Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Expression of CDK11 and CK2 protein complex members in untransformed and malignant breast cells. (A) Immunoblot analysis of cultured breast cell lines, as indicated above the blots. Proteins detected are indicated on the right side of the blots. Actin signal was used as the loading control. (B) Indirect immunofluorescent detection of CDK11, CK2α, and CK2α′ (red color) in breast cell lines. Cell lines are indicated above each set of images and proteins detected are indicated on the left side of the images. Blue, 4′,6-diamidino-2-phenylindole-stained nuclei. Scale bar: 100 μm. (C) Immunohistochemical detection of CDK11 proteins in human normal and malignant breast tissue. Type of breast tissue indicated on the left side of the images. Magnification indicated above the images; dotted ellipse, portion of the 100× image that is shown at 400×. Scale bars: 400 μm for 100× and 100 μm for 400× images. (D) Human microarray tissues stained for CDK11 were scored by two independent observers. The average value was taken and the results plotted for normal ( n = 16) versus triple-negative breast cancer (TNBC; n = 44) tissues. Box, first to third (Q1 to Q3) quartiles; diamond, mean; line inside box, median; whiskers, minimum and maximums of data range. CDK, cyclin-dependent kinase; CK2, casein kinase 2.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Cell Culture, Control, Staining, Immunohistochemical staining, Microarray

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: mRNA expression levels in nontransformed and malignant breast cells a

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: RNA expression levels in normal breast and breast cancer subtypes. Normalized RNAseq read count data for PAM50 breast cancer subtypes and normal breast from The Cancer Genome Atlas were analyzed for CDK11 and CK2 protein complex genes as shown above each plot. Box, first to third (Q1 to Q3) quartiles; line inside box, median; whiskers, 1.5 maximum interquartile range. Normal, n = 95; basal, n = 141; Her2, n = 67; LumA, n = 421; LumB, n = 192. CDK, cyclin-dependent kinase; CK2, casein kinase 2; Her2, human epidermal growth factor receptor 2; LumA, luminal A; LumB, luminal B.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: RNA Expression

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Immunoblot analyses following small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells. Immunoblot analysis of MDA-MB-231 and SUM-149 cell lysates following small interfering RNA (siRNA) transfection. Transfected siRNAs are indicated above the blots, proteins detected are indicated on the right side of the blots. CDK11 p110 , cyclin L1α, cyclin L2α, and CK2αα′β lysates are 72 hours post transfection; caspase 3, Bcl-xL, and survivin lysates are 96 hours post transfection. Actin signal was used as the loading control. CDK, cyclin-dependent kinase; CK2, casein kinase 2.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Small Interfering RNA, Transfection, Control

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Protein expression levels following small interfering RNA transfection

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Small Interfering RNA

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: mRNA expression levels in small interfering RNA transfected cells

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Small Interfering RNA, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Preclinical evaluation of cyclin dependent kinase 11 and casein kinase 2 survival kinases as RNA interference targets for triple negative breast cancer therapy

doi: 10.1186/s13058-015-0524-0

Figure Lengend Snippet: Small interfering RNA-mediated downregulation of CDK11 and CK2 in breast cancer cells decreases cell viability and inhibits clonal survival. (A) Breast cancer cells were transfected with 30 nM single small interfering RNA (siRNA) or 15 nM each of the two siRNAs combined as indicated. After 96 hours, cell viability was determined relative to the untreated cells. Means ± standard errors (SEs) are presented. * P <0.05. ** P <0.01, *** P <0.001 relative to untreated. ^ P = 0.055, # P <0.05, ## P <0.01, ### P <0.001 relative to siCtrl. (B) Triple-negative breast cancer (TNBC) cells were transfected twice with 30 nM single siRNAs or 15 nM each of the two siRNAs combined as indicated and as described in . Seven days after the second transfection, cell colonies were stained and counted. Means ± SE are presented. $ P <0.0001 relative to siCtrl and untreated. (C) Representative crystal violet stained colonies on 35 mm plates 7 days after the second siRNA transfection as described in (B). Cell lines are indicated above the plate images and siRNA transfections are indicated to the left of the plate images. CDK, cyclin-dependent kinase; CK2, casein kinase 2; si, small interfering.

Article Snippet: Antibodies used were: CDK11 (A300-311A), cyclin L1 (A302-058A), CK2α (A300-197A) and CK2α′ (A300-199A) from Bethyl Laboratories; cyclin L2 (600-401-878) from Rockland Immunochemicals (Limerick, PA, USA); CK2β (sc-12739 and sc-46666) and actin (sc-1616) from Santa Cruz Biotechnology; CDK11 (5524), caspase 3 (9661, 9662), lamin A/C (2032), and Bcl-xL (2762) from Cell Signaling (Beverly, MA, USA); and survivin (AF886) from R&D Systems (Minneapolis, MN, USA).

