Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with HPV genomes and the next day treated either with vehicle or different concentrations of CX4945 for 48 h. Total DNA was extracted from cells, digested with DpnI and restriction enzymes to linearize the HPV genomes, and analyzed using Southern blot (SB). B, C. U2OS cells were transfected with HPV18 genome, next day treated with different concentrations of CX4945 and incubated for the indicated periods of time. Cell cycle profile was analyzed using propidium iodide by flow cytometry. Viability of the cells was examined using MTT assay. D. U2OS cells were transfected with different HPV genomes and siRNAs simultaneously using electroporation. Treatment with 6 μM CX4945 was started 24 h post transfection. Levels of the linearized replicated HPV genomes were analyzed using SB. E. U2OS cells were transfected with HPV18 and siRNAs specific for CK2α and CK2α’ or scrambled Neg. siRNA simultaneously and incubated for 3 or 5 days. The cells incubated for 5 days, were transfected with the same siRNAs on the 3 rd day of incubation. Levels of CK2α, CK2α’ and tubulin proteins were detected using WB. F. left panel: U2OS cells were transfected with HPV18 genome, the next day treated with 6 μM CX4945, incubated for 3, 6, 9 and 12 days and analyzed using SB (lanes 1, 2, 3, 4, 7, 8, 9 and 10). One set of the cells treated with CX4945 for 6 days was switched to treatment with DMSO for additional 3 and 6 days that corresponded to 9 and 12 days post transfection (lanes 5 and 6). Right panel: SB signals from three independent experiments were quantified and set as 100% in the sample treated with DMSO for 3 days. Data are presented as an average mean +/- SD.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Southern Blot, Incubation, Flow Cytometry, MTT Assay, Electroporation

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with different HPVNLuc genomes, propagated for 2, 3 and 4 days and subjected to luciferase assay. Nluc activity was normalized to alkaline phosphatase activity. Normalized Nluc activity was set as 100% in the cells propagated for 2 days. B. U2OS cells were transfected with 250 or 400 ng of the HPV18NLuc genome and incubated for 3, 4 and 5 days. Total DNA was isolated and treated with DpnI and BglI. Replication of HPV18NLuc genome was analyzed using SB. C. U2OS cells were transfected with different amounts of HPV18NLuc genome and FFLuc encoding plasmid and incubated for the indicated periods of time. Signals of HPV18NLuc replication were quantified using ImageQuant software; the average means of three experiments +/- SD are shown (left panel). The NLuc activity was measured and normalized to FFLuc activity (right panel). Signals of HPV18NLuc replication or normalized NLuc activity were set as 1 in the sample transfected with 250 ng of HPV18NLuc and incubated for 3 days. D. U2OS cells were transfected with HPV18NLuc genome, FFLuc encoding plasmid and siRNAs if indicated. The next day, cells were treated with different concentrations of CX4945 or vehicle. Cells were incubated for 3, 4 or 5 days and subjected to luciferase assay. NLuc activity was normalized either to alkaline phosphatase activity in the samples treated with CX4945 or to FFLuc and alkaline phosphatase activities in the samples treated with siRNAs. Normalized NLuc activity in the cells treated with DMSO and incubated for 3 days was set as 1. E. U2OS cells were transfected with different amounts of HPV18NLuc genome and siRNAs. The next day, the cells were treated with 6 μM CX4945 or vehicle. Total DNA was extracted, treated with DpnI and BglI and analyzed using SB. F. NHEKs were transfected with different HPVNLuc genomes, incubated for 48 h and treated with 1 μM CX4945, if indicated. Nluc activity was normalized to total protein concentrations and set as 100% in the samples incubated for 2 days after transfection. NA–not analyzed. All panels: normalized NLuc data are presented as an average mean +/- SD of at least 3 replicates measured in 3 independent experiments.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Luciferase, Activity Assay, Incubation, Isolation, Plasmid Preparation, Software

