cdk11  (Santa Cruz Biotechnology)

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: 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: The primary antibodies for CDK11 (1:1000 dilution) and β-actin (1:2000 dilution) were purchased from Santa Cruz and Sigma-Aldrich, respectively.

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: The primary antibodies for CDK11 (1:1000 dilution) and β-actin (1:2000 dilution) were purchased from Santa Cruz and Sigma-Aldrich, respectively.

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: The primary antibodies for CDK11 (1:1000 dilution) and β-actin (1:2000 dilution) were purchased from Santa Cruz and Sigma-Aldrich, respectively.

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: The primary antibodies for CDK11 (1:1000 dilution) and β-actin (1:2000 dilution) were purchased from Santa Cruz and Sigma-Aldrich, respectively.

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: The primary antibodies for CDK11 (1:1000 dilution) and β-actin (1:2000 dilution) were purchased from Santa Cruz and Sigma-Aldrich, respectively.

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: The primary antibodies for CDK11 (1:1000 dilution) and β-actin (1:2000 dilution) were purchased from Santa Cruz and Sigma-Aldrich, respectively.

Techniques: Expressing, Immunohistochemical staining, Staining, Derivative Assay