Techniques: Small Interfering RNA, Transfection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional activation of CBFβ by CDK11 p110 is necessary to promote osteosarcoma cell proliferation

doi: 10.1186/s12964-019-0440-5

Figure Lengend Snippet: Global gene expression profiling reveals gene regulation in response to CDK11 knockdown. a Venn diagram of genes overexpressed and underexpressed in CDK11 siRNA treated cells compared with control group. Genes overexpressed/underexpressed in two cell lines are indicated in the overlapping regions of the circles. b Differentially expressed genes from gene expression profiling were grouped by biological process; the percentages of genes that are involved in each particular biological process are shown. c Distribution of molecular function was determined by differentially expressed genes (over 1.5-fold). d Heat map of significantly regulated genes in U-2OS and KHOS cell lines transfected with CDK11 siRNA. The genes listed in the hierarchical clustering represent the average log 2 fold change

Article Snippet: Protein G Magnetic beads (SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads), Cell Signaling Technology, Danvers, MA) were washed with blocking solution and pre-coated overnight with anti-CDK11 antibody (sc-928, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (SimpleChIP®Kit, Cell Signaling Technology) as a positive control, or IgG as a negative control.

Techniques: Gene Expression, Knockdown, Control, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional activation of CBFβ by CDK11 p110 is necessary to promote osteosarcoma cell proliferation

doi: 10.1186/s12964-019-0440-5

Figure Lengend Snippet: CBFβ expression depends on CDK11 expression on transcriptional and translational levels. a Verification of the altered gene expression observed in the gene array for selected genes, including CBFβ and CDK11, using quantitative RT-PCR. Two different cell lines (KHOS and U-2OS) were analyzed, expression was normalized using the KHOS or U-2OS untreated control, and results were depicted as relative expression in these cells. b CDK11 siRNA downregulated the expression of CDK11 and CBFβ in a dose-dependent manner. KHOS or U-2OS cells were transfected with CDK11 siRNA with increasing concentrations. The protein expression of CBFβ decreased dependently with CDK11 expression. c Expressions of CDK11 and CBFβ in osteosarcoma cell lines and in normal osteoblast cell lines. d Expressions of CDK11 and CBFβ in osteosarcoma cell lines and metastatic cell lines. e Expressions of CDK11 and CBFβ in osteosarcoma tissues. * P < 0.05

Article Snippet: Protein G Magnetic beads (SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads), Cell Signaling Technology, Danvers, MA) were washed with blocking solution and pre-coated overnight with anti-CDK11 antibody (sc-928, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (SimpleChIP®Kit, Cell Signaling Technology) as a positive control, or IgG as a negative control.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional activation of CBFβ by CDK11 p110 is necessary to promote osteosarcoma cell proliferation

doi: 10.1186/s12964-019-0440-5

Figure Lengend Snippet: CDK11 p110 localizes to the nucleus and exhibits high expression in tumor cells. a Schematic showing CDK11 chromosome exon locations, specifically the translated site that codes two protein isoforms. The CDK11 p58 isoform is generated by an internal ribosome entry site sequence (IRES) in the same mRNA encoding the CDK11 p110 isoform. b GFP-CDK11 p110 and GFP-CDK11 p58 transient transfection revealed that CDK11 p110 is localized only in the nucleus, while CDK11 p58 is expressed in the cytoplasm in three different cell lines (original magnification, 200×). c U-2OS-GFP-CDK11 p110 and U-2OS- GFP-CDK11 p58 selective stable cell lines show that CDK11 p110 is localized only in the nucleus, while CDK11 p58 is mainly expressed in the cytoplasm (original magnification, 200×). d CDK11 p110 is highly expressed in U-2OS and KHOS cell lines; on the contrary, CDK11 p58 is almost undetectable in these cell lines

Article Snippet: Protein G Magnetic beads (SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads), Cell Signaling Technology, Danvers, MA) were washed with blocking solution and pre-coated overnight with anti-CDK11 antibody (sc-928, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (SimpleChIP®Kit, Cell Signaling Technology) as a positive control, or IgG as a negative control.