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. CIN612 cells were transfected with siRNAs one or two times and propagated for 3 or 5 days. Levels of CK2α and CK2α’ proteins were detected using WB. B. CIN612 cells were transfected with siRNAs as is described in the panel A. Levels of CK2α and CK2α’ mRNA expression were measured using qPCR, normalized to GAPDH expression level and set as 100% in a sample transfected with Neg. siRNA. Data from other samples were calculated relative to that. Data are presented as the average mean +/- SD of three independent experiments performed in triplicate. C. CIN612 cells were subjected to single or double transfection with siRNAs and incubated for 3, 5 and 6 days, respectively. Total DNA was extracted, treated with HindIII restriction enzyme and subjected to SB analysis (left panel). SB signals obtained from three independent experiments were quantified, and data from the sample treated with Neg. siRNA and incubated for 3 days was set as 100% (right panel). D. CIN612 cells were treated with the indicated concentrations of CX4945 for 3 or 6 days. Total DNA was digested with HindIII restriction enzyme to linearize the HPV31b genome and subjected to SB analysis. E. Viability of CIN612 cells incubated in the presence of different concentrations of CX4945 for 3 or 6 days was tested using MTT assay. F. CIN612 cells were treated with 0.5 μM CX4945 for 3 or 6 days. The levels of the respective gene mRNA expression were measured by qPCR using 2 different pairs of primers, normalized with GAPDH mRNA expression levels and set as 1 in the samples treated with DMSO for 3 days. G. CIN612 cells were transfected with different HPVNLuc genomes, incubated for 2 days and treated with 0.5 0.5 μM CX4945 for additional 2 and 3 days. NLuc activity was measured, normalized to total protein concentrations and set as 100% in the samples incubated for 2 days. NA–not analyzed. The linearized HPV31b genome was analyzed using SB in the respectively treated samples (right panel). All panels: data are presented as an average mean +/- SD.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Expressing, Incubation, MTT Assay, Activity Assay

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with HPV18E1HA and HPV18wt genomes, the next day treated with 6 μM CX4945 or vehicle and propagated for 3, 4 and 5 days. Total DNA was extracted, digested with DpnI and BglI restriction enzymes and analyzed using SB. Low molecular weight bands correspond to DpnI-sensitive input DNA. B. U2OS cells were transfected with the HPV18E1HA genome and propagated for 2, 3, 5 and 6 days. CX4945 was added to cells 2 days after transfection. Cells were fractionated for isolation of total DNA and WCEs. Total DNA was treated with DpnI and BglI restriction enzymes and analyzed using SB. HA-tagged E1 protein was immunoprecipitated from WCEs and analyzed using WB. Tubulin was used as a loading control. C. U2OS cells were transfected with HPV18E1HA construct and siRNAs, if indicated, incubated for 3 days and treated with 6 μM CX4945 for 24 h. Cells were detached using trypsin-EDTA and fractionated for nuclear (Nuc) and cytoplasmic (Cyt) extracts. Nuclear and cytoplasmic HA-tagged E1 protein was immunoprecipitated using r-a-HA antibody and analyzed by immunoblotting using m-a-HA antibody. The nuclear and cytoplasmic markers lamin B and GAPDH were used. D. U2OS cells were transfected with the HPV18 genome, incubated for 2 days and treated with 6 μM CX4945 for 24 h (left panel). Alternatively, U2OS cells were cotransfected with the HPV18 genome and indicated siRNAs (right panel). Total RNA was extracted, treated with Turbo DNase and used for cDNA synthesis. Levels of E1 , E2 , E1^E4 and E8^E2 transcripts were measured using qPCR, normalized to GAPDH expression level and set as 100% in samples treated with DMSO or transfected with Neg. siRNA. Data are presented as the average means +/- SD of three independent experiments performed in triplicate.

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Molecular Weight, Isolation, Immunoprecipitation, Control, Construct, Incubation, Western Blot, cDNA Synthesis, Expressing

Journal: PLoS Pathogens

Article Title: Activity of CK2α protein kinase is required for efficient replication of some HPV types

doi: 10.1371/journal.ppat.1007788

Figure Lengend Snippet: A. U2OS cells were transfected with the HPV18E1HA genome and incubated for 3 days. Cells were treated with 6 μM CX4945 and/or 10 μM MG132 for 6 h. HA-tagged E1 protein was immunoprecipitated from whole cell extracts (WCEs) using r-a-HA antibody and analyzed by immunoblotting using m-a-HA-HRP antibody (left panel). WB signals from three independent experiments were quantified. E1 level was set as 100% in the cells treated with DMSO (right panel). B. U2OS cells were transfected with the HPV18E1HA genome, incubated for 3 days and treated with 6 μM CX4945 (lanes 3–12) and 10 μM MG132 (lanes 7 and 8) for the indicated periods of time. Nuclear (Nuc) and cytoplasmic (Cyt) extracts were isolated. Nuclear and cytoplasmic HA-tagged E1 protein was immunoprecipitated using r-a-HA antibody and analyzed by immunoblotting using a-HA-HRP antibody. The nuclear and cytoplasmic markers lamin B and GAPDH were used (left panel). WB signals obtained from at least three independent experiments were quantified. The E1 protein level was set as 100% in the NE of cells treated with DMSO. All panels: data are presented as the average means +/- SD of at least three independent experiments (*—p<0.05; ***—p<0.001).

Article Snippet: CX4945 was purchased from Santa Cruz Biotechnology and MG132 (kind gift of Dr. Reet Kurg) was purchased from Sigma-Aldrich.

