Journal: PLOS Biology

Article Title: Casein kinase 2-mediated phosphorylation of the splicing factor SF3B3 plays a key role in esophageal squamous cell carcinoma progression

doi: 10.1371/journal.pbio.3003729

Figure Lengend Snippet: (A) Kinase-substrate enrichment analysis (KSEA) was performed based on phosphoproteomic data in ESCC. Kinases are considered hyper-activated when enrichment score > 0 (red) and P < 0.05, while they are regarded as inhibited when the enrichment score < 0 (blue) and P < 0.05. (B) KEGG pathway enrichment analysis results are shown for differential phosphoproteins. Pathways enriched in the upregulated ( n = 1,038) and down-regulated ( n = 574) phosphoproteins are indicated by pink and blue bars, respectively. (C) Fold change (FC) in the abundance of phosphosites conforming to the CK2 consensus motif (S/T X X E/D) is shown (BH adjusted P value < 0.01) (D) The abundance of p-SF3B3 (T1200) in ESCC tumor (T) and adjacent normal (N) tissues ( n = 21). (E) Kaplan–Meier plots of Disease-Free Survival (DFS) in ESCC patients of p-SF3B3 (T1200). (F) The amino acid sequences surrounding T1200 in SF3B3 in different species are shown. The consensus motif for CK2 phosphorylation is shown at the bottom. (G) The specificity of p-SF3B3 (T1200) antibody was examined by using both wild-type (WT, EELDRTPPEVS) and p-T1200 (EELDRT(p)PPEVS) peptides. (H) Bacterially expressed, purified His-SF3B3 (WT) and His-SF3B3 (T1200A) proteins were examined by Coomassie blue staining (C.B.S) and indicated by the red arrow. (I) In vitro phosphorylation assay was performed by mixing bacterially expressed, purified His-SF3B3 (WT) or His-SF3B3 (T1200A) with or without CK2 kinase, followed by immunoblotting (IB) analysis using antibodies as indicated or Ponceau Staining (P.S). (J) KYSE140 cells treated with or without CX-4945 (10 μM, 24 h) were subjected to immunoprecipitation (IP) with anti-SF3B3 antibody, followed by IB analysis with antibodies as indicated. (K) HEK293T cells were transfected with GFP-tagged SF3B3 and then treated with or without CX-4945 (10 μM, 24 h), followed by IP analysis with anti-GFP antibody and IB analysis with antibodies as indicated. (L) KYSE140 cells were subjected to IP analysis with control IgG or anti-SF3B3 antibody, followed by IB analysis with antibodies as indicated. (M) HEK293T cells transfected with FLAG-tagged CSNK2A1 and GFP-tagged SF3B3 were subjected to IP analysis with anti-Flag M2 agarose, followed by IB analysis with antibodies as indicated. (N) HEK293T cells transfected with FLAG-tagged SF3B3 and MYC-tagged CSNK2A1 were subjected to IP analysis with anti-Flag M2 agarose, followed by IB analysis with antibodies as indicated. The data underlying the graphs shown can be found in .

Article Snippet: The proteasome inhibitor MG-132 (T2154), the CK2 inhibitor CX-4945 (T2259), and the USP7 inhibitor P5091 (T6925) were purchased from TargetMol.

Techniques: Phospho-proteomics, Purification, Staining, In Vitro, Western Blot, Immunoprecipitation, Transfection, Control

Journal: PLOS Biology

Article Title: Casein kinase 2-mediated phosphorylation of the splicing factor SF3B3 plays a key role in esophageal squamous cell carcinoma progression

doi: 10.1371/journal.pbio.3003729

Figure Lengend Snippet: (A) KYSE140 and KYSE30 cells were treated with CX-4945 for 0, 12, and 24 h (10 μM), followed by IB analysis with antibodies as indicated. (B) KYSE30 cells were treated with CX-4945 (10 μM) for 12 h and then treated with cycloheximide (CHX) (50 μg/ml) for 0, 4, 8, or 12 h, followed by IB analysis with antibodies as indicated. (C) The quantification of SF3B3 proteins in (B) is shown (mean ± SD; * P < 0.05, Student t test). (D) KYSE30 cells were infected with lentivirus expressing SF3B3 (WT), SF3B3 (T1200A), or SF3B3 (T1200D) for 48 h and then treated with CHX (50 μg/mL) for 0, 4, 8, or 12 hours, followed by IB analysis with antibodies as indicated. (E) The quantification of SF3B3 proteins in (D) is shown (mean ± SD; * P < 0.05, ** P < 0.01, Student t test). (F) The rela t ive expression of SF3B3 in ESCC tumor (T) and adjacent normal (N) tissues (n = 124) from CPPA database is shown. (G) Kaplan–Meier plots of overall survival of SF3B3 in ESCC patients using the CPPA dataset. (H) The correlation between the expression of SF3B3 and the abundance of p-SF3B3 (T1200) in ESCC clinical samples as described in is shown. (I) The correlation between PTMSEA score of CK2 and the expression of SF3B3 in ESCC clinical samples as described in is shown. The data underlying the graphs shown can be found in .

