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: 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: bioRxiv

Article Title: CK2 inhibitor, CX-4945, enhances BH3 priming and promotes apoptosis of venetoclax-resistant AML by targeting antiapoptotic proteins

doi: 10.64898/2025.12.24.696284

Figure Lengend Snippet: A) AML cell lines including both VEN-susceptible and resistant (Molm-13, Molm-13/VR, HL-60, HL-60/VR, U937), and AML PDX cells (2016-1, 2016-7) were treated with different concentrations of CX-4945 and VEN alone or in combination with indicated doses for 48 h. Cell viability was assessed using WST reagent and cell viability was calculated relative to vehicle-treated cells as 100%. B) ZIP synergy scores for CX-4945 and VEN combination in different AML cell lines and PDX cells were shown. ZIP scores greater than 10 indicates ‘synergy’, and 0-10 indicate ‘additive’ activity between CX-4945 and VEN. C-D) AML cells (Molm-13, Molm-13/VR, PDX 2016-7) were treated with CX-4945 and VEN alone or in combination for 24 h and stained with Annexin V/7AAD for analyzing apoptosis by flow cytometry ( C ). Relative apoptosis was calculated by normalizing to vehicle treated cells ( D ). The data are presented as mean ± SD (n=2-4). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by one-way ANOVA (Holm-Sidak’s multiple comparisons test) denotes statistical significance. E) AML cell lines (Molm-13, Molm-13/VR) were treated with CX-4945 and VEN alone or in combination for 24 h and the surface expression of chemoresistance markers (CD47 and CD123) was analyzed by flow cytometry. F) Schematic showing the workflow for dynamic BH3 profiling to measure drug-induced priming of BH3 peptides in AML cells. The difference in Cyt c release (%) with and without drug treatment was presented as delta priming. Created with BioRender.com . G) Molm-13 and Molm-13/VR cells were tested for Cyt C release by priming with BH3 peptides with and without CX-4945 treatment and calculated the delta priming. The data are presented as mean ± SEM (n=2) and analyzed by two-way ANOVA (Sidak’s multiple comparisons test).

Article Snippet: CX-4945 (#HY-50855B) and venetoclax (#HY-15531) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Activity Assay, Staining, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: CK2 inhibitor, CX-4945, enhances BH3 priming and promotes apoptosis of venetoclax-resistant AML by targeting antiapoptotic proteins

doi: 10.64898/2025.12.24.696284

Figure Lengend Snippet: A) The common mechanisms that govern cellular MCL-1 levels at transcription, translation, and post-translation levels are depicted. The signaling cascade mediated through CK2, PI3K/AKT, and mTOR regulate transcription and translation of MCL-1 isoforms (L-large, S-short, ES-extra short). MCL-1L (anti-apoptotic) undergoes caspase-mediated cleavage to form pro-apoptotic shorter MCL-1 isoforms (S & ES) that can induce cellular apoptosis independent of BAX and BAK. Created using BioRender.com . B) The levels of MCL-1 isoforms (L and ES) were measured by immunoblotting in AML cell lines and PDX cells after 24h of treatment with CX-4945 and VEN alone or in combination. C) Quantification of MCL-1 transcript levels after treatment with CX-4945 and VEN alone or in combination for 24h in indicated AML cell lines and PDX cells by qRT-PCR. The data are presented as mean ± SD (n=3) and analyzed by two-way ANOVA (Tukey’s multiple comparisons test). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 are considered statistically significant. D) Immunoblotting analysis of AML cell lines (Molm-13, Molm-13/VR, U937) and PDX (2016-1, 2016-7) cells after 24h of treatment with CX-4945 and VEN alone or in combination. β-Actin was used as a loading control. Representative blots from two to three independent experiments were shown.

Article Snippet: CX-4945 (#HY-50855B) and venetoclax (#HY-15531) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: CK2 inhibitor, CX-4945, enhances BH3 priming and promotes apoptosis of venetoclax-resistant AML by targeting antiapoptotic proteins

