pcdna3 cdk8 ha  (Addgene inc)

Journal: International Journal of Biological Sciences

Article Title: The Cyclin-Dependent Kinase 8 Inhibitor E966-0530-45418 Attenuates Pulmonary Fibrosis In Vitro and In Vivo

doi: 10.7150/ijbs.105826

Figure Lengend Snippet: E966-0530-45418 inhibited the activation of EMT and the collagen I protein in TGFβ1-evoked human primary AECs. ( A - F ) Human primary AECs were transfected with the pcDNA3 CDK8-HA plasmid (1 μg) for 24 h, treated with E966-0530-45418 (5 μM), pirfenidone (1 mM) or not for 1 h, and then incubated with TGFβ1 (10 ng/mL) for an additional 24 h (A-D) or 3 h (E and F). Immunofluorescence analysis using a high-content imaging system (ImageXpress Micro confocal microscope) was used to evaluate E-cadherin and CDK8 protein expression. Images were taken at 200 × magnification (Scale bar: 50 μm) (n = 5 independent samples per group) (A and B). The protein levels of COL1A1 and CDK8 were determined by western blotting (n = 5 independent samples per group) (C and D). The protein levels of p-Smad3 T179 and CDK8 were assessed by flow cytometry (n = 6 independent samples per group) (E and F). The results are shown as the mean ± SEM. P values were determined using one-way ANOVA followed by Tukey's post hoc test (B, D, and F).

Article Snippet: Recombinant human TGFβ1 was obtained from PeproTech (Cranbury, NJ, USA). pcDNA3 CDK8-HA (P#634) was a gift from Matija Peterlin (Addgene plasmid #14649; http://n2t.net/addgene:14649 ; RRID: Addgene_14649); the 7TFP CDH1 reporter was a gift from Bob Weinberg (Addgene plasmid #91704; http://n2t.net/addgene:91704 ; RRID: Addgene_91704); and the pGL3- TGFB1 reporter was a gift from Yuh-Shan Jou (Addgene plasmid #101762; http://n2t.net/addgene:101762 ; RRID: Addgene_101762).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Incubation, Immunofluorescence, Imaging, Microscopy, Expressing, Western Blot, Flow Cytometry

Journal: Cell reports

Article Title: Transcriptome-based chemical screens identify CDK8 as a common barrier in multiple cell reprogramming systems.

doi: 10.1016/j.celrep.2023.112566

Figure Lengend Snippet: Figure 2. CDK8 was a barrier of MEF-XEN reprogramming (A) CDK8 activity inhibition assays in vitro for SEL120-34A HCl (control), MSC2530818, E-616452, and A-83-01. (B) qPCR of Sox17, Gata4, Sall4, and Foxa2 in samples with MEF medium, base, CFAE+8+DMSO/BI/MSC, and CFAE+6 treatment and base control for 12 days, respectively (C, CHIR99021; F, forskolin; A, AM580; E, EPZ04777; 6, E-616452; 8, A-83-01; S, SB431542; L, LY364947). Base, XEN basal medium. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 between CFAE+8+DMSO and indicated samples. Numbers in brackets are the concentrations (mM) of each small molecule.

Article Snippet: For the construction of FU-tet-o-GFP, FU-tet-o-mouse Cdk8-2HA, FU-tet-o-mouse Hnf4a, FU-tet-o-mouse Foxa3, FU-tet-o-human CDK8 plasmids, full-length cDNAs encoding GFP, mouse Cdk8, mouse Hnf4a/Foxa3, and human CDK8 were amplified by PCR and subcloned into the FU-tet-o-hOct4 backbone (Addgene Catalog #19778)37 after EcoR I digestion.

Techniques: Activity Assay, Inhibition, In Vitro, Control

Journal: Cell reports

Article Title: Transcriptome-based chemical screens identify CDK8 as a common barrier in multiple cell reprogramming systems.