Article Snippet: The proteasome inhibitor MG-132 (T2154), the CK2 inhibitor CX-4945 (T2259), and the USP7 inhibitor P5091 (T6925) were purchased from TargetMol.

Techniques: Infection, Expressing

Journal: PLOS Biology

Article Title: Casein kinase 2-mediated phosphorylation of the splicing factor SF3B3 plays a key role in esophageal squamous cell carcinoma progression

doi: 10.1371/journal.pbio.3003729

Figure Lengend Snippet: (A) KYSE140 cells transfected with siCTL or two independent siSF3B3 (siSF3B3-1 and siSF3B3-2) were subjected to RT-qPCR analysis to assess mRNA expression levels of SF3B3 (mean ± SD; *** P < 0.001, Student t test). (B) RNA extracted from cells as described in (A) were subjected to library preparation and Nanopore sequencing, and the number of alternative splicing (AS) events regulated by SF3B3 is shown (|ΔPSI| ≥ 0.2 and FDR ≤ 0.05). Cassette Exons or Exon Skipping (ES); Intron Retain (IR); Alternative 5′ Splice Site (5′ ASS); Alternative 3′ Splice Site (3′ ASS). (C) The number of exon inclusion and skipping induced by SF3B3 is shown by pie chart. (D) The Pearson’s correlation was analyzed between the expression levels of SF3B3 and genes containing SF3B3-regulated cassette exons that are highly expressed in ESCC (Log 2 FC ≥ 2, P ≤ 0.01). Red dots indicate genes with r ≥ 0.3. (E) GO analysis results for genes with SF3B3-regulated cassette exons. The top 6 most enriched GO terms are shown. (F) Kaplan–Meier analyses of progression-free interval in ESCC patients using EXOSC2 cassette exon 4 as an input in the OncoSplicing dataset. (G) KYSE140 cells transfected with siCTL, siSF3B3-1, or siSF3B3-2 were subjected to standard PCR analysis to examine the expression of both the short and long isoforms of EXOSC2 as indicated at the bottom. Percentage spliced in (PSI) values were measured by Image J. The position of the cassette exon in EXOSC2 is as follows: EXOSC2 ( NM_001114122 , exon 4). DNA marker is indicated on the left. (H) KYSE140 cells were transfected with siCTL or siSF3B3 in the presence or absence of control vector, SF3B3 (WT), or SF3B3 (T1200A), followed by standard PCR analysis to examine the short and long isoforms of EXOSC2 as described in (G). (I) KYSE140 cells treated with or without CX-4945 (10 μM) were subjected to standard PCR analysis to examine the short and long isoforms of EXOSC2 as described in (G). (J) HEK293T cells transfected with GFP control vector or GFP-tagged SF3B3 (WT), SF3B3 (T1200A), or SF3B3 (T1200D) were subjected to IP analysis with anti-GFP magnetic beads, followed by IB analysis with antibodies as indicated. (K) HEK293T cells transfected with GFP control vector or GFP-tagged SF3B3 (WT), SF3B3 (T1200A), or SF3B3 (T1200D) were subjected to RIP analysis with anti-GFP magnetic beads, followed by RT-qPCR analysis to examine the binding of SF3B3 protein with genes as indicated (mean ± SD; *** P < 0.001, ** P < 0.01, Student t test) . (L–N) KYSE140 cells s t ably expressing control lentiviral vector, EXOSC2 long isoform (EXOSC2-L), or EXOSC2 short isoform (EXOSC2-S) were subjected to IB analysis (L), cell proliferation assay (M), and colony formation assay (N) (mean ± SD; ns: not significant, *** P < 0.001, Student t test). (O) The quantifica t ion of the crystal violet dye in (N) is shown (mean ± SD; ns: not significant, ** P < 0.01, *** P < 0.001, Student t test). (P–R) KYSE140 cells transfected with siCTL or siSF3B3, followed by infec t ion with lentivirus expressing control vector, EXOSC2-L, or EXOSC2-S were subjected to IB analysis (P), cell proliferation assay (Q), and colony formation assay (R) (mean ± SD; * P < 0.05, ** P < 0.01, Student t test). (S) The quantification of the number of colonies in (R) (mean ± SD; *** P < 0.001, Student t test). (T) KYSE140 cells infected with lentivirus expressing shCTL or shSF3B3 in the presence or absence of lentivirus expressing control vec t or, EXOSC2-L, or EXOSC2-S were injected into BALB/c nude mice for 4 weeks, and the tumors were collected and photographed. (U, V) The tumor volume (U) and weight (V) as shown in (T) (mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, Student t test). (W) The expression of the short and long isoforms of EXOSC2 were measured by standard PCR as described in (G) in paired ESCC and adjacent normal tissues ( n = 6). (X) The quantification of the PSI values in (W) (mean ± SD; * P < 0.05, Student t test). The data underlying the graphs shown can be found in .

