Journal: Science immunology

Article Title: Conversion of antigen-specific effector/memory T cells into Foxp3-expressing T reg cells by inhibition of CDK8/19.

doi: 10.1126/sciimmunol.aaw2707

Figure Lengend Snippet: Fig. 3. Interaction of CDK8/19 and STAT5 in inducing Foxp3 expression. (A) Mouse CD4+ T cells were mock-infected or infected with retrovirus harboring the WT CDK8 and STAT5b genes, stimulated with anti-CD3/CD28 and IL-2, and subjected to immunoprecipitation and immunoblotting for CDK8, STAT5b, and MED12. Data are representative of two independent experiments. (B) STAT5 serine phosphorylation by CDK8. Recombinant GST-STAT5b incubated with recombinant WT or KD CDK8 in the presence or absence of 1.0 M AS with 100 M ATP and 10 mM MgCl2 was subjected to immunoblotting for phosphoserine (pS) of STAT5b. Data are representative of two independent experiments. (C) Control of serine and tyrosine phos- phorylation by AS in activated T cells. Mouse CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of TGF- for 22 hours and in the absence (DMSO) or presence of 100 nM AS, lysed, and subjected to immunoblot analysis for STAT5b, pS-STAT5b, or pY-STAT5. Signal intensity was quantified and normalized by GAPDH (n = 3 or 4). **P < 0.01 (Student’s t test). AU, arbitrary units. (D and E) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/28 for 22 hours, and PLA was performed to assess interaction between CDK8 and STAT5. Images were obtained using an LSM710 confocal microscope. Data were presented as maximum intensity projection (n = 3). Each red spot represents a single interaction, and DNA was stained with DAPI. (F) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/CD28 for 22 hours and examined for expression of CDK8 and STAT5. DNA was stained with Hoechst33342. Images were obtained using an LSM710 confocal microscope. Data are representative of two independent experiments. (G) Mouse CD4+ T cells infected with retrovirus encoding WT or S730A mutant STAT5b were stimulated with anti-CD3/CD28 and IL-2, without TGF-, and subjected to immunoblotting for STAT5b (left), or assessed for the percentage of Foxp3+ cells among live virus–infected (i.e., GFP+) CD4+ T cells by flow cytometry (n = 7) (right). ***P < 0.001 (Student’s t test).

Article Snippet: A fragment encoding mouse CDK8, CDK19, or STAT5b cDNA was obtained from pCMV6-CDK8 (OriGene, MR218219), pCMV6-CDK19 (OriGene, MR215712), or pCMV6-STAT5b (OriGene, MR210649) and subcloned into a retroviral plasmid pMCs-IRES-GFP (Cell Biolabs).

Techniques: Expressing, Infection, Immunoprecipitation, Western Blot, Phospho-proteomics, Recombinant, Incubation, Control, Microscopy, Staining, Mutagenesis, Virus, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of Cdk8/Cdk19 Activity Promotes Treg Cell Differentiation and Suppresses Autoimmune Diseases

doi: 10.3389/fimmu.2019.01988

Figure Lengend Snippet: Cdk8/Cdk19 inhibition promotes the differentiation of Treg cells. (A) Purified CD4 + T cells were cultured under iTreg cell polarizing condition (1 ng/ml of TGF-β and 20 U/ml of rIL-2) in the presence of DMSO or different concentrations of CCT251921 as indicated. Treg population was assessed by Foxp3 intracellular staining and flow cytometry. The FACS plots are representative for three independent experiments. (B) Quantification of Foxp3 + cell percentage after DMSO or 0.05 μM CCT251921 treatment. Composite data of 7 mice per group from three independent experiments, Mean ± SD, *** P < 0.001, by two-sided t -test. (C) CFSE-labeled CD4 + T cells were activated with the treatment of mock (blue line) or CCT251921 (0.5 μM, red line) for 4 days. Cell proliferation was analyzed by CFSE dilution and division index (right) by flow cytometry. Result is summary of 4 mice per group from two independent experiments, Mean ± SD, n.s. not significant, by two-sided t -test. (D) Purified CD4 + T cells were cultured under iTreg cell polarizing condition in the presence of DMSO or different doses of Senexin A as indicated. Treg population was assessed by Foxp3 intracellular staining and flow cytometry. The FACS plots are representative for three independent experiments. (E) Quantification of Foxp3 + cell percentage after DMSO or 0.5 μM Senexin A treatment. Composite data of 6 mice per group from three independent experiments, Mean ± SD, *** P < 0.001, by two-sided t -test. (F) CFSE-labeled CD4 + T cells were activated with the treatment of mock (blue line) or Senexin A (0.5 μM, red line) for 4 days. Cell proliferation was analyzed by CFSE dilution and division index (right) by flow cytometry. Result is summary of 4 mice per group from two independent experiments, Mean ± SD, n.s. not significant, by two-sided t -test.

