condition. (D–E) Numbers of CK2 interacting proteins (D) and enriched Reactome pathways (E) in gastrocnemius muscle. (F–G) Numbers of CK2 interacting proteins (F) and enriched Reactome pathways (G) in epididymal fat. (H) Interacting proteins and significantly enriched Reactome pathways in MOTS-c-administered gastrocnemius muscle compared to the control group. The protein-protein interactions and enrichment analysis were assessed by using the STRING database. (I) Summary of the interactome analysis in gastrocnemius muscle and epididymal fat.

Article Snippet: First, 70 nM of CK2α (GenScript) or CK2β (IBL) was flowed over to check the bingeing between MOTS-c and CK2α or CK2β.

Techniques: Immunoprecipitation, Control

Journal: iScience

Article Title: MOTS-c modulates skeletal muscle function by directly binding and activating CK2

doi: 10.1016/j.isci.2024.111212

Figure Lengend Snippet: A naturally occurring K14Q MOTS-c is a bio-inactive form of MOTS-c due to its reduced binding to CK2 alpha (A) Nucleotide and amino acid substitutions of naturally occurring MOTS-c variant K14Q MOTS-c, which modulates skeletal muscle function and increases type 2 diabetes risk. (B) Comparison of reciprocal K D between WT and K14Q MOTS-c assessed by the surface plasmon resonance (Biacore) assay. (C) Comparison of CK2 activating effects between WT MOTS-c and K14Q MOTS-c in cell-free condition ( n = 3 per group). MOTS-c concentrations are 0, 0.8, 1.6, 3.1, 6.3, 12.5, and 25 μM ∗ p < 0.05, ∗∗ p < 0.01 versus control. # p < 0.05 versus same concentration of K14Q MOTS-c. (D–E) Comparison of gastrocnemius muscle CK2 activity between WT MOTS-c- and K14Q MOTS-c-administered young mice (2.5 mg/kg, n = 5 per group). ∗ p < 0.05 versus control group. (F) Protective effect of MOTS-c administration (WT or K14Q MOTS-c, 15 mg/kg/day, IP injection) against 8 days of immobilization-induced skeletal muscle atrophy ( n = 8 per group). Skeletal muscle mass was assessed by a total mass of gastrocnemius, plantaris, and soleus muscles. ∗ p < 0.05, ∗∗ p < 0.01. (G)Skeletal muscle 2-deoxy-d-glucose (2DG) uptake after MOTS-c administration (WT or K14Q, 7.5 mg/kg) with/without CK2 inhibitor (CX-4945, 25 mg/kg) ( n = 10–11 per group). ∗ p < 0.05. Data are represented as mean ± SEM for (C, E, F, and G).

Article Snippet: First, 70 nM of CK2α (GenScript) or CK2β (IBL) was flowed over to check the bingeing between MOTS-c and CK2α or CK2β.

Techniques: Binding Assay, Variant Assay, Comparison, SPR Assay, Control, Concentration Assay, Activity Assay, Injection, Muscles

Journal: iScience

Article Title: MOTS-c modulates skeletal muscle function by directly binding and activating CK2

doi: 10.1016/j.isci.2024.111212

Figure Lengend Snippet:

Article Snippet: First, 70 nM of CK2α (GenScript) or CK2β (IBL) was flowed over to check the bingeing between MOTS-c and CK2α or CK2β.

Techniques: Recombinant, RNA Sequencing Assay, Software, Immunoprecipitation, Blocking Assay, Western Blot, Bicinchoninic Acid Protein Assay

Journal: Molecular Biology of the Cell

Article Title: Altered Lipid Droplet Dynamics in Hepatocytes Lacking Triacylglycerol Hydrolase Expression

doi: 10.1091/mbc.E09-05-0364

Figure Lengend Snippet: Ablation of TGH leads to TG accumulation in OA treated hepatocytes. WT and TGH KO hepatocytes were incubated overnight with DMEM containing 0.4 mM oleic acid complexed to 0.5% BSA. Three dishes of each cell type were analyzed. (A) Cytosol were isolated from cell homogenates, and TG mass was determined by gas chromatography. (B) Cell homogenates were analyzed for ADRP, calnexin (CNX), and TGH levels by Western blotting. Twenty micrograms of cell protein from cell homogenates was analyzed. ADRP levels were determined by densitometry and normalized to calnexin. Note that anti-TGH sera recognize both TGH (bottom band) and a protein highly homologous to TGH, Es-x (top band). *p < 0.05; **p = 0.001; NS, not significant.

