cdk8 sirna  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: LncRNA EILA promotes CDK4/6 inhibitor resistance in breast cancer by stabilizing cyclin E1 protein

doi: 10.1126/sciadv.adi3821

Figure Lengend Snippet: ( A ) Luciferase reporter assays showing the transcriptional activity in MCF7-palR cells transfected with truncated fragments of the EILA promoter region. ( B ) Predicted transcriptional factors of EILA in the JASPAR online software based on the −500- to 0-bp sequence. ( C ) qRT-PCR analysis of CTCF and EILA expression in MCF7-palR cells after CTCF knockdown. ( D ) Luciferase reporter assays showing the EILA transcriptional activity in MCF7-palR cells after CTCF knockdown. ( E ) Luciferase reporter assays performed in MCF7-palR cells transfected with mutated fragments of the EILA promoter region (−500 to 0 bp). ( F ) Predicted CTCF-binding site in the EILA promoter region. ( G ) ChIP-qPCR analysis revealing the localization of CTCF at EILA promoter in MCF7-palR cells. ( H ) Analysis of ChIP-seq binding peaks in the Cistrome Database and UCSC Genome Browser. ( I ) ChIP-qPCR analysis showing the localization of CDK8 at EILA promoter in MCF7-palR cells. ( J ) Co-IP assays and Western blot analysis showing the interaction between CTCF and CDK8/TFII-I in MCF7-palR cells. In ( K to M ), CDK8 was silenced in MCF7-palR cells. (K) qRT-PCR detection of CTCF and EILA in MCF7-palR cells after CTCF knockdown. (L and M) Cell proliferation assays (L) and EdU incorporation (M) assays showing the growth of transfected cells under palbociclib treatment. For (A), (C) to (E), (G), (I), and (K) to (M), data from three independent experiments were expressed as means ± SD and the P values were calculated by Student’s t test for two-group comparison and one-way ANOVAs for multiple-group comparison. **** P < 0.0001, *** P < 0.001, ** P < 0.01, and N.S. for P > 0.05.

Article Snippet: Antibodies against CTCF (3418, CST, 1:50), CDK8 (sc-1521, Santa Cruz Biotechnology, 1:50), and rabbit/mouse IgG were used for immunoprecipitation, and the precipitated DNA fragments were subjected to qPCR amplification.

Techniques: Luciferase, Activity Assay, Transfection, Software, Sequencing, Quantitative RT-PCR, Expressing, Knockdown, Binding Assay, ChIP-qPCR, ChIP-sequencing, Co-Immunoprecipitation Assay, Western Blot, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Transfection efficiency determined by flow cytometry . The transfection efficiency was 97.2% 6 h after transfecting with CDK8-siRNA of HCT116. The ratio of Lipofectin 2000 to siRNA was 4 μL: 4 μL, and the concentration of CDK8-siRNA is 80 pmol/L.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Transfection, Flow Cytometry, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin mRNA expression of CDK-siRNA transfected HCT116 cells detected by RT-PCR . 48 h later of CDK8-siRNA transfection, RT-PCR was performed to detect CDK8 and β-catenin mRNA expression. A: CDK8-siRNA group; B: scrambled siRNA group; C: non-siRNA group; D, E and F represented corresponding internal reference, and M: marker. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Representative Western blots of CDK8 and β-catenin expression level in CDK-siRNA transfected HCT116 cells . 72 h later of CDK8-siRNA transfection of HCT116 cells, protein expression of CDK8 (A) and β-catenin (B) was determined by western blot assay. a: non-siRNA group; b: scrambled siRNA group; c: CDK-siRNA group. Results are given as average value of the gray in three target genes and interal controls from three independent experiments.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Western Blot, Expressing, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Cell proliferation activity after transfection of CDK8-siRNA assessed by MTT assay . Curves of cell growth after transfection for 24, 48 and 72 h by MTT assay. Results are given as means ± SD from three independent experiments. P < 0.05.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Activity Assay, Transfection, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: Effect of CDK8-siRNA transfection on the apoptosis and cell cycle of HCT116 cells . 48 h after transfection, cell apoptosis (A) and cell cycle (B) were determined by flow cytometry. Quadrants D2-D4 represent necrotic/late apoptotic cells, viable cells, and early apoptotic cells, respectively. Results are given as means ± SD from three independent experiments.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Transfection, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin mRNA expression in colon tumor and adjacent normal tissues detected by real-time PCR . Fresh tumor and corresponding adjacent tissues from 12 patients were resected under sterile conditions and then snapfrozen in liquid nitrogen immediately. 200 mg tissue was taken out from liquid nitrogen and plused 1 ml Trizol when RNA was extaracted. Real-time PCR is performed for the expression levels of CDK8 (A) and β-catenin (B). Results are given as means ± SD from three independent experiments. P < 0.05.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells

doi: 10.1186/1756-9966-30-109

Figure Lengend Snippet: CDK8 and β-catenin protein expression in colon tumor and adjacent normal tissues detected by IHC . The expression of CDK8 (left) and β-catenin (right) was stained brown and present in tumor tissue and adjacent normal tissues. Representative sites with negative (a, 400 X ), moderate positive (c, 400 × ), strongly positive (e, 400 ×) expression of CDK8 and corresponding weakly positive (b, 400 ×), moderate positive (d, 400 ×), strongly positive (f, 400 ×) expression of β-catenin.

Article Snippet: Annexin V apoptosis kit (Keygentec, China) and siRNA-CDK8 (Genepharma, China) were used in the present study.

Techniques: Expressing, Staining