Article Snippet: Protein extracts were separated by 4–15% SDS-PAGE gel (Bio-Rad) and transferred to the PVDF membrane (Millipore) and analyzed by immuno-blotting with following antibodies: β-actin (I-19, Santa Cruz), CDK8 (P455, CST), pStat1-Y701 (58D6, CST), pStat1-S727 (D3B7, CST), pSmad2 linker (Ser245/250/255, CST), pSmad2 (Ser465/467)/Smad3 (Ser425/427) (D27F4, CST), and FoxP3 (eBio7979, eBioscience).

Techniques: Inhibition, Purification, Cell Culture, Staining, Flow Cytometry, Labeling

Journal: Frontiers in Immunology

Article Title: Inhibition of Cdk8/Cdk19 Activity Promotes Treg Cell Differentiation and Suppresses Autoimmune Diseases

doi: 10.3389/fimmu.2019.01988

Figure Lengend Snippet: Blockade of Cdk8/Cdk19 promotes the expression of Treg signature genes and enhances the Treg suppressive function in vitro . (A) The relative mRNA expression level of Foxp3, Ctla4, Tnfrsf18 (GITR), and IL2Ra compared to β -actin from CD4 T cells after 3 days in vitro iTreg differentiation treated with DMSO or CCT251921 (0.1 μM). (B) The expression of CTLA-4, GITR, and CD25 in CD4 + Foxp3 + Treg cells as shown in (A) . The FACS plots are representative for three independent experiments. (C) The in vitro suppressive function of Treg cells from DMSO or CCT251921 treated groups analyzed by Tresp (responder T) cell proliferation (left) and statistical summary of suppression efficiency (right). ( N = 3 mice; representative results are shown; means ± SD, ** p < 0.01, *** p < 0.001, per two-sided t -test). (D) The mRNA expression level of Foxp3 compared to β-actin from CD4 T cells at indicated hours after in vitro iTreg differentiation treated with DMSO or CCT251921 (0.1 μM). (E) The mRNA expression level of Foxp3 compared to β-actin from CD4 T cells at indicated days after in vitro iTreg differentiation treated with DMSO or CCT251921 (0.1 μM). All the realtime results are summary of 3 mice per group from three independent experiments, Mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s . not significant, by two-sided t -test.

Article Snippet: Protein extracts were separated by 4–15% SDS-PAGE gel (Bio-Rad) and transferred to the PVDF membrane (Millipore) and analyzed by immuno-blotting with following antibodies: β-actin (I-19, Santa Cruz), CDK8 (P455, CST), pStat1-Y701 (58D6, CST), pStat1-S727 (D3B7, CST), pSmad2 linker (Ser245/250/255, CST), pSmad2 (Ser465/467)/Smad3 (Ser425/427) (D27F4, CST), and FoxP3 (eBio7979, eBioscience).

Techniques: Expressing, In Vitro

Journal: Frontiers in Immunology

Article Title: Inhibition of Cdk8/Cdk19 Activity Promotes Treg Cell Differentiation and Suppresses Autoimmune Diseases

doi: 10.3389/fimmu.2019.01988

Figure Lengend Snippet: Blocking Cdk8/Cdk19 activity sensitizes TGF-β signaling to promote Treg differentiation. (A) Purified CD4 + T cells were cultured under Treg cell polarizing condition with different dose of TGF-β in the presence of DMSO, or CCT251921 (0.1 μM), with/out SB431542 (1 μM). Treg population was assessed by Foxp3 intracellular staining and flow cytometry. The FACS plots are representative for three independent experiments. (B) Statistical analysis of Treg population in (A) . Mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, by two-sided t -test.