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Isolation

Journal: Cells

Article Title: Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells

doi: 10.3390/cells12030506

Figure Lengend Snippet: ECE1c K6R mutant was highly stable in GBM cells. Flag-tagged ECE1c WT - or ECE1c K6R -expressing U87MG ( A ), T98G ( B ), and U251 ( C ) cells were treated with 20 μg/mL cycloheximide (CHX) in the absence or presence of 25 μM silmitasertib for 6 h. ECE1c protein levels were evaluated using Western blots with an anti-Flag antibody, using β-actin as a loading control. Representative blots from three independent experiments are shown. Representative Western blots of ECE1c WT or ECE1c K6R and β-actin from three independent cell lines ( n = 3).

Article Snippet: Then, cells were incubated in the presence of 25 μM silmitasertib (a selective CK2 inhibitor, formerly known as CX-4945; Apexbio, Houston, TX, USA) or vehicle only (0.001% DMSO) for 6 h. Cells were then harvested and lysed, and the total protein was analyzed using Western blots.

Techniques: Mutagenesis, Expressing, Western Blot, Control

Journal: ASN NEURO

Article Title: Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia

doi: 10.1177/17590914231158218

Figure Lengend Snippet: Pathologic deposition of TDP-43 and casein kinase 2 (CK2) immunoreactivity in the basal ganglia of HIV+ individuals. (a,a’ and b,b’) Representative confocal images of TDP-43, casein kinase 1δ (CK1δ), pTDP-43, and CK2 immunofluorescence in HIV− (a,b) and HIV+ (a’,b’) basal ganglia. Note the overall paucity of CK1δ antigenicity and the increased nuclear-to-cytoplasmic redistribution of TDP-43 antigenicity in the neural cells of HIV+ individuals. (c) A high magnification (63×) image showing pTDP-43 and CK2 immunofluorescence colocalization in cells within post-mortem HIV+ basal ganglia.

Article Snippet: Cells were either treated with vehicle (DNase/RNase-free non-pyrogenic Ultrapure water), co-treated with Tat (100 nM) and morphine (500 nM), or a combination of Tat, morphine, and 0.5, 1, or 2 μM concentrations of the highly selective CK2 antagonist CX-4945 (#A11060, AdooQ Bioscience, Irvine, CA).

Techniques: Immunofluorescence

Journal: ASN NEURO

Article Title: Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia

doi: 10.1177/17590914231158218

Figure Lengend Snippet: Assessment of pathologic TDP-43 and casein kinase 2 (CK2) immunoreactivity in the basal ganglia of HIV− and HIV+ individuals. (a) Colocalization of pTDP-43, CK2, and DARPP32 immunoreactivity in cells of post-mortem HIV+ brains confirming the identity of the cells as medium spiny neurons. (b,c) Cytoplasmic TDP-43 (b) and pTDP-43 (c) fluorescence intensities were increased in HIV+ compared to HIV− basal ganglia. (d) Cytoplasmic-to-nuclear ratio of TDP-43 fluorescent intensity was increased in HIV+ tissues indicating mislocalization. (e) CK1δ fluorescent intensity was slightly above background levels and not changed in any of the groups evaluated. (f) Cytoplasmic CK2 intensity was increased in HIV+ compared to HIV− basal ganglia. Pearson correlations revealed no significant relationship between the nuclear (g) and cytoplasmic (h) intensities of pTDP-43 (pooled across groups) vs CK1δ fluorescence. On the contrary, nuclear (i) and cytoplasmic (j) intensities of pTDP-43 fluorescence (pooled across groups) correlated positively with CK2 fluorescence intensities. Mean intensity values indicate the mean fluorescence pixel intensities for each respective protein acquired in optical sections using confocal microscopy and analyzed using CellProfiler TM 6.1 software (see Materials and Methods 2.1.2). Data are expressed as the mean + the SEM ( n = 4-7/group). * indicates a significant difference by Student's t -test ( p < 0.05).

Article Snippet: Cells were either treated with vehicle (DNase/RNase-free non-pyrogenic Ultrapure water), co-treated with Tat (100 nM) and morphine (500 nM), or a combination of Tat, morphine, and 0.5, 1, or 2 μM concentrations of the highly selective CK2 antagonist CX-4945 (#A11060, AdooQ Bioscience, Irvine, CA).