Article Snippet: The proteasome inhibitor MG-132 (T2154), the CK2 inhibitor CX-4945 (T2259), and the USP7 inhibitor P5091 (T6925) were purchased from TargetMol.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Nanopore Sequencing, Alternative Splicing, Marker, Control, Plasmid Preparation, Magnetic Beads, Binding Assay, Proliferation Assay, Colony Assay, Infection, Injection

Journal: PLOS Biology

Article Title: Casein kinase 2-mediated phosphorylation of the splicing factor SF3B3 plays a key role in esophageal squamous cell carcinoma progression

doi: 10.1371/journal.pbio.3003729

Figure Lengend Snippet: (A, B) KYSE140 cells were treated with CX-4945 (A) or P5091 (B) at concentrations as indicated for 72 h before cell viability measurement, and the IC50 is shown. (C, D, F, G) KYSE140 cells were treated with CX-4945 or P5091 at concentrations as indicated, followed by cell proliferation (C, F) and colony formation (D, G) assay (mean ± SD; *** P < 0.001, Student t test). (E, H) The quantification of the crystal violet dye in (D) (E) and (G) (H) is shown (mean ± SD; *** P < 0.001, Student t test). (I) An inhibitory dose-response matrix of CX-4945 and P5091 was conducted in KYSE140 cells. (J) Synergy heatmaps representing combination treatment with CX-4945 and P5091 for 3 days in KYSE140. Red color denotes drug synergy. HSA: Highest Single Agent. (K, L) KYSE140 cells were treated with CX4945 (10 μM) or P5091 (20 μM) alone or in combination followed by cell proliferation (K) and colony formation (L) assay (mean ± SD, *** P < 0.001, Student t test). (M) The quan t ification of the crystal violet dye in (L) is shown (mean ± SD, *** P < 0.001, Student t test). (N, O) KYSE140 cells were trea t ed with CX4945 (10 μM) or P5091 (20 μM) alone or in combination followed by IB analysis (N) to examine the protein levels of SF3B3 and standard PCR analysis (O) to examine the alternative splicing of EXOSC2. (P) Male BALB/c nude mice were subcutaneously injected with KYSE30 cells for 20 days and then randomized (3 mice/group) and treated with CX4945 (25 mg/kg) or P5091 (25 mg/kg) alone or in combination as indicated by intraperitoneal (i.p.) injection daily before tumor collection. (Q) Images of excised tumors in (P) are shown. (R, S) The tumor growth curve (R) and tumor weight (S) as described in (P) are shown (mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, Student t test). The data underlying the graphs shown can be found in .

Article Snippet: The proteasome inhibitor MG-132 (T2154), the CK2 inhibitor CX-4945 (T2259), and the USP7 inhibitor P5091 (T6925) were purchased from TargetMol.

Techniques: Alternative Splicing, Injection

Journal: PLOS Biology

Article Title: Casein kinase 2-mediated phosphorylation of the splicing factor SF3B3 plays a key role in esophageal squamous cell carcinoma progression

doi: 10.1371/journal.pbio.3003729

Figure Lengend Snippet: In ESCC, aberrantly activated CK2 phosphorylates SF3B3 at Thr1200, enhancing its interaction with the deubiquitinase USP7. This interaction leads to deubiquitination and stabilization of SF3B3, ultimately promoting a large cohort of alternative splicing events, including the inclusion of exon 4 in EXOSC2, and thereby driving ESCC progression. Targeting both CK2 and USP7, either individually or in combination, using CX-4945 and P5091, respectively, effectively suppresses ESCC progression.

Article Snippet: The proteasome inhibitor MG-132 (T2154), the CK2 inhibitor CX-4945 (T2259), and the USP7 inhibitor P5091 (T6925) were purchased from TargetMol.