Article Snippet: Protein extracts were separated by 4–15% SDS-PAGE gel (Bio-Rad) and transferred to the PVDF membrane (Millipore) and analyzed by immuno-blotting with following antibodies: β-actin (I-19, Santa Cruz), CDK8 (P455, CST), pStat1-Y701 (58D6, CST), pStat1-S727 (D3B7, CST), pSmad2 linker (Ser245/250/255, CST), pSmad2 (Ser465/467)/Smad3 (Ser425/427) (D27F4, CST), and FoxP3 (eBio7979, eBioscience).

Techniques: Blocking Assay, Activity Assay, Purification, Cell Culture, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of Cdk8/Cdk19 Activity Promotes Treg Cell Differentiation and Suppresses Autoimmune Diseases

doi: 10.3389/fimmu.2019.01988

Figure Lengend Snippet: Cdk8/Cdk19 inhibition promotes Treg differentiation by suppressing IFN-γ signaling and activating TGF-β-Smad signaling. (A) The phosphorylation of Stat1 (pStat1-Y701, pStat1-S727), Smad2/3 (pSmad2, pSmad3) and Smad2 linker (pSmad2 Ser245/250/255), and protein expression of CDK8 and Foxp3 were analyzed by western blot at day 1 and day 2 of iTreg differentiation in the presence of DMSO, Senexin A (0.5 μM, SA), or CCT251921 (0.1 μM, CCT). The image was representative for at least three independent experiments. (B) The phosphorylation of Stat1 (pStat1-S727), Smad2/3 (pSmad2, pSmad3) and Smad2 linker (pSmad2 Ser245/250/255), and protein expression of CDK8 at early time points (0, 3, 6, and 12 h) of iTreg differentiation in the presence of DMSO, Senexin A (0.5 μM, SA), or CCT251921 (0.1 μM, CCT). The image was representative for at least three independent experiments. (C) Purified CD4 + T cells were cultured under iTreg cell polarizing condition (1 ng/ml of TGF-β and 20 U/ml of IL-2) in the presence of DMSO, or CCT251921 (0.1 μM), with/out IFN-γ (20 ng/ml). Treg population was assessed by Foxp3 intracellular staining and flow cytometry. (D) Purified CD4 + T cells were cultured under iTreg cell polarizing condition (1 ng/ml of TGF-β and 20 U/ml of IL-2) in the presence of DMSO, or CCT251921 (0.1 μM), with/out anti-IFN-γ (5 μg/ml). Treg population was assessed by Foxp3 intracellular staining and flow cytometry. For (C,D) , the FACS plots are representative for more than three independent experiments. Mean ± SD, n.s . not significant, ** p < 0.01, by two-sided t -test.

Article Snippet: Protein extracts were separated by 4–15% SDS-PAGE gel (Bio-Rad) and transferred to the PVDF membrane (Millipore) and analyzed by immuno-blotting with following antibodies: β-actin (I-19, Santa Cruz), CDK8 (P455, CST), pStat1-Y701 (58D6, CST), pStat1-S727 (D3B7, CST), pSmad2 linker (Ser245/250/255, CST), pSmad2 (Ser465/467)/Smad3 (Ser425/427) (D27F4, CST), and FoxP3 (eBio7979, eBioscience).

Techniques: Inhibition, Phospho-proteomics, Expressing, Western Blot, Purification, Cell Culture, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Inhibition of Cdk8/Cdk19 Activity Promotes Treg Cell Differentiation and Suppresses Autoimmune Diseases

doi: 10.3389/fimmu.2019.01988

Figure Lengend Snippet: Treatment with Cdk8/Cdk19 inhibitor ameliorates EAE symptoms. (A–C) The disease incidence (A) and the recorded clinical scores (B) and the linear-regression analysis (C) of mice with indicated treatments at different time points after EAE elicitation. N = 10 mice for DMSO group; N = 9 mice for CCT251921 group. Mean ± SEM, *** p < 0.001, by two-way multiple-range ANOVA. (D,E) The population of CD4 + Foxp3 + Treg cells (D) and IFN-γ, IL-17A-producing CD4 + T cells (E) in draining lymph nodes (dLN, left), spleen (middle) and spinal cord (right) at day 18 of EAE treated either with DMSO or CCT251921. (F) Total lymphocyte number recovered in the spinal cord of EAE mice with indicated treatment. N = 5 mice, Mean ± SD, * p < 0.05, by two-sided student's t -test.