doi: 10.1016/j.celrep.2023.112566

Figure Lengend Snippet: Figure 3. Overexpression of CDK8 repressed MEF-XEN reprogramming, and knockdown of CDK8 promoted MEF-XEN reprogramming (A) Immunofluorescence staining of XEN master regulators SOX17, GATA4, SALL4, and FOXA2 of Tet-on Cdk8-2HA with and without Dox under CFAE+6 medium induction for 12 days. Scale bar, 50 mm. (B) The number of SOX17+ GATA4+, SOX17+ SALL4+, and SOX17+ FOXA2+ colonies in (A). Data are presented as mean ± SEM. ****p < 0.0001 between with and without Dox samples. (C and D) Immunofluorescence co-staining of XEN master regulators SOX17 and SALL4 (C) or FOXA2 (D) in scramble, shCDK8-1, and shCDK8–2 under CFAE+8 induction, respectively (C, CHIR99021; F, forskolin; A, AM580; E, EPZ04777; 8, A-83-01). Scale bar, 200 mm. (E) The numbers of XEN-like colonies. Data are presented as mean ± SEM. **p < 0.01. (F) qPCR of Cdk8 in scramble and shCdk8. Base, CFAE+8 medium. Data are presented as mean ± SEM. ****p < 0.0001. (G) qPCR of Sox17, Gata4, Sall4, and Foxa2 in scramble and shCdk8. Base, CFAE+8 medium. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 between scramble in CFAE+8 and indicated samples.

Article Snippet: For the construction of FU-tet-o-GFP, FU-tet-o-mouse Cdk8-2HA, FU-tet-o-mouse Hnf4a, FU-tet-o-mouse Foxa3, FU-tet-o-human CDK8 plasmids, full-length cDNAs encoding GFP, mouse Cdk8, mouse Hnf4a/Foxa3, and human CDK8 were amplified by PCR and subcloned into the FU-tet-o-hOct4 backbone (Addgene Catalog #19778)37 after EcoR I digestion.

Techniques: Over Expression, Knockdown, Staining

Journal: Cell reports

Article Title: Transcriptome-based chemical screens identify CDK8 as a common barrier in multiple cell reprogramming systems.

doi: 10.1016/j.celrep.2023.112566

Figure Lengend Snippet: Figure 6. CDK8 suppression enhances mouse hepatocyte reprogramming (A) Schematic diagram of Hnf4a+Foxa3-mediated mouse hepatocyte reprogramming. (B) Immunofluorescence staining of ALB in Hnf4a+Foxa3-mediated hepatocyte reprogramming in hepatocyte culture medium (HCM) + C8B (C, CHIR99021; 8, A-38-01; B, BMP4) plus DMSO, MSC, or BI. Scale bar, 100 mm. (C) Number of ALB+ colonies in (B). Data are presented as mean ± SEM. (D) qPCR of Alb mRNA in indicated samples. (E) Immunofluorescence staining of ALB in Hnf4a+Foxa3-mediated hepatocyte reprogramming in HCM + DMSO, MSC, or BI. Scale bar, 100 mm. (F) Number of ALB+ colonies in (E). Data are presented as mean ± SEM. (G) Periodic acid-Schiff (PAS) or indocyanine green (ICG) staining of Hnf4a+Foxa3-mediated hepatocyte reprogramming in HCM + DMSO and MSC. *p < 0.05, ***p < 0.001, ****p < 0.0001 between indicated samples.

Article Snippet: For the construction of FU-tet-o-GFP, FU-tet-o-mouse Cdk8-2HA, FU-tet-o-mouse Hnf4a, FU-tet-o-mouse Foxa3, FU-tet-o-human CDK8 plasmids, full-length cDNAs encoding GFP, mouse Cdk8, mouse Hnf4a/Foxa3, and human CDK8 were amplified by PCR and subcloned into the FU-tet-o-hOct4 backbone (Addgene Catalog #19778)37 after EcoR I digestion.

Techniques: Staining

Journal: Nature

Article Title: The histone variant macroH2A suppresses melanoma progression through regulation of CDK8.

doi: 10.1038/nature09590

Figure Lengend Snippet: Figure 3 | Microarray and ChIP analyses identify CDK8 as a direct mH2A- regulated gene in melanoma. a, Heat map representing gene expression changes (at least twofold) in B16-F1 mH2A-deficient cells. qRT–PCR of CDK8 (bottom); mean 6 s.d. (n 5 3). b, Immunoblots of CDK8 in murine shRNA lines; actin for loading. c, Immunoblot and qRT–PCR of CDK8 in human shRNA lines; mean 6 s.d. (n 5 3). d, CDK8 and DsRed immunoblots of mH2A2_24 line expressing H2A- and mH2A2-cherry; asterisk depicts fusion proteins. e, CDK8 qRT–PCR analysis in A375 subcutaneous tumours; mean 6 s.d. (n 5 3). f, mH2A1 ChIP analysis of the 21 kb position from TSS for CDK8, PACS2, ATP5G1 and GAPDH; intergenic control. IgG used as control antibody; mean 6 s.d. (n 5 3).

Article Snippet: Plasmids expressing CDK8 (pBabe.puro.CDK8 and CDK8-KD7) were obtained from Addgene.

Techniques: Microarray, Gene Expression, Quantitative RT-PCR, Western Blot, shRNA, Expressing, Control