Article Snippet: Anti-protein disulfide isomerase (PDI) and anti-calnexin polyclonal antibodies were from AKELA Pharma (Montreal, QC, Canada).

Techniques: Incubation, Isolation, Gas Chromatography, Western Blot

Journal: Plant Physiology

Article Title: The Acidic A-Domain of Arabidopsis Toc159 Occurs as a Hyperphosphorylated Protein ^{1 [W] [OA]}

doi: 10.1104/pp.110.158048

Figure Lengend Snippet: Phosphorylation of recombinant A-domains by CK2. A, One microgram of purified Toc159A-His-6x or casein as a control was incubated without (lanes 1 and 4) or with (lanes 2 and 5) recombinant maize CK2 α-subunit and ATP/[γ-33P]ATP as the phosphate donor. In addition, phosphorylation of Toc159A-His-6x with CK2 was performed in the presence of 15 μg mL−1 heparin as an inhibitor (lane 3). The samples were separated by SDS-PAGE followed by Coomassie Brilliant Blue staining (lanes 1′–5′) and phosphorimager analysis (lanes 1–5). B, One microgram of purified Toc33G-His-6x, Toc120A-His-6x, Toc132A-His-6x, Toc159A-His-6x, or GST-Toc159G was incubated without (even lanes) or with (odd lanes) recombinant CK2 and ATP/[γ-33P]ATP as the phosphate donor. The samples were separated by SDS-PAGE followed by Coomassie Brilliant Blue staining (lanes 1′–10′) and phosphorimager analysis (lanes 1–10).

Article Snippet: Phosphorylation Assays One microgram of recombinant protein or casein was incubated with or without 37.5 units of recombinant maize ( Zea mays ) CK2 α -subunit (Biaffin) in the presence of 25 m m Tris-HCl, pH 8.0, 5 m m MgCl 2 , 1 m m dithiothreitol (DTT), 50 μ m ATP, and 1 μ Ci of [ γ - 33 P]ATP for 30 min at 25°C.

Techniques: Phospho-proteomics, Recombinant, Purification, Control, Incubation, SDS Page, Staining

Journal: Plant Physiology

Article Title: The Acidic A-Domain of Arabidopsis Toc159 Occurs as a Hyperphosphorylated Protein ^{1 [W] [OA]}

doi: 10.1104/pp.110.158048

Figure Lengend Snippet: Inhibition of in vitro chloroplast protein import by heparin. A, Isolated Arabidopsis chloroplasts were preincubated without or with 15 μg mL−1 heparin, 6 μm DRB, 3 units μL−1 recombinant maize CK2 α-subunit, 50 μm apigenin, or 10 mm glycerol-2-phosphate for 20 min at 25°C in the dark. Then, in vitro-translated, [35S]Met-labeled preprotein of the small subunit of Rubisco (pSSu) was added and import was allowed to proceed for 0, 7.5, and 15 min. B, Quantification of the effect of heparin on chloroplast protein import. The graph shows the quantification of the amount of imported SSu at 15 min in three independent experiments. In both panels, the amount of SSu imported into wild-type (WT) chloroplasts without the addition of inhibitor at 15 min was set to 100%. IVT, In vitro translate; M, molecular mass standard. Asterisks indicate pSSu modified in the course of the import reactions.

Article Snippet: Phosphorylation Assays One microgram of recombinant protein or casein was incubated with or without 37.5 units of recombinant maize ( Zea mays ) CK2 α -subunit (Biaffin) in the presence of 25 m m Tris-HCl, pH 8.0, 5 m m MgCl 2 , 1 m m dithiothreitol (DTT), 50 μ m ATP, and 1 μ Ci of [ γ - 33 P]ATP for 30 min at 25°C.