Article Snippet: Protein extracts were separated by 4–15% SDS-PAGE gel (Bio-Rad) and transferred to the PVDF membrane (Millipore) and analyzed by immuno-blotting with following antibodies: β-actin (I-19, Santa Cruz), CDK8 (P455, CST), pStat1-Y701 (58D6, CST), pStat1-S727 (D3B7, CST), pSmad2 linker (Ser245/250/255, CST), pSmad2 (Ser465/467)/Smad3 (Ser425/427) (D27F4, CST), and FoxP3 (eBio7979, eBioscience).

Techniques:

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: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a, A representative Western blot analysis of CDK8 immunoprecipitation (n = 2) from nuclear extracts from Med13/13l fl/fl (WT) and Med13/13l −/− (CKM-Mediator KO) ESCs, probed with the indicated antibodies. b, Quality control metrics of the Hi-C data, showing total sequenced read-pairs in millions, total valid contacts in millions and percentages in cis contacts for WT and CKM-Mediator KO ESCs. c, Aggregate analysis of super enhancer interactions in WT and CKM-Mediator KO ESCs. The difference between WT and KO is shown. d, Aggregate analysis of Hi-C signal (10 kb resolution) at pairs of Polycomb domains at the indicated distance ranges in Med13/13l fl/fl (WT) and Med13/13l −/− (CKM-Mediator KO) ESCs, with 200 kb flanking regions. Interactions of inactive non-Polycomb gene promoters subsampled to match regions as in Fig. (n = 2096), are included as a negative control (bottom). The difference between WT and KO is shown. e , Capture-C interaction scores for interactions between Polycomb domains in WT and CKM-Mediator KO ESCs (number of promoters = 51, number of interactions = 148). f, Boxplot analysis of Capture-C interaction scores from WT and CKM-Mediator KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters with active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Control, Hi-C, Negative Control, Capture-C

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A Venn diagram showing the overlap between CDK8 peaks and Polycomb domains. Number of peaks and percent overlap are indicated. b, Metaplot analysis of CDK8 enrichment at Polycomb domains (n = 2097) in WT and CKM-Mediator (CKM-MED) KO ESCs. c , Heatmaps showing CDK8 ChIPseq signal at Polycomb domains (n = 2097) in WT and CKM-Mediator KO ESCs, sorted by decreasing RING1B signal. d, A representative Western blot analysis (n = 6) of nuclear extracts from WT and CKM-MED KO ESCs probed with the indicated antibodies. TBP and HDAC1 are used as loading controls. e , Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and loss of cPRC1 (PCGF2) binding (log2 fold change) at Polycomb domains. Polycomb domains were divided into equal bins (261 domains each) based on log2 fold change in cPRC1 binding. f, Comparison between loss of Hi-C signal (difference between CKM-MED-KO and WT) and levels of CDK8 binding in WT cells (log2RPKM). Domains were divided into eight bins based on CDK8 RPKM levels.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: Western Blot, Comparison, Hi-C, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , Heatmaps showing RING1B (PRC1) and CDK8 ChIP–seq signals at Polycomb domains ( n = 2097), sorted by decreasing RING1B signal. b , A genomic snapshot of a Polycomb-bound locus, showing CDK8, RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal in WT (+) and CKM–MED KO (-) ESCs. c , Heatmaps showing RING1B, PCGF2, CBX7 and H3K27me3 ChIP–seq signal at Polycomb domains ( n = 2,097) in WT (+) and CKM–MED KO (-) ESCs, sorted by decreasing RING1B signal. d , Metaplot analysis of RING1B, PCGF2, CBX7 and H3K27me3 enrichment at Polycomb domains ( n = 2,097) in WT and CKM–MED KO ESCs.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: ChIP-sequencing