Techniques: Fluorescence, Confocal Microscopy, Software

Journal: ASN NEURO

Article Title: Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia

doi: 10.1177/17590914231158218

Figure Lengend Snippet: Effects of morphine and Tat on the concentrations of TDP-43 and casein kinases in the mouse striatum. (a) Nuclear TDP-43 levels were not changed in any of the groups evaluated. (a,b) Alternatively, morphine exposure increased cytoplasmic TDP-43 levels (a), and nuclear and cytoplasmic levels of pTDP-43 (b), while Tat increased cytoplasmic pTDP-43 concentrations (b). (b) Post hoc comparisons showed differences in nuclear pTDP-43 between saline-treated (Tat+ and Tat−) mice vs mice co-exposed to Tat and morphine, and in cytoplasmic pTDP-43 levels between saline-treated Tat− mice vs mice co-exposed to Tat and morphine. (c) Representative immunoblots of cytoplasmic pTDP-43, TDP-43, CK1δ, CK2α, and CK2α’, and GAPDH. (d) The cytoplasmic-to-nuclear ratios of TDP-43 levels were not changed in any of the treatment groups. (e) Nuclear and cytoplasmic CK1δ levels were not changed in any of the groups evaluated. (f) When CK2 levels were evaluated, Tat increased nuclear CK2α levels, and either Tat or morphine alone increased cytoplasmic CK2α levels. (f) Post hoc comparisons of cytoplasmic CK2α levels showed differences between saline-treated Tat− mice vs Tat+ mice that were exposed to morphine. (g) Nuclear and cytoplasmic levels of the CK2α’ subunit were unaffected by Tat or morphine exposure. Data are expressed as the mean + the SEM ( n = 5-6/group). # indicates significant main effects by two-way ANOVA; * indicates planned contrasts with post hoc Tukey's test ( p < 0.05).

Article Snippet: Cells were either treated with vehicle (DNase/RNase-free non-pyrogenic Ultrapure water), co-treated with Tat (100 nM) and morphine (500 nM), or a combination of Tat, morphine, and 0.5, 1, or 2 μM concentrations of the highly selective CK2 antagonist CX-4945 (#A11060, AdooQ Bioscience, Irvine, CA).

Techniques: Saline, Western Blot

Journal: ASN NEURO

Article Title: Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia

doi: 10.1177/17590914231158218

Figure Lengend Snippet: Effects of morphine and Tat on the relationship between the concentrations of pTDP-43 and TDP-43 kinases. (a,b) No significant correlations were observed between levels of nuclear (a) or cytoplasmic (b) CK1δ and pTDP-43 . (c) No significant correlations were observed between levels of nuclear CK2α vs nuclear pTDP-43 . (d) By contrast, levels of cytoplasmic CK2α positively correlated with cytoplasmic pTDP-43 concentrations ( p < 0.05). (e,f) No significant correlations were observed between levels of nuclear (e) or cytoplasmic (f) CK2α’ ( Figure 3 g ) and pTDP-43 .

Article Snippet: Cells were either treated with vehicle (DNase/RNase-free non-pyrogenic Ultrapure water), co-treated with Tat (100 nM) and morphine (500 nM), or a combination of Tat, morphine, and 0.5, 1, or 2 μM concentrations of the highly selective CK2 antagonist CX-4945 (#A11060, AdooQ Bioscience, Irvine, CA).

Techniques:

Journal: ASN NEURO

Article Title: Casein Kinase 2 Mediates HIV- and Opioid-Induced Pathologic Phosphorylation of TAR DNA Binding Protein 43 in the Basal Ganglia

doi: 10.1177/17590914231158218

Figure Lengend Snippet: The effects of Tat and morphine on pTDP-43 levels and CK2 activity in striatal neurons in vitro . (a) Tat or morphine exposure did not affect the concentration of cytoplasmic TDP-43. (b) By contrast, Tat and morphine co-exposure significantly increased levels of cytoplasmic pTDP-43, and these increases in pTDP-43 were fully reversible by the CK2 antagonist (CX-4945) in a concentration-dependent manner. (c) Tat and morphine co-exposure increased CK2 enzymatic activity in cytoplasmic extracts of striatal neurons. (d) Representative immunoblots of cytoplasmic pTDP-43, TDP-43, and GAPDH. Symbols indicate planned contrasts with post hoc Tukey's test ( p < 0.05). $ vs the control group (no active treatment), $$ vs the control group at the 20 min reaction time, $$$ vs the control group at 40 min, ǂ vs Tat+/morphine group, ^ vs Tat+/morphine and Cx-4945 at 20 min, ^^ vs Tat+/morphine and Cx-4945 at 40 min, * vs the 0 min reaction time. Data are expressed as the mean + or ± the SEM ( n = 4 experiments/group).

Article Snippet: Cells were either treated with vehicle (DNase/RNase-free non-pyrogenic Ultrapure water), co-treated with Tat (100 nM) and morphine (500 nM), or a combination of Tat, morphine, and 0.5, 1, or 2 μM concentrations of the highly selective CK2 antagonist CX-4945 (#A11060, AdooQ Bioscience, Irvine, CA).

Techniques: Activity Assay, In Vitro, Concentration Assay, Western Blot, Control