doi: 10.64898/2025.12.24.696284

Figure Lengend Snippet: A) Schematic depicting the treatment plan in AML patient-derived xenograft (PDX) mouse model. Similar scheme followed for cell line-derived xenograft (CDX) mice except the analysis after two-weeks of treatment. The NRG-S mice were irradiated and intravenously injected with AML cell lines Molm-13VR (0.25×10 6 cells/mouse), U937 (1×10 4 cells/mouse), and PDX 2016-7 (0.5×10 6 cells/mouse) and randomized into experimental groups (Vehicle, CX-4945, VEN, and CX+VEN Como). The drug treatment started after one week of cell transplantation and followed up for survival analysis or analysis after two-weeks of treatment (only for PDX). B-F) After two weeks of drug treatment, mice injected with PDX 2016-7 cells were sacrificed and spleen weight was recorded ( B ). The cells collected from spleens were analyzed by flow cytometry to analyze percent hCD45 positive cells ( C ). D-F) CBC analysis was performed on PDX 2016-7 mice after two-weeks of drug treatment by Hemavet analyzer. The data for B-F are presented as mean ± SD (n=7-8) and analyzed by one-way ANOVA (Tukey’s multiple comparisons test). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 indicates statistical significance, while ‘ns’ denotes ‘not significant’. G) Immunoblotting analysis of spleen cells collected from PDX 2016-7 mice after two-weeks of treatment with CX-4945, VEN, and CX+VEN combo. The data for three different experimental animals was presented. H-J) Kaplan-Meyer survival analysis of 2016-7 PDX ( H ), Molm-13/VR CDX ( I ), and U937 CDX ( J ) mice after treatment with CX-4945, VEN and CX+VEN combo. The overall median survival (MS) days for each experimental group of PDX and CDX mice were provided next to the legend. *p<0.05, **p<0.01 and ***p<0.001 by Gehan-Breslow-Wilcoxon test indicates statistical significance.

Article Snippet: CX-4945 (#HY-50855B) and venetoclax (#HY-15531) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Derivative Assay, Irradiation, Injection, Transplantation Assay, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: CK2 inhibitor, CX-4945, enhances BH3 priming and promotes apoptosis of venetoclax-resistant AML by targeting antiapoptotic proteins

doi: 10.64898/2025.12.24.696284

Figure Lengend Snippet: A) AML cells from patients were surfaced stained with different fluorescent labeled antibodies and analyzed for the expression of markers for LSCs (CD34, CD38, TIM3) and chemoresistance (CD47 and CD123) by flow cytometry. B) The basal level expression of CK2α, BCL-XL, and CK2 activity (p-CK2 substrate) was measured in primary AML cells by immunoblotting analysis. C) The basal level apoptosis and viability of primary AML patient cells was assessed after 12-24h of post-thaw using Annexin V & dead cell kit for Muse cell analyzer. The patient cells with high basal level apoptosis were omitted and the ones used for further testing were highlighted using red square box. D & E) AML patient cells were treated with CX-4945 and VEN alone and in combination for 24 h and surface stained for different cell surface markers along with annexin V. The relative apoptosis in bulk cells ( D ), LSCs (CD34+CD38−) and chemoresistant (CD47+CD123-) subpopulations were calculated by normalizing the percent apoptosis in vehicle treated cells ( E ). The data are presented as mean ± SEM (n=6-7) and analyzed via one-way ANOVA (Tukey’s multiple comparisons test). *p<0.05, **p<0.01, and ***p<0.001 considered as statistically significant.

Article Snippet: CX-4945 (#HY-50855B) and venetoclax (#HY-15531) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Staining, Labeling, Expressing, Flow Cytometry, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: CK2 inhibitor, CX-4945, enhances BH3 priming and promotes apoptosis of venetoclax-resistant AML by targeting antiapoptotic proteins

doi: 10.64898/2025.12.24.696284

Figure Lengend Snippet: Inhibition of CK2-mediated signaling through CX-4945 negatively affect cell survival pathways (PI3K/AKT/mTOR, NF-κB pathways) and anti-apoptotic proteins (MCL-1L and BCL-XL). Downregulation of anti-apoptotic BCL2 members (MCL-1L, BCL-XL) further enhance BCL-2 dependency and enhance VEN-mediated apoptosis in VR-AML cells. Overexpression of pro-apoptotic MCL-1ES isoform that can mediate mitochondrial depolarization independent of BAX/BAK contribute to potentiate VEN activity. Moreover, the transcriptome of VR-AML cells followed by CX-4945 and VEN combination treatment showed reversal of molecular gene signatures associated with VEN resistance and result in potentiated apoptosis in VR-AML cells. The signaling targeted by CX-4945 and VEN are represented by dotted lines. Created using BioRender.com .

Article Snippet: CX-4945 (#HY-50855B) and venetoclax (#HY-15531) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Inhibition, Over Expression, Activity Assay

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