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A schematic of the integrated TetO site and experimental setup. b , A snapshot showing Capture-C read count signal from TetR-PCGF2, TetR-CDK8 and TetR-GFP lines at the TetO array. CDK8 and PCGF2 (cPRC1) ChIP–seq signal is given as a reference. The TetO bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. c , A schematic of the cPRC1 ( Pcgf4 −/− Pcgf2 fl/fl ) conditional KO line. d , A snapshot showing Capture-C read count signal from WT and cPRC1 KO ESCs. Interactions between the Nkx2-1 promoter bait (triangle) and surrounding Polycomb domain sites are shown with arrowheads. cPRC1 binding (PCGF2 ChIP–seq) is shown as a reference. e , Boxplot analysis of normalized read counts from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Boxes show IQR, center lines represent the median, whiskers extend 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt( n ), giving a roughly 95% confidence interval for comparing medians.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: Capture-C, ChIP-sequencing, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A representative Western blot analysis (n = 3) of nuclear extracts from the TetR-fusion lines used for Capture-C analysis probed with anti-Flag antibody to detect expression of the fusion proteins. HDAC1 is used as a loading control. b , ChIP-qPCR analysis of binding of the different TetR-fusion lines to the TetO array. Data are presented as mean value (n = 2) ±SD. Data points for individual replicates are shown. c, ChIP-qPCR analysis of binding of the CKM-Mediator complex to the TetO array in the TetR-CDK8, TetR-PCGF2, and TetR-GFP lines.. Data are presented as mean value (n = 2 for TetR-CDK8 and n = 3 for TetR-PCGF2 and TetR-GFP) ± SD. Data points for individual replicates are shown. d , Boxplot analysis of Capture-C mean normalised read counts and interaction scores in the TetR-fusion lines, looking at interactions with Polycomb domains (PCGF2-bound). Number of interactions is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. e , Snapshots showing Capture-C read count signal from TetR-CDK8, TetR-PCGF2 and TetR-GFP lines at a control locus. CDK8 and PCGF2 (cPRC1) ChIPseq signal is given as a reference. The Fli1 promoter bait is shown as a triangle and interactions created with surrounding cPRC1-bound sites are represented with arrowheads. f , A representative Western blot analysis of nuclear extracts (n = 3) from WT and cPRC1 KO ESCs probed with the indicated antibodies. TBP is used as a loading control. g , Metaplot analysis of CDK8 enrichment at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs. h , Heatmaps showing CDK8 ChIPseq signal at CDK8 peaks (n = 24275) and Polycomb domains (n = 2097) in WT and cPRC1 KO ESCs, sorted by decreasing CDK8 or RING1B signal, respectively. i, Boxplot analysis of Capture-C interaction scores from WT and cPRC1 KO ESCs showing interactions between Polycomb gene promoters and other Polycomb-domains (left), or non-Polycomb gene promoters and active sites (H3K27ac, right). Number of promoters (P) and interactions (int) is shown. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: Western Blot, Capture-C, Expressing, Control, ChIP-qPCR, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A schematic of the differentiation of WT and CKM–MED KO ESCs used for cnRNA-seq. b , Boxplot analysis of the expression of CKM–MED-dependent genes ( n = 631) in WT ESCs and following RA (retinoic acid) induction (WT and CKM–MED KO). Boxes show IQR, center lines represent the median, whiskers extend by 1.5 × IQR or the most extreme point (whichever is closer to the median), whereas notches extend by 1.58 x IQR/sqrt( n ), giving a roughly 95% confidence interval for comparing medians. c , A schematic of the differentiation of WT and cPRC1 KO ESCs for cnRNA-seq. d , As in b but for cPRC1 cKO cells. e , A screenshot showing the expression of genes within the HoxB cluster following RA induction of CKM–MED cKO or cPRC1 KO cells. Forward strand is shown on top and reverse strand is shown at the bottom of each track. ChIP–seq tracks for CDK8 and cPRC1 (PCGF2) enrichment are shown. f , Boxplot analysis of the expression of RA-induced (RA-ind) genes from the Polycomb (PcG) network (top, n=482) and CKM–Med-dependent genes from the PcG network (bottom, n=184) following RA induction of CKM–MED cKO or cPRC1 KO cells. Boxes are defined as in a .