Techniques: Expressing, Generated, Sequencing, Transfection, Stable Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional activation of CBFβ by CDK11 p110 is necessary to promote osteosarcoma cell proliferation

doi: 10.1186/s12964-019-0440-5

Figure Lengend Snippet: CDK11 p110 , not CDK11 p58 , regulates promoter activity of CBFβ genes. a Schematic representation of the promoter-luciferase construct showing the CBFβ promoter region. b CDK11 p110 upregulates CBFβ promoter-luciferase in a dose-dependent manner in the U-2OS cell line. U-2OS cells were transfected with the CBFβ promoter-luciferase construct. Co-expression was conducted with an empty expression vector (control) or CDK11 expression vectors. Error bars indicate standard deviation and are from at least three replicates. c CDK11 p110 upregulates CBFβ promoter-luciferase in a dose-dependent manner in the KHOS cell line. KHOS cells were transfected with the CBFβ promoter-luciferase construct. Co-expression was conducted with an empty expression vector (control) or CDK11 expression vectors. Error bars indicate standard deviation and are from at least three replicates. d CDK11 p110 , not CDK11 p58 , activates the CBFβ promoter-luciferase in the KHOS cell line. e CDK11 p110 significantly increases CBFβ promoter-luciferase in KHOS cell line. f Schematic showing major structural features of the CDK11 p110 protein. CDK11 p110 kinase-dead or kinase-active mutations were generated. The asterisk represents the amino acid that was mutated to create kinase-dead or active mutations. g In the U-2OS cell line, the C-terminal kinase domain mutation of CDK11 p110 failed to affect CDK11 p110 -mediated CBFβ activation. h The C-terminal kinase domain mutation of CDK11 p110 also did not change CBFβ promoter activity in the KHOS cell line. * P < 0.05., ** P < 0.01

Article Snippet: Protein G Magnetic beads (SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads), Cell Signaling Technology, Danvers, MA) were washed with blocking solution and pre-coated overnight with anti-CDK11 antibody (sc-928, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (SimpleChIP®Kit, Cell Signaling Technology) as a positive control, or IgG as a negative control.

Techniques: Activity Assay, Luciferase, Construct, Transfection, Expressing, Plasmid Preparation, Control, Standard Deviation, Generated, Mutagenesis, Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional activation of CBFβ by CDK11 p110 is necessary to promote osteosarcoma cell proliferation

doi: 10.1186/s12964-019-0440-5

Figure Lengend Snippet: CDK11 p110 upregulates CBFβ expression directly by associating with its promoter. a Schematic representation of potential CDK11 binding sites in the CBFβ promoter and primer sets (p1, p2, p3, p4) indicating amplified regions encompassing the four primer sites along with the transcription start site (TSS). Chromatin immunoprecipitations were analyzed using a 2% input of KHOS sample treated with CDK11 siRNA by PCR. PCR products were only observed with p3 and p4 primer. b ChIP analysis was performed by CDK11 antibodies or 2% input sample and by measuring enrichment at p3 in human CBFΒ promoter by RT-PCR. The amount of immunoprecipitated DNA by CDK11 antibodies are represented as ratio of input DNA (1:50) and presented as mean of three independent experiments ( n = 3, mean ± SD). * P < 0.05; ** P < 0.01, Student’s t-test. c Electrophoretic mobility shift assay of CDK11- CBFβ binding activity in nuclear extracts from different cell lines. Metastatic cell lines MNNH/HOS and 143B demonstrated notable high binding activity (lane 3 and 4, purple arrow) compared with KHOS and U-2OS non-metastatic cell lines. d The formation of CDK11-DNA complexes was determined by incubation with labeled CBFβ. Decreased CDK11 DNA-binding activity was present in CDK11 siRNA knockdown KHOS and MNNH/HOS cells (purple arrow)

Article Snippet: Protein G Magnetic beads (SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads), Cell Signaling Technology, Danvers, MA) were washed with blocking solution and pre-coated overnight with anti-CDK11 antibody (sc-928, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (SimpleChIP®Kit, Cell Signaling Technology) as a positive control, or IgG as a negative control.

Techniques: Expressing, Binding Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Activity Assay, Incubation, Labeling, Knockdown

