cdk8  (Cell Signaling Technology Inc)

Journal: Respiratory Research

Article Title: Runx2 acetylation enhances pulmonary inflammation in chronic obstructive pulmonary disease through activating CDK8

doi: 10.1186/s12931-025-03475-4

Figure Lengend Snippet: Runx2 activation boosted CDK8 transcription in BEAS-2B cells. A The downstream target genes of Runx2 were predicted through CHEA, hTFTarget, and KnockTF2.0. B The effects of acute CSE incubation on the top 3 target genes were evaluated in BEAS-2B cells by RT-PCR. C SiRunx2 plasmids were established and transfected to repress Runx2 expression in BEAS-2B cells. Then, the influences of siRunx2 on CSE-induced the changes of the top 3 target genes were estimated using RT-PCR. D , E CDK8 nuclear protein was measured in mouse lungs after CS exposure using western blotting ( D ) and densitometry analysis ( E ). F , G The effect of acute CSE on CDK8 nuclear protein was determined in BEAS-2B cells using western blotting ( F ) and densitometry analysis ( G ). H - K The effects of siRunx2 plasmids transfection on CSE-activated CDK8 and inflammatory cytokines were explored in BEAS-2B cells. H , I The proteins expression of Runx2 and CDK8 were detected using western blotting ( H ) and densitometry analyses ( I ). J The levels of inflammatory cytokines were measured using RT-PCR. K The effects of CDK8 inhibition on CSE-upregulated inflammatory cytokines were evaluated by RT-PCR. L , M Potential binding sequences for Runx2 were predicted in the CDK8 promoter ( L ), Runx2 andluciferase reporter plasmids were transfected and luciferase activity was detected ( M ). All the data were expressed as the means± S.E.M. ( N =6).* P < 0.05, ** P < 0.01

Article Snippet: Information on the primary antibodies used in this experiment were as follows: β-actin (Proteintech, 81115-1-RR, 1:10000), Lamin A/C (Bioword, BS6019, 1:5000), Sirt1 (CST, #8469, 1:1000), Sirt2 (CST, D4O5O, 1:1000), Sirt3 (Affinity, AF5135, 1:1000), Sirt4 (CST, #69786, 1:2000), Sirt5 (Abcam, Ab259967 , 1:2000), Sirt6 (CST, D8D12, 1:3000), Sirt7 (Abcam, Ab259968 , 1:2000), Runx2 (D1L7F) (CST, #12556, 1:1000), Runx2 (SANTA CRUZ, sc-101145, 1:200), CDK8 (CST, 4101 S, 1:1000), Acetylated-Lysine (CST, #9441, 1:1000), Ubiqutin (Abcam, Ab140601 , 1:1000).

Techniques: Activation Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Western Blot, Inhibition, Binding Assay, Luciferase, Activity Assay

Journal: Respiratory Research

Article Title: Runx2 acetylation enhances pulmonary inflammation in chronic obstructive pulmonary disease through activating CDK8

doi: 10.1186/s12931-025-03475-4

Figure Lengend Snippet: CS exposure induced Runx2 acetylation through repressing Sirt3. A - L The effects of acute CSE on acetylation and Sirts family were analyzed in BEAS-2B cells, as well as the levels of acetylation in animal models exposed to CS and in patients with COPD. The overall acetylation levels were observed in BEAS-2B cells exposed to acute CSE ( A , B ), COPD-like animal models exposed to CS ( C , D ), and patients with COPD ( E , F ) using western blotting. ( G , H ) The effects of acute CSE exposure on Sirts family were evaluated by western blotting ( G ) and densitometry analyses ( H ). I , J Sirt3 nuclear protein was determined in CSE-exposed BEAS-2B cells using western blotting ( I ) and densitometry analysis ( J ). K , L The effect of CS exposure on Sirt3 nuclear translocation was analyzed through IHC in lung tissues of COPD mice ( K ) and quantitative analysis ( L ). M , N The colocalization between Sirt3 and Runx2 was detected in mouse lungs using IF ( M ) and Mander colocalization coefficient was analyzed ( N ). O , P The impact of CSE exposure on Runx2 acetylation was determined by western blotting ( O ) and densitometry analysis ( P ). Q Molecular docking of Runx2 with Sirt3 was determined via the HDOCK method. R , S The impact of Sirt3 decrease on CSE-elevated Runx2 protein stability was analyzed by western blotting ( R ) and densitometry analysis ( S ). T , U The effect of Sirt3 decline on CSE-upregulated Runx2/CDK8 was estimated via western blotting ( T ) and densitometry analyses ( U ). V - Y The effect of Sirt3 downregulation on CSE-mediated Runx2 acetylation was explored with Co-IP. V IP: Runx2; Western blotting: Sirt3. W Densitometry analyses. X IP: Sirt3; Western blotting: Runx2. Y Densitometry analyses. All the data were expressed as the means± S.E.M. ( N =6).* P < 0.05, ** P < 0.01

Article Snippet: Information on the primary antibodies used in this experiment were as follows: β-actin (Proteintech, 81115-1-RR, 1:10000), Lamin A/C (Bioword, BS6019, 1:5000), Sirt1 (CST, #8469, 1:1000), Sirt2 (CST, D4O5O, 1:1000), Sirt3 (Affinity, AF5135, 1:1000), Sirt4 (CST, #69786, 1:2000), Sirt5 (Abcam, Ab259967 , 1:2000), Sirt6 (CST, D8D12, 1:3000), Sirt7 (Abcam, Ab259968 , 1:2000), Runx2 (D1L7F) (CST, #12556, 1:1000), Runx2 (SANTA CRUZ, sc-101145, 1:200), CDK8 (CST, 4101 S, 1:1000), Acetylated-Lysine (CST, #9441, 1:1000), Ubiqutin (Abcam, Ab140601 , 1:1000).

