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: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: Effect of CDK8 inhibitor Q12 treatment of MDA-MB-468 cells on E2F1 protein and E2F1-dependent luciferase expression. ( A ) Effect of treatment with 10 µM Q12 (6 h and 24 h) on E2F1 ( left ) and pE2F1(Ser375) ( right ) expression in MDA-MB-468 cells compared to vehicle-treated control (DMSO) cells ( n = 3–6). ( B ) Relative luciferase expression in MDA-MB-468 cells transfected with an E2F1-responsive luciferase plasmid following treatment with Q12 (10 µM) or SEL120 (1µM) compared to vehicle-treated control (DMSO) cells ( n = 4). Unpaired Student’s t -test was used to determine significance. * ρ < 0.05, ** ρ < 0.01, **** ρ < 0.0001.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Luciferase, Expressing, Control, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) p73 protein expression following treatment with 10 µM Q12 (1, 6, 12, 24 h) compared to vehicle-treated (DMSO) control ( n = 3). ( B ) CDK8 protein expression in MDA-MB-468 cell line following 24 h treatment with 10 µM Q12 ( n = 3). Images are representative of three independent experiments. Error bars represent mean ± SD. Unpaired Student’s t -test was used to determine significance. ** ρ < 0.01.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) Western blot assessment of siRNA-mediated CDK8 KD. ( B ) Western blot assessment of siRNA-mediated E2F1 KD. ( C ) Comparison of cell viability of wild-type, siRNA control-transfected, CDK8 siRNA-transfected, and E2F1 siRNA-transfected MDA-MB-468 cells treated with Q12 (20 µM) for 0.5, 1, 2, 6, and 24 h ( n = 3). Only significant differences are shown. * ρ ≤ 0.01.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Comparison, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) Expression of Mcl-1 protein in MDA-MB-468 cells treated with Q12 (10 µM, 24 h), cryptotanshinone (CPT, 10 µM, 24 h), or co-treatment with Q12 (10 µM, 24 h) and CPT (10 µM, 24 h) compared to vehicle-treated (DMSO) control cells ( n = 3). ( B ) Proposed mechanism by which CDK8 inhibitor decreases viability of MDA-MB-468 triple-negative breast cancer cells. Images are representative of three independent experiments. Error bars represent mean ± SD. Unpaired Student’s t -test was used to determine significance. * ρ < 0.05, *** ρ < 0.001, ns = not significant.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Identification of target genes for the CDK subunits of the Mediator complex.

doi: 10.1111/j.1365-2443.2011.01565.x

Figure Lengend Snippet: Figure 1 Purification of HA ⁄ FLAG-tagged (HF:) CDK8- and CDK19-formed Mediator complexes. (A) SDS–PAGE of purified two CDK-formed Mediator complexes. Nuclear extracts were prepared from either HF:CDK8- or HF:CDK19-expressing HeLa cells as previously described. Prepared extracts were loaded onto anti-FLAG M2 Agarose (Sigma-Aldrich) column and eluted by FLAG peptide. Arrows indicate HF:CDK8 and HF:CDK19 protein bands. (B) Western blot analysis of purified CDK8 and CDK19-formed Mediator complexes. Arrows indicate HA ⁄ FLAG-tagged (HF:) and endogenous (Endo) CDK8 and CDK19 protein bands.

Article Snippet: To prepare CDK8-specific antibody, 20 lg of anti-CDK8 rabbit polyclonal antibody (Santa Cruz, sc-1521) was loaded onto 100 lg of GST-CDK19-associating glutathione- Genes to Cells (2011) 16, 1208–1218 2011 The Authors Journal compilation 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. 1216 sepharose column (GE) and incubated for 4 h at 4 C then correct flow through fraction.

Techniques: SDS Page, Expressing, Western Blot

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Identification of target genes for the CDK subunits of the Mediator complex.

doi: 10.1111/j.1365-2443.2011.01565.x

Figure Lengend Snippet: Figure 3 Northern blot analysis of expression profiles of both CDKs in various human tissues. Commercially available preb- lotted membrane which poly-A RNAs obtained from each indicated tissues were already blotted was used for this assay. Upper and middle columns show CDK19 and CDK8 expres- sion profiles, respectively. b-actin was used as a control.

Article Snippet: To prepare CDK8-specific antibody, 20 lg of anti-CDK8 rabbit polyclonal antibody (Santa Cruz, sc-1521) was loaded onto 100 lg of GST-CDK19-associating glutathione- Genes to Cells (2011) 16, 1208–1218 2011 The Authors Journal compilation 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. 1216 sepharose column (GE) and incubated for 4 h at 4 C then correct flow through fraction.