Techniques: Alternative Splicing

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: 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: HIF-1α levels in w.t. and KO clones of SK-N-BE and U2OS cells. ( A ) w.t. SK-N-BE cells were treated with CX-4945 at the indicated time and concentration conditions. 30 μg proteins from lysates were analyzed by WB for the HIF-1α levels. Actin was used as loading control. ( B ) SK-N-BE cells (w.t or KOα clone 1, or KOα’ clone 1) or ( C ) U2OS cells (w.t or KOα clone 1 or KOα’ clone 2) were treated, where indicate (+), with 2mM dimethyloxallyl glycine (DMOG) for 20 h. 30 μg proteins from cell lysates were analyzed by WB for the indicated proteins. Actin was used as loading control. At least three independent experiments (also with different clones) were performed. Representative WB are shown. The bar graphs report quantification of HIF-1α, HK2 and LDH, as normalized mean values ± SEM of all experiments, in arbitrary units, obtained by analyses with Kodak 1D Image software. Significance refers to the corresponding condition in w.t. cells. (**) 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: Clone Assay, Concentration Assay, Control, Software

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: 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: Seahorse glycolytic stress test of w.t. and KO clones of SK-N-BE and U2OS cells. The glycolysis stress tests were performed using a Seahorse extracellular flux analyzer. ECAR (extracellular acidification rate) was monitored following the indicated additions and normalized to protein concentrations. Each experiment was performed at least in quadruplicate. Values are expressed as the mean ± SEM. At least two independent experiments were performed; representative profiles are shown. ( A ) SK-N-BE cells glycolytic stress test profile (left) (KOα clone 1, KOα’ clone 2) and quantification of the experiment (right), performed with the XF software. ECAR values are expressed in AUC (Area Under Curves) units, quantifying the area of the curves corresponding to the different phases of the experiment (see ). Significance refers to w.t. cells; ( B ) U2OS cells glycolytic stress test profile (left) (w.t. cells or the indicated clones) and quantification of the experiment (right), performed with the Wave software. ECAR values are expressed in mpH/min, as detected at the last measure point of each phase of the experiment (see ). Significance refers to w.t. cells; ( C ) SK-N-BE cells glycolytic stress test profile, upon treatment with vehicle (control) or the indicated concentration of CX-4945 for 5 h before running the Seahorse experiment (left). Quantification of the experiment is shown on the right, performed with the XF software. ECAR values are expressed in AUC (Area Under Curves) units, quantifying the area of the curves under three points of each phases of the experiment (see ). Significance refers to vehicle-treated control cells. (*) p < 0.05, (**) p < 0.01, and (***) 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: Clone Assay, Software, Control, Concentration Assay

Journal: Clinical Cancer Research

Article Title: Extracellular HMGA1 Promotes Tumor Invasion and Metastasis in Triple-Negative Breast Cancer

doi: 10.1158/1078-0432.ccr-18-0517

Figure Lengend Snippet: Figure 1. HMGA1 is overexpressed and secreted in a regulated fashion in TNBC-invasive cells. A, IHC analysis from the Protein Atlas database of HMGA1 in different breast tumors shows a clear cytoplasmic staining. B, Graph representing the fold changes of a group of selected proteins differentially secreted between MDA231i and MCF- 7 cells. Among them, we chose to further validate HMGA1. C, WB showing HMGA1 expression (cell lysate and secretome) in MDA231i and MCF-7 cells. Tubulin was used as loading control when suitable. D, IF (20X) showing HMGA1 expression (in green) in MCF-7, MCF10A, HMEC, and MDA231i cells. E, WB of MDA231i cells secretomes done in the presence or absence of brefeldin (BFA), an inhibitor of ER to Golgi transport. Fibronectin, a classically secreted protein, TSG101, an exosomal protein, and HMGA1 levels are shown. HMGA1 secretion is not affected by BFA treatment. F, WB of MDA231i cells secretomes treated with elastase (serine protease), triton X-100 (nonionic surfactant that can permeabilize vesicles), or the combination of both. Fibronectin, TSG101, and HMGA1 levels are shown. Elastase alone or in combination with triton X-100 reduces HMGA1 levels. G, WB showing HMGA1 expression levels in MDA231i cells after the pharmacologic inhibition of CK2, by treatment with CX4945 and TBB, both selective inhibitors of CK2. The two treatments reduce the secretion of HMGA1. H (Left plot), WB of MDA231i cells expressing shCK2 sequences and control cells. Levels of CK2 (intracellular) and HMGA1 (intracellular and secretome) are shown. Tubulin was used as loading control when suitable. Right plot, Graphs showing intracellular WB ratios of CK2 and HMGA1, as well as the quantification of the secretion of HMGA1 in MDA231/shCK2 cells versus MDA231i cells. In both cases, the data obtained from the four different shCK2 sequences were average.

Article Snippet: The CK2 inhibitors CX4945 (Santa Cruz Biotechnology) and TBB (Santa Cruz Biotechnology) were used at 5 mmol/L and 0.05 mmol/L, respectively.

Techniques: Staining, Expressing, Control, Inhibition

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