Techniques: Western Blot, Translocation Assay, Co-Immunoprecipitation Assay

Journal: Respiratory Research

Article Title: Runx2 acetylation enhances pulmonary inflammation in chronic obstructive pulmonary disease through activating CDK8

doi: 10.1186/s12931-025-03475-4

Figure Lengend Snippet: NR supplementation alleviated CS-activated Runx2/CDK8 axis in mouse lungs. A - R The impact of NR supplementation on CS-activated Runx2/CDK8 axis activation was explored in mouse lungs. A The content of NAD+ was detected in BEAS-2B cells after CSE treatment. B The effect of NR supplementation on CSE-downregulated NAD+ was evaluated in BEAS-2B cells. C , D The effect of NR supplementation on CSE-activated Runx2/CDK8 axis was estimated in BEAS-2B cells by western blotting ( C ) and densitometry analyses ( D ). E The concentration of NAD + was analyzed in mouse lungs after CS exposure. F The influence of NR pretreatment on CSE-evoked downregulation of NAD + was evaluated in mouse lungs. G , H The effect of NR supplementation on CS-activated Runx2/CDK8 axis was explored in mouse lungs via western blotting ( G ) and densitometry analyses ( H ). I , J Runx2-positive nuclei was determined in mouse lungs by IHC ( G ) and quantitative analysis ( H ). K , L ) Sirt3-positive nuclei was assessed in mouse lungs through IHC ( I ) and quantitative analysis ( J ). M - P The effects of NR supplementation on CS-promoted the production of inflammatory cytokines were estimated in mouse lungs via RT-PCR, including Cxcl1 ( M ), Cxcl2 ( N ), Il-6 ( O ), Cxcl15 ( P ). All the data were expressed as the means± S.E.M. ( N =6).* P < 0.05, ** P < 0.01

Article Snippet: Information on the primary antibodies used in this experiment were as follows: β-actin (Proteintech, 81115-1-RR, 1:10000), Lamin A/C (Bioword, BS6019, 1:5000), Sirt1 (CST, #8469, 1:1000), Sirt2 (CST, D4O5O, 1:1000), Sirt3 (Affinity, AF5135, 1:1000), Sirt4 (CST, #69786, 1:2000), Sirt5 (Abcam, Ab259967 , 1:2000), Sirt6 (CST, D8D12, 1:3000), Sirt7 (Abcam, Ab259968 , 1:2000), Runx2 (D1L7F) (CST, #12556, 1:1000), Runx2 (SANTA CRUZ, sc-101145, 1:200), CDK8 (CST, 4101 S, 1:1000), Acetylated-Lysine (CST, #9441, 1:1000), Ubiqutin (Abcam, Ab140601 , 1:1000).

Techniques: Activation Assay, Western Blot, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet:

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques:

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Superposition of docked Sorafenib (cyan carbon atoms) onto its corresponding crystallographic structure. Sorafenib from 3RGF is depicted with light gray carbon atoms. As no structure of CDK8 with ATP is available, the ATP (dark gray carbon atoms) of the superposed structure of the human CDK2/Cyclin A complex (PDB ID: 4EOQ) is shown to highlight its potential binding site in CDK8.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Compounds were tested in duplicate at 10 µM in kinase assays with recombinant CDK8/Cyc C complex, in the presence of 15 µM [γ- 33 P] ATP. The graph represents the residual CDK8 activity (%) for each compound tested (blue dots); background controls (red dots) and negative control (DMSO 1%, green dots). 36 compounds inhibit CDK8 to below 50%.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Recombinant, Activity Assay, Negative Control

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Thirty six selected compounds and Senexin A (SNX A) were tested in dose-response on CDK8/Cyc C in kinase assays. IC 50 is expressed in µM and calculated from dose-response curves (each curve was performed in duplicate). The graph represents the distribution of IC 50 obtained, with two threshold lines: at 100 nM (red line) and 1 µM (green line).

