Journal: Journal of Biological Chemistry

Article Title: Mediator Subunit MED28 (Magicin) Is a Repressor of Smooth Muscle Cell Differentiation

doi: 10.1074/jbc.m706592200

Figure Lengend Snippet: FIGURE 3. Med28 suppression resulting in SMC differentiation involves other Mediator subunits. A, nuclear extracts of siRNA-trans- fected C2C12 cells demonstrate that Med28KD leads to down-regulation of Mediator subunits Cdk8, Med6, Med8, and Med18. B, in C2C12, similar to Med28KD, suppression of Med6 (Med6KD) revealed an up-regulation of SM22 and SM-Actin. C, RT-PCR also revealed increased expression of other SMC contractile markers upon Med6KD in C2C12 cells. Non-target- ing siRNA (Control) was used (A–C), and GAPDH was used as a loading/RT- PCR control (A–C). D, phase-contrast images of Med6 RNAi (panel b) dem- onstrate a SM phenotype (resembling Med28KD) when compared with non-targeting siRNA (panel a) in C2C12 cells. Bar, 75 m.

Article Snippet: Primary antibodies used included the anti-MED28 antibodies Tim3 (rabbit polyclonal) and 7E1 (mouse monoclonal), which have been previously described (1); M2 (anti-FLAG monoclonal; Sigma), MED6, CDK8, and SM22 /transgelin (Santa Cruz Biotechnology); SM- Actin (MP Biomedicals); GAPDH (Chemicon); and SM- Actin, -tubulin, and normal rabbit serum (Sigma).

Techniques: Reverse Transcription Polymerase Chain Reaction, 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: 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: International Journal of Molecular Sciences

Article Title: Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ER T2 System

doi: 10.3390/ijms232214077

Figure Lengend Snippet: Detection of the tamoxifen-induced Cdk8 knockout in different organs of mice: ( A ) feeding method of tamoxifen administration; ( B ) injection method of tamoxifen administration. ( C ) Detection of the tamoxifen-induced Cdk8 knockout in the mouse brain and uterus (original oral and improved injection methods of tamoxifen administration).

Article Snippet: The membranes were blotted with primary anti CDK8 specific (sc-1521, SantaCruz, Santa Cruz, CA, USA) and anti β-actin specific antibodies (A2228, Sigma-Aldrich, Burlington, MA, USA) diluted in Tris buffered saline with Tween 20 (TBS-T; 1:1000) supplemented with 1% BSA overnight at 4 °C.

Techniques: Knock-Out, Injection

Journal: International Journal of Molecular Sciences

Article Title: Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ER T2 System

doi: 10.3390/ijms232214077

Figure Lengend Snippet: ( A )—Schemes of loxP-sites localization in Cdk8 gene of Cdk8 fl/fl mice (upper scheme) and Cdk8 −/− mice (lower scheme) and annealing positions of PCR primers used for genotyping of animals. ( D , E )—PCR detection of 2nd Cdk8 exon excision. Panels ( B , C ) represent CDK8-F + R2 PCR product, which is 242 bp ( A ) in case of floxed exon 2 presence and is absent when the exon is completely excised. ( D , E )—from PCR with F + R primer pair that flanks the whole floxed exon (~890 bp). Panels ( B , D ) correspond to a set of genomic PCRs of organs from tamoxifen-treated Cdk8 fl/fl female mice. ( C , E )—those of Cdk8 fl/fl control mouse. Organs are shown in the following order: 1—heart, 2—ovary, 3—uterus, 4—liver, 5—lung, 6—brain, 7—muscle (quadriceps), 8—kidney, 9—small intestine, 10—thymus, 11—spleen, 12—bone marrow. “wt”—wildtype mouse tail sample; “mQ”—no template control.

Article Snippet: The membranes were blotted with primary anti CDK8 specific (sc-1521, SantaCruz, Santa Cruz, CA, USA) and anti β-actin specific antibodies (A2228, Sigma-Aldrich, Burlington, MA, USA) diluted in Tris buffered saline with Tween 20 (TBS-T; 1:1000) supplemented with 1% BSA overnight at 4 °C.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ER T2 System

doi: 10.3390/ijms232214077

Figure Lengend Snippet: Genotyping of Cdk8 fl/fl mice. 208 bp band corresponds to the wild type allele and 242 band to the floxed allele. “fl/wt”—untreated heterozygous for a floxed exon mouse sample. “wt”—wildtype mouse. mQ—no template control (milliQ water). Lanes 1–4 with 242 bp bands give an example of homozygous for floxed exon samples.

Article Snippet: The membranes were blotted with primary anti CDK8 specific (sc-1521, SantaCruz, Santa Cruz, CA, USA) and anti β-actin specific antibodies (A2228, Sigma-Aldrich, Burlington, MA, USA) diluted in Tris buffered saline with Tween 20 (TBS-T; 1:1000) supplemented with 1% BSA overnight at 4 °C.

Techniques: Control