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

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 1. Optimised compounds for exploring CDK8 and CDK19 function. (A) Chemical structure and activity of compounds 2, 3 and 4 (n > 2, mean ± s.d.). (B) Overlay of 3 (grey; ocd 5HBJ) and 4 (pink; Pdb code: 5IDN) bound to CDK8/CCNC. Key interactions (yellow) and residues are shown. Residues 23 – 39 and 359 – 361 have been cropped for clarity. (C) Amino acid sequence alignment for human CDK8 and CDK19. Red, sequence differences; yellow, ATP binding; green, CCNC binding; gray, activation loop; blue, inhibitor binding. (D) Luciferase activity in COLO205-cl4 cells containing a TCF/ LEF reporter gene construct following 24 hr compound treatment (n = 4, mean ± s.d.). (E) Colony assay. Plates were seeded with LS513 or insensitive RKO cells and treated for 14 d. DOI: 10.7554/eLife.20722.003 The following source data and figure supplements are available for figure 1:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: Activity Assay, Sequencing, Binding Assay, Activation Assay, Luciferase, Construct, Colony Assay

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 2. Target inhibition and antitumor activity of CDK8/19 ligands 3 and 4 in established human colorectal cancer cell line xenografts. (A) Level of p-STAT1SER727 in SW620 human colorectal cancer xenografts following a single dose of 4, relative to the p-STAT1SER727 level in vehicle-treated mice. Significance was determined by Kruskal-Wallis test and Dunn’s post-test (*p=<0.001; ). (B, C) Modelling of experimental data, including data from (A) and Figure 2—figure supplement 2D, of STAT1SER727 phosphorylation following (B) single or (C) twice daily doses of 30 mg/kg 3 (black) or 30 and 100 mg/kg 4 (orange and purple). (D) Volume of SW620 xenografts in mice treated with 4 or a vehicle control. (E) Level of p-STAT1SER727, relative to control, in SW620 tumor xenografts at the stated time following the final dose of 4 (from D). DOI: 10.7554/eLife.20722.007 The following source data and figure supplements are available for figure 2:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Control

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 3. In vitro and in vivo activity of CDK8/19 ligands in patient-derived tumour xenograft models. (A) GI50 values for 2 in PDX soft agar colony cultures. (B) Exemplar dose-response profiles for selected colorectal cancer clonogenic assays treated with 2. (C) Volume of human colorectal cancer CXF 1034 (CTNNB1MUT, PIK3CAMUT, PTENMUT) PDXs in mice treated with vehicle, 3 and / or irinotecan (mean values ± s.e.m., n = 10 per cohort). Tumor volume was significantly different (p=<0.001; 2 way ANOVA and Tukey’s multiple comparison test) in mice receiving the combination treatment, compared with the monotherapy groups. (D) Level of p-STAT1SER727, relative to control, in CXF 1034 xenografts in mice treated with 3 measured 1 hr after the final dose (p=<0.0001, Mann-Whitney test; mean values ± s.d., n = 10 per cohort). DOI: 10.7554/eLife.20722.013 The following figure supplements are available for figure 3:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: In Vitro, In Vivo, Activity Assay, Derivative Assay, Comparison, Control, MANN-WHITNEY

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 4. Microarray gene expression profiling following in vivo treatment of human colorectal cancer xenografts with CDK8/19 ligands. Mice were treated with 70 mg/kg po 1 (SW620 and COLO205), 20 mg/kg po 3 (COLO205). (A) Venn plots of transcription factor-associated genesets or those encoding or regulating pathways enriched in genes whose expression was significantly altered by treatment (Supplementary Dataset). (B) GSEA of CDK8 or BRD4-associated super-enhancer genes in treated human tumor xenografts. (C) Scatterplot of false discovery rate (FDR-q) versus normalized enrichment score (NES) for indicated gene sets evaluated by GSEA (n=10,218), signatures include those from MSigDB, dbSUPER and the ChIP-seq data from Pelish and colleagues (Pelish et al., 2015). DOI: 10.7554/eLife.20722.016 The following source data and figure supplement are available for figure 4:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: Microarray, Gene Expression, In Vivo, Expressing, ChIP-sequencing

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 5. Treatment with CDK8/19 ligand 1 reduces the hyperplastic crypt stem cell population. Gene expression, measured by RT-PCR, in the intestinal epithelial stem and TA cells isolated from mice expressing a Dox-inducible activated b-catenin transgene. (A) Transcript abundance, relative to control, in stem and TA cells following induction with 2 mg/ml Dox. (B) Abundance of different cell types following treatment with Dox and compound 1. (C) Proportion of stem versus TA cells following treatment. (D) Fold changes in transcript abundance in stem (D) and TA (E) cells following treatment. All data are mean values ± s.d., n = 3. DOI: 10.7554/eLife.20722.019 The following source data and figure supplement are available for figure 5:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Control

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 6. Effect of CDK8/19 chemical ligands on bone development and the immune response in model systems. Mouse KS483 osteoprogenitor cells were treated with LGK974 (red) or compound 3 (black) for 13 d and bone matrix formation determined by measuring (A) N-terminal propeptide of type I procollagen (PINP) and (B) calcium, in the external medium (mean ± s.d., n = 6). Blue region, level following 50 ng/ml BMP-2 (positive control); red region, basal level. (C) Heat map showing the 10 biomarkers most affected by compound treatment in cell co- culture models. Data are log2 ratios of biomarker levels following compound treatment relative to control (range: 0.874 to 0.396). Blue, decreased ratio; red, increased ratio. (D) Plasma IL-12 levels in Wistar rats treated with 5 – 20 mg/kg po qd 3. Two rats per cohort. DOI: 10.7554/eLife.20722.022 The following source data and figure supplement are available for figure 6:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: Positive Control, Co-Culture Assay, Biomarker Discovery, Control, Clinical Proteomics

Journal: eLife

Article Title: Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases

doi: 10.7554/elife.20722

Figure Lengend Snippet: Figure 7. Examples of degenerative and proliferative lesions in rats treated with CDK8/19 ligands 3 or 4. (A) Intact proliferative zone in the bone growth plate of a control rat. (B) Dysplastic proliferative zone, showing disturbance of regular endochondrial ossification, from a rat treated with 20 mg/kg 3. Scale bar in A and B = 100 mm. (C) Intact epididymides, with epididymal cells, isolated from a control rat. (D) Epididymides with epithelial hyperplasia (distal corpus) isolated from a rat treated with 60 mg/kg 4. Scale bar in C and D = 50 mm. DOI: 10.7554/eLife.20722.025 The following figure supplement is available for figure 7:

Article Snippet: Proteins were also detected using an automated capillary immunoassay system (Protein Simple) with antibodies specific for CDK8 (Cell Signaling, Danvers Massachusetts, USA #4106, RRID:AB_1903936), CDK19 (SigmaAldrich, UK, HPA007053, RRID:AB_1233803) phospho-STAT1SER727 (Cell Signaling, #8826), total STAT1 (Santa-Cruz Biotechnology, Dallas, Texas, USA, #346, RRID:AB_632435), and B-actin (Cell Signalling, #4970, RRID:AB_2223172), subsequently immunodetected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrate.

Techniques: Control, Isolation