Techniques: Northern Blot, Expressing, Membrane, Control

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Identification of target genes for the CDK subunits of the Mediator complex.

doi: 10.1111/j.1365-2443.2011.01565.x

Figure Lengend Snippet: Figure 2 C-terminal domain (CTD) phosphorylation specific- ities of two CDK-formed Mediator complexes. (A) Wild-type and mutant heptapeptide sequences. Synthetic oligonucleotides encoding three repeats of wild-type CTD hepta peptide (WT) and each mutant (S2A, S5A) were subcloned into GST-fusion protein expression vector and expressed in E. coli. (B) GST- CTD phosphorylation assay. Wild-type and mutant GST- CTD (triplet repeats) were incubated with either HF:CDK8 complex or HF:CDK complex in the presence of [c-32P] ATP and then separated by SDS–PAGE. Top column shows autora- diography. Bottom shows CBB-staining of each input. Relative intensities obtained from autoradiography bands are presented in the middle. (C) GST-tagged full-length murine CTD was expressed, purified from E. coli and incubated with either HF:CDK8- or HF:CDK19-formed complex in the presence of ATP. Phosphorylation was then detected by anti- phosphorylated serine-specific antibodies. An arrow shows phosphorylated GST-CTD, and asterisks show unphosphory- lated GST-CTD.

Article Snippet: To prepare CDK8-specific antibody, 20 lg of anti-CDK8 rabbit polyclonal antibody (Santa Cruz, sc-1521) was loaded onto 100 lg of GST-CDK19-associating glutathione- Genes to Cells (2011) 16, 1208–1218 2011 The Authors Journal compilation 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. 1216 sepharose column (GE) and incubated for 4 h at 4 C then correct flow through fraction.

Techniques: Phospho-proteomics, Mutagenesis, Expressing, Plasmid Preparation, Incubation, SDS Page, Staining, Autoradiography

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Identification of target genes for the CDK subunits of the Mediator complex.

doi: 10.1111/j.1365-2443.2011.01565.x

Figure Lengend Snippet: Figure 4 Transcriptome analysis of CDK8 or CDK19 knocked-down HeLa S3 cells by microarray. (A) siRNA speci- ficity against either CDK8 or CDK19. siRNAs were transfect- ed into HeLa S3 cells and incubated for 60 h. Cell extracts were subjected to Western blotting. (B) Transcripts whose expression levels were altered at least 1.5-fold in two indepen- dent siRNAs against the same CDK were clustered using a hierarchical clustering algorithm. Knockdown and microarray analyses were carried out as three independent experiments and analyzed using GeneSpring software. The whole data were deposited in the NCBI GEO repository (GSE32108). (C) Venn diagrams of CDK8 and CDK19 knockdown-affected genes.

Article Snippet: To prepare CDK8-specific antibody, 20 lg of anti-CDK8 rabbit polyclonal antibody (Santa Cruz, sc-1521) was loaded onto 100 lg of GST-CDK19-associating glutathione- Genes to Cells (2011) 16, 1208–1218 2011 The Authors Journal compilation 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. 1216 sepharose column (GE) and incubated for 4 h at 4 C then correct flow through fraction.

Techniques: Microarray, Incubation, Western Blot, Expressing, Knockdown, Software

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Identification of target genes for the CDK subunits of the Mediator complex.

doi: 10.1111/j.1365-2443.2011.01565.x

Figure Lengend Snippet: Figure 5 Confirmation of microarray results by RT-qPCR analyses. Six genes were picked up from microarray analyses. ARRB2 (A) and SLC22A5 (B): specifically CDK8-knockdown-affected genes. CXCR4 (C) and MARKS (D): specifically CDK19-knock- down-affected genes. PPARG1A (E) and EIF4EBP2 (F): both CDK knockdown affected genes. Bar graph shows RT-qPCR results, and line graph shows microarray results. RT-qPCR experiments were carried out independently from microarray analyses.

Article Snippet: To prepare CDK8-specific antibody, 20 lg of anti-CDK8 rabbit polyclonal antibody (Santa Cruz, sc-1521) was loaded onto 100 lg of GST-CDK19-associating glutathione- Genes to Cells (2011) 16, 1208–1218 2011 The Authors Journal compilation 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. 1216 sepharose column (GE) and incubated for 4 h at 4 C then correct flow through fraction.

Techniques: Microarray, Quantitative RT-PCR, Knockdown

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