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques:

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. Left, DepMap analysis of dependecy of cancer cell lines on CDK8 and CDK1 (an example of an essential CDK), from genome-wide CRISPR screens. Score 0 indicates a non essential gene; score of -1 corresponds to a median of all common essential genes. Right, number of cancer cell lines dependent on the indicated gene according to the DepMap CRISPR screen database. All CDKs and kinases used to screen for CDK8/19 inhibitor specificity are shown. B. Expression levels (from RNAseq data, TPM-normalised and represented as log2(TPM+1)) and relative copy number (based on whole exome sequencing) in cancer cell lines. C. cBioPortal mutation analysis of the indicated genes in pancancer TCGA data.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Genome Wide, CRISPR, Expressing, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Induced fit docking experiments were performed using either CDK8 (PDB: 3RGF) or CDK2 (PDB: 4GCJ) structures as protein target. Upper panels: ID and structural formula of the seven selected compounds. Lower panels: 3D arrangement of the best docking pose (white carbon atoms) into CDK8 or CDK2 active site with its corresponding docking score. Amino acids from the CDK8 DMG loop are represented in ball and stick; corresponding residues (DFG) in CDK2 structure are also highlighted.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques:

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. MEFs seeded at 10 6 in 10 cm plates were grown for 5 days at 10 μM of drugs and imaged. B. Colony formation assay (CFA) with WT SV-40-immortalised MEFs, seeded at 15 000 in 10cm plates, treated with inhibitors at 10 μM for 8 days. N=2. C. CFA with immortalised MEFs, WT, CDK8 -/- , CDK19 -/- , or double knock-out, treated with inhibitors at 10μM for 6d. N = 2. D. 3T3-immortalised MEFs were treated for 2h with the indicated inhibitors, after which γIFN (at 100 ng/ml) was added for 1h, without changing the media. Cells were collected and samples prepared for WB analysis. CDK8/19 double knockout MEF cells were used as a positive control (for the absence of STAT1-S727ph signal). N=2. E. HCT116 cells grown in spheroids were pre-incubated with γIFN (at 20 ng/ml) for 3h and treated with selected inhibitors at 10 μM or DMSO at 0.1% for 3h. Spheroids were lysed and analysed by Western blotting for the indicated proteins. Normalised STAT1-S727ph/STAT1 signal ratio are shown. N=2.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Colony Assay, Knock-Out, Double Knockout, Positive Control, Incubation, Western Blot

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: SV-40-immortalised MEF cell lysates (A, in vitro N=4) or MEF cells (B, in vivo , N=6) were treated with the indicated CDK8/19 inhibitors, or DMSO as a control, followed by heat treatment at the indicated temperatures and Western Blot analysis with anti-CDK8 antibody.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: In Vitro, In Vivo, Control, Western Blot

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. CDK8 was immunoprecipitated from interphase egg extract and the beads were blotted for the indicated proteins. B. Interphase egg extract was fractionated by sucrose gradient and fractions were blotted for the indicated proteins. C. Left, chromosomal DNA replication quantified by 32 P-dCTP incorporation assay, in control conditions, or after addition of recombinant Geminin, to block replication licencing. Right, chromatin loading of pre-replication complex (ORC2, MCM5 and CDC6) and pre-initiation complex (PCNA) replication factors, CDK8 and Cyclin C during DNA replication time course. N=4. D. Left, immunodepletion of CDK8 (but not using Mock beads) removes majority of the kinase from the interphase egg extract; GADPH was used as loading control. Right, chromatin fractions from CDK8- or Mock-immunodepleted egg extract, or CDK8-depleted extract supplemented with recombinant CDK8-Cyclin C complex were analysed by WB with the indidated proteins. Slower migrating recombinant GST-CDK8 and His-Cyclin C are indidated. E. Chromosomal DNA replication in immunodepleted extracts, as in D, was quantified. N=3. Error bars show standard deviation. F,G. Chromosomal DNA replication quantified by 32 P-dCTP incorporation assay, in control conditions (DMSO), or in the presense of the indicated inhibitors.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Immunoprecipitation, Control, Recombinant, Blocking Assay, Immunodepletion, Standard Deviation

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. Alignment of the 20 CDK sequences. Only the regions containing the amino acids responsible for interactions with the compounds are shown. Interacting amino acids and their conservation are highlighted. B. View of the CDK8 amino acid residues involved in contact with compounds. The seven compounds are superposed and shown with gray balls. Interacting residues are depicted by colored balls and sticks according to their percentage of conservation (red: highly conserved residues (71-100%); pink: moderately conserved residues (35-70%); green: poorly conserved resides (10-34%)). C. Highlight of virtually mutated residues. Left panel: wild-type CDK8 showing the seven residues selected (ball and stick and dark grey carbon) to assess the effect of point mutations on compound binding. The induced fit pose of molecule #82 is shown (ball and stick and light grey carbon). Right panel: superposition of the poses obtained for each mutant following induced-fit docking. For clarity, only one of the two side chains is shown (ball and stick and dark grey carbon) in the case of the A100M/L and L158R/W mutants. In contrast, all the positions obtained for compound #82 are shown (ball and stick and light grey carbon).

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Overlay of structures of selected inhibitors with the inactive (DMG-out, PDB 3RGF; A, cyan) and active (DMG-in) conformations, without (PDB 5XS2; A and B, blue) and with MED12 (PDB 8TQ2; B, cyan). Zoom-in on ATP-binding pocket of CDK8, with side chains of D173 and M174 shown. In red, inhibitor docked into 5XS2; in yellow, inhibitor docked into 3RGF, A, and 8TQ2, B. Induced fit docking scores for compared structures are indicated.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Binding Assay