Techniques: Inhibition, In Vitro, Isolation, Recombinant, Labeling, Modification

Journal: Brain Pathology

Article Title: Brain‐Derived Neurotrophic Factor Enhances Bcl‐xL Expression Through Protein Kinase Casein Kinase 2‐Activated and Nuclear Factor Kappa B‐Mediated Pathway in Rat Hippocampus

doi: 10.1111/j.1750-3639.2010.00431.x

Figure Lengend Snippet: Casein kinase 2 (CK2) increases nuclear factor kappa B (NF‐κB) phosphorylation and Bcl‐xL mRNA expression. Effects of CK2αWT DNA transfection and CK2α156A mutant DNA transfection on (A) NF‐κB phosphorylation at Ser529 and (B) Bcl‐xL mRNA expression in CA1 area. Animals received plasmid DNA transfection (1.5 µg) in CA1 area bilaterally and were sacrificed 48 h later. n = 6–10 each group. Statistical significance was evaluated by one‐way analysis of variance followed by Dunnett's t‐test. **P < 0.01. C. Immunohistochemical staining showing CK2αWT DNA transfection and expression in CA1 area (left panel, arrow indicates the area of plasmid expression). The anti‐HA‐tagged antibody and fluorescein isothiocyanate (FITC)‐conjugated IgG secondary antibody were used for visualization of plasmid transfection and expression in individual cells in CA1 area at a higher magnification (right panel). Dotted lines indicate the CA1 area. Scale bar equals 250 µm for the left panel and scale bar equals 25 µm for the right panel. D. Confocal images showed the co‐expression of CK2α and 4′,6‐diamidino‐2‐phenylindole (DAPI) in the same cells in CA1 area (upper panels). Arrow indicates the right half of the tissue showing coexpression and was used for viewing at a higher magnification (right three panels). Confocal images also showed the co‐expression of CK2α and NeuN in the same neurons in CA1 area (lower panels). Similarly, arrow indicates the right half of the tissue showing co‐expression and was further viewed at a higher magnification (right three panels). Scale bar of the lower left panel equals 100 µm and scale bar of the lower right panel equals 25 µm.

Article Snippet: Both sets of CK2α siRNA were synthesized and conjugated with Cy3 by MDBio Inc. (Taipei, Taiwan).

Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, Immunohistochemical staining, Staining

Journal: Brain Pathology

Article Title: Brain‐Derived Neurotrophic Factor Enhances Bcl‐xL Expression Through Protein Kinase Casein Kinase 2‐Activated and Nuclear Factor Kappa B‐Mediated Pathway in Rat Hippocampus

doi: 10.1111/j.1750-3639.2010.00431.x

Figure Lengend Snippet: CK2α small interfering RNA (siRNA) antagonized the effects of brain‐derived neurotrophic factor (BDNF) on nuclear factor kappa B (NF‐κB) phosphorylation and Bcl‐xL expression. A. Effect of CK2α siRNA transfection on basal casein kinase II (CK2) protein level in CA1 area (n = 4 each group). B. Representative immunohistochemical staining against Cy3 and confocal image showing the co‐expression of CK2α siRNA and 4′,6‐diamidino‐2‐phenylindole (DAPI) in the same neurons in CA1 area (color in pink). Arrow indicates the right half of the CA1 tissue showing the co‐expression and was further viewed at a higher magnification. Dotted lines indicate the CA1 area. CK2α siRNA (upper right) and DAPI (lower left) staining alone at a higher magnification was shown. The merged image at a higher magnification showed the co‐expression of CK2α siRNA and DAPI in the same neurons in CA1 area. Scale bar equals 360 µm for the upper left panel and scale bar equals 36 µm for the lower right panel. Effects of prior CK2α siRNA transfection (8 pmol) on BDNF‐induced (C) NF‐κB phosphorylation at Ser529 (D) Bcl‐xL mRNA level and (E) Bcl‐xL protein level in CA1 area. Animals received bilateral transfections of CK2α siRNA (or negative siRNA) 60 h prior to 0.4 µg BDNF infusion in CA1 area and were sacrificed 4 h after BDNF infusion. N = 4–5 each group. Data are expressed as mean ± standard error of the mean. Statistical significance was evaluated by Student's t‐test or one‐way analysis of variance followed by Newman–Keul's comparison. *P < 0.05, **P < 0.01.

Article Snippet: Both sets of CK2α siRNA were synthesized and conjugated with Cy3 by MDBio Inc. (Taipei, Taiwan).