Journal: Cell Communication and Signaling : CCS

Article Title: Transcriptional activation of CBFβ by CDK11 p110 is necessary to promote osteosarcoma cell proliferation

doi: 10.1186/s12964-019-0440-5

Figure Lengend Snippet: High CBFβ expression correlates with CDK11 expression and contributes to metastasis and reduced overall survival in patients. a Representative images of different immunohistochemical staining intensities of CBFβ and CDK11 staining are shown in osteosarcoma tissues. CBFΒ and CDK11 immunoreactivity was found mostly in the nucleus of tumor cells. The percentage of cells showing positive nuclear staining for CBFβ or CDK11 was calculated by reviewing the entire spot (original magnification, 200×). b The correlation coefficient (R 2 ) between CBFβ expression and CDK11 expression was derived by GraphPad PRISM 5 (correlation coefficient R 2 = 0.7729, P < 0.001). c Kaplan-Meier survival curve of patients with osteosarcoma were subgrouped as either CDK11 low staining (CDK11 staining ≤3) or high staining (CDK11 staining ≥4). d Kaplan-Meier survival curve of patients with osteosarcoma were subgrouped as either CBFβ low staining (CBFΒ staining ≤3) or high staining (CBFβ staining ≥4). e Distribution of CDK11 staining scores from the same patients with metastatic and primary specimens. f Distribution of CBFβ staining scores from the same patients with metastatic and primary specimens

Article Snippet: Protein G Magnetic beads (SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads), Cell Signaling Technology, Danvers, MA) were washed with blocking solution and pre-coated overnight with anti-CDK11 antibody (sc-928, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Histone H3 (SimpleChIP®Kit, Cell Signaling Technology) as a positive control, or IgG as a negative control.

Techniques: Expressing, Immunohistochemical staining, Staining, Derivative Assay

Journal: Oncotarget

Article Title: CDK8/19 inhibition induces premature G1/S transition and ATR-dependent cell death in prostate cancer cells

doi: 10.18632/oncotarget.24414

Figure Lengend Snippet: ( A ) Compound structure, potency, and kinase selectivity of T-474 or T-418. Kinase selectivity profiling was performed using 300 nmol/L T-474 or T-418. ( B ) VCaP cells were treated with T-474 or T-418 together with 10 ng/mL IFN-γ as indicated for 30 minutes. Cell lysates were analyzed by western blot. ( C ) mRNA expression of CDK8 or CDK19 in prostate cancer cell lines (CCLE). ( D ) Western blot of CDK8 or CDK19 in prostate cancer cell lines. VCaP cells were transfected with siRNA as indicated for 72 hours. Cell lysates were analyzed by western blot. The relative band intensities of CDK8 or CDK19 were quantified and are indicated as percentage (%) of control (non-treated VCaP cells). An arrow indicates the expected position of bands derived from CDK19. ( E ) LNCaP or 22Rv1 cells were treated with T-474 as indicated for 9 days ( N = 3, mean with SD ). ( F ) PC-3 or DU 145 cells were treated with T-474 as indicated for 6 days ( N = 3, mean with SD ). ( G ) VCaP cells were treated with T-474 or T-418 as indicated for 7 days ( N = 2, mean). Cell viability was measured.

Article Snippet: Ligand displacement assays were conducted with Tb-labeled anti-GST antibody (CisBio), 20 nmol/L Kinase Tracer 236 (Thermo Fisher), 20 nmol/L GST-CDK8/Cyclin C (CarnaBio) or GST-CDK19/Cyclin C (CarnaBio), and each compound.

Techniques: Western Blot, Expressing, Transfection, Derivative Assay

Journal: AIDS Research and Human Retroviruses

Article Title: HIV Transcription Is Independent of Mediator Kinases

doi: 10.1089/aid.2019.0039

Figure Lengend Snippet: Knockdowns of MDKs do not inhibit HIV transcription. HeLa-CD4 cells were transfected with siRNAs against CDK9, CDK8, and CDK19, or scrambled non-specific control (SCR). Twenty-four hours post knockdown, cells were transfected with NL4.3 Luc. At 24 h post NL4.3-Luc transfection (48 h post knockdown), lysates were harvested and examined for luciferase activity or Western blotting. (A) Luciferase activity is presented as a fold change in luciferase activity over untreated control cells (set to 1). (B) Whole-cell lysates were run on 10% SDS-PAGE and blotted with anti-CDK9, anti-CDK8, anti-CDK19 antibodies, with β-actin serving as loading control. Samples from cells treated with scrambled and CDK8/19 siRNAs were run on the same gel. Triplicate stimulations were performed. Error bars represent standard errors of the mean (**p < .01). CDK, cyclin-dependent kinase; MDK, mediator kinase.

Article Snippet: After incubation for 24 h at 37°C, plasmid transfection of NL4.3-Luc was performed as described above. siRNAs used in this study were: control siRNA-A (sc-37007; SCBT), human CDK9 siRNA (sc-29268; SCBT), human cdk19 siRNA (sc-72844; SCBT), and human cdk8 siRNA (6438S; Cell Signaling).

Techniques: Transfection, Control, Knockdown, Luciferase, Activity Assay, Western Blot, SDS Page