Techniques: Small Interfering RNA, Derivative Assay, Expressing, Transfection, Immunohistochemical staining, Staining

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Status of B23 in the cytoplasm and NM fractions of prostate cancer cells in response to CK2 specific inhibitor TBB mediated induction of apoptosis. A, TUNEL staining of ALVA-41 and PC-3 cells treated with varying doses of TBB (from 50 to 150 µM). Induction of apoptosis is shown. Cells were counterstained with Hoechst 33342 to identify nuclear component. B, cytoplasmic and NM associated B23 in ALVA-41 and PC-3 cells treated with TBB at varying concentrations as under A. The relative change in the protein bands is calculated compared to the respective control and normalized to the β-actin in the sample (e.g., lanes 2 and 3 from left in Fig. 1B had a higher amount of protein to ensure visualization of B23 signal in the nuclear matrix). C, effect of TBB and apigenin on CK2 activity at the concentrations of the inhibitors shown. D, measurement of the message expression for CK2α in ALVA-41 and PC-3 cells under the same conditions as for C. E, measurement of B23 message in ALVA-41 and PC-3 cells under the same conditions as for C.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: TUNEL Assay, Staining, Control, Activity Assay, Expressing

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Status of B23 in the cytoplasm and NM fractions of prostate cancer cells in response to siRNA-mediated downregulation of CK2. A, cell viability was determined by WST-1 assay in ALVA-41 and PC-3 cells transfected with varying concentrations of CK2••′ siRNA for the periods of time shown. B, ALVA-41 and PC-3 cells were transfected with varying concentrations of CK2αα′ siRNA as under A. Cyclophilin B (Dharmacon, Catalog: D-001136-01-05) was employed as a control. All other details were as described under Experimental Procedures. TUNEL staining shows the induction of apoptosis in cells treated with CK2 siRNA. Cells were counterstained with Hoechst 33342 to identify nuclei. C, immunoblot analysis of cytoplasmic and NM associated B23 and CK2••′ was carried out in ALVA-41 and PC-3 cells transfected with varying concentrations of CK2••′ siRNA. Lane a, untreated control; lane b, 5 nM CK2••′ siRNA; lane c, 10 nM CK2••′ siRNA; lane d, 50 nM CK2αα′ siRNA; lane e, untreated control; lane f, DharmaFECT control, lane g, 10 nM CK2αα′ siRNA; lane h, 100 nM CK2αα′ siRNA. The relative change in the protein bands is calculated compared to the respective control.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: WST-1 Assay, Transfection, Control, TUNEL Assay, Staining, Western Blot

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Effect of apoptosis inducing conditions on NM-associated B23. A, prostate cancer cells (ALVA-41) were treated with doses of etoposide, or CK2 inhibitors (TBB or Apigenin) at doses that are sub-optimal for induction of apoptosis. The results show a minimal effect on cell death under these conditions. When the sub-optimal doses of the same inhibitors are combined it results in induction of apoptosis, as shown in the chart. Illustrated in A are the data employing etoposide (an apoptosis-inducing agent) with and without CK2 inhibitors TBB or apigenin. Similar effects have previously been reported using TRAIL (an apoptosis-inducing agent) with and without TBB at sub-optimal doses (27) (the latter conditions were employed in the experiment shown under C. B, NM-associated B23 immunoreactive protein in cells treated with TBB, apigenin and etoposide, as shown. Lane a, control; lane b, 20 µM etoposide (sub-optimal for induction of apoptosis); lane c, TBB at 40 µM (sub-optimal for induction of apoptosis); lane d, etoposide plus TBB (combined effect to produce apoptotic activity); lane e, apigenin at 20 µM (insufficient for induction of apoptosis); lane f, apigenin plus etoposide (combined effect to produce apoptotic activity). Densitometric values are calculated relative to the level in control cells. C, NM-associated B23 immunoreactive protein in cells treated with TBB, apigenin and TRAIL as shown. Lane a, control; lane b, TRAIL 2 ng/ml (sub-optimal dose which is insufficient for induction of apoptosis); lane c, TBB at 40 µM (sub-optimal dose for induction of apoptosis); lane d, TBB plus TRAIL (combined effect evokes apoptotic activity); lane e, apigenin at 20 µM (insufficient dose for induction of apoptosis); lane f, TRAIL plus apigenin (conditions to induce apoptotic activity). Relative densitometric values are based on level in the control cells. D, combined effect of TBB and TRAIL (both at sub-optimal doses) in causing induction of apoptosis is illustrated by cleavage of lamin A in treated cells, as shown. Lane a, control; lane b, 60 µM TBB (insufficient for induction of apoptosis) showing no cleavage of Lamin A; lane c, 2 ng/ml of TRAIL (insufficient for apoptosis induction) as indicated by a minimal cleavage of Lamin A; lane d, 60 µM TBB plus 2 ng/ml of TRAIL resulting in potent induction of apoptosis as indicated by extensive cleavage of Lamin A. These results accord with the corresponding effect on NM-associated B23 as shown under C. In all cases, equal amount of protein was loaded in the gels, and each experiment was confirmed at least three times.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Control, Activity Assay

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Effect of overexpression of CK2α on the status of NM-associated B23 in prostate cancer cells treated with apoptotic inducers. ALVA-41 and PC-3 cells were treated with etoposide or TRAIL at apoptosis inducing concentrations in the absence or presence of forced overexpression of the catalytic subunit CK2α as described under Experimental Procedures. A, treatment with TRAIL at 10 ng/ml for ALVA-41 and 20 ng/ml for PC-3 cells (for 24 h) was carried out to induce apoptotic condition in cells. pcDNA6-CK2α expression vector (2.0 µg/ml) was employed to achieve overexpression of CK2α in ALVA-41 and PC-3 cells, as indicated. NM-associated B23 is shown for each experimental condition; there was no change in cytoplasmic B23 immunoreactive B23 under these conditions (not shown). Lane a, control cells treated with pcDNA6; lane b, pcDNA6 plus TRAIL; lane c, pcDNA6-CK2α; and lane d, pcDNA6-CK2α plus TRAIL. B, treatment of ALVA-41 and PC-3 cells with 50 µM etoposide (apoptosis-inducing level). Overexpression of CK2α was achieved as described under A. NM-associated immunoreactive B23 is shown. Lane a, pcDNA6 control; lane b, pcDNA6 plus 50 µM etoposide for 48 h; lane c, pcDNA6-CK2α; and lane d, pcDNA6-CK2α plus etoposide. C, a representative experiment showing confirmation of CK2α overexpression in PC-3 cells by transient transfection with pcDNA6-CK2α is illustrated by analysis of the message and protein levels.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Transfection

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Colocalization of CK2α and B23 in the nucleus. A, double immunofluorescence staining for anti-CK2α and anti-B23 was carried out in ALVA-41 cells treated with varying doses of TBB, as shown. Merged image of the immunofluorescence stains for anti-CK2α and anti-B23 shows co-localization of the two proteins in the nucleus. Downregulation of CK2 by TBB treatment (producing apoptotic condition) shows co-ordinate loss of B23 and CK2α in the nucleus. B, double immunofluorescence staining for anti-CK2α and anti-B23 was carried out in PC-3 cells treated with varying doses of TBB. All details are the same as for A. Results confirm the co-localization of CK2α and B23 in the nucleus and its reduction on induction of apoptotic condition induced by TBB mediated inhibition of CK2 as observed for ALVA-41 cells under A.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Double Immunofluorescence Staining, Immunofluorescence, Inhibition

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Effect of altered androgenic status on CK2α and B23 levels in rat ventral prostate tissue. Lysate, cytoplasm and nuclear matrix fractions were isolated from prostatic tissue of rats subjected to various treatments as shown. Each fraction was analyzed by Western blotting for the presence of immunoreactive CK2α, B23 and lamin A. A, level of B23 in total lysates from prostatic tissues isolated from rats with altered androgenic status. Lane a, control rats; lanes b to d, rats orchiectomized for 1, 2, and 3 days, respectively. B, detection of protein CK2α, B23 and lamin A in cytoplasm and NM fractions isolated from prostate tissue of normal rats, or rats subjected to altered androgenic status. Lane a, control rats; lane b, 3-day orchiectomized rats; lane c, 6-day orchiectomized rats; lane d, 6-day orchiectomized rats given 5α-DHT for 4 days; lane e, same as lane d except that 5α-DHT was administered for 7 days. Breakdown of lamin A (to 28 kDa fragment) in the NM fraction indicates apoptotic condition. All other details are as described under Experimental Procedures.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Isolation, Western Blot, Control