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: Expressing, ChIP-sequencing

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a, A schematic illustration of the generation of the T7-MED14 expressing Med13/13l fl/fl ESC line. b, PCR showing amplification of homozygously-tagged T7-Med14 alleles (n = 2). c, A representative Western blot analysis of nuclear extracts from the T7-MED14 Med13/13l fl/fl ESC line, following tamoxifen (TAM) treatment (n = 3). Extract from an untagged ESC line was used as a control. HDAC1 was used as a loading control. d, A representative immunoprecipitation (IP) of endogenously T7-tagged MED14 with T7 antibody using nuclear extracts from Med13/13l fl/f ESCs before (UNT) and after tamoxifen (TAM) treatment (n = 2). The IPs were probed with the indicated antibodies. An IP from an untagged ESC line was performed as a negative control and a Western blot for SUZ12 was included as a control protein that does not interact with Mediator. e, Heatmaps of CDK8 and T7-MED14 ChIPseq signal at Polycomb domains (n = 2097) and H3K27ac peaks (n = 4037), sorted by decreasing CDK8 signal. f, Boxplots showing gene expression change (log2FC) of CKM-Mediator-dependent (n = 631) and CKM-Mediator-independent (n = 2689) RA-induced genes following RA differentiation of WT ESCs. Boxes show interquartile range, center line represents median, whiskers extend by 1.5x IQR or the most extreme point (whichever is closer to the median), while notches extend by 1.58x IQR/sqrt(n), giving a roughly 95% confidence interval for comparing medians. g, Boxplots showing T7-MED14 ChIPseq signal at the TSS (1000 bp) of the different classes of RA-induced gene classes as defined in e in ESCs and RA-induced cells (WT and CKM-Mediator KO). Boxes are defined as in f . Signal is an average from three independent biological experiments.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: Expressing, Amplification, Western Blot, Control, Immunoprecipitation, Negative Control, Gene Expression

Journal: Nature Structural & Molecular Biology

Article Title: Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

doi: 10.1038/s41594-022-00840-5

Figure Lengend Snippet: a , A genomic snapshot of two CKM–Mediator-dependent genes, showing CDK8 and T7-MED14 ChIP–seq and cnRNA-seq in WT (+) and CKM–MED KO (-) ESCs (top) and following RA induction (bottom). b , Heatmaps showing CDK8 and T7-MED14 ChIP–seq signal at promoters (TSS±2.5 kb) of CKM–MED-dependent genes in ESCs and following RA induction ( n = 631). T7-MED14 signals are shown for WT and CKM–Mediator KO RA-induced cells. Genes are sorted by decreasing T7-MED14 signal in RA-treated cells. Metaplots showing read density are shown on the top of each heatmap.

Article Snippet: Samples were incubated with 5 μg CDK8 antibody (A302-500A, Bethyl laboratories) and 25 units benzonase nuclease (Millipore) overnight at 4 °C.

Techniques: ChIP-sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: Hotair promotes the migration and proliferation in ovarian cancer by miR-222-3p/CDK19 axis

doi: 10.1007/s00018-022-04250-0

Figure Lengend Snippet: miR-222-3p suppresses OC tumor growth in vivo by targeting CDK19. A Subcutaneous xenograft model. The equivalent numbers of LV-miR-222-3p and GFP-labeled OE-CDK19 transfected stably in OC cells (1 × 10 7 ) were subcutaneously (s.c.) injected into each mouse ( n = 5). Tumor growth was monitored every 5 days. B Fluorescence efficiency of four groups of stable cell lines. C The volume curve of tumor growth in mice for 30 days. D Representative images and bioluminescence images of tumors in mice 30 days after implantation ( n = 5 mice per group, right ). Bar , 0.5 cm. The tumor weights in nude mice at the 30 days were determined ( left ). E Western blotting analysis of CDK19 levels in OC xenografted tumor. Image J calculated the relative expression rate. F Representative HE staining of the tumor tissues was obtained from 30 days after implantation. Bar, 50 µm ( left ) and 100 µm ( right ). G IHC staining for CDK19 in the tumor tissues of mice 30 days after implantation. Bar , 100 µm. Results are presented as mean ± SD. The results of ( C, D ) are presented as mean ± SD and determined by unpaired two-way ANOVA

Article Snippet: The expression levels of CDK19 in ovarian tumors/normal ovarian tissues and the mice’ section of cancerous tissue were evaluated by IHC using an anti-CDK19 antibody (Proteintech, 13761–1-AP, 1:1000).

Techniques: In Vivo, Labeling, Transfection, Stable Transfection, Injection, Fluorescence, Western Blot, Expressing, Staining, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences

Article Title: Hotair promotes the migration and proliferation in ovarian cancer by miR-222-3p/CDK19 axis

doi: 10.1007/s00018-022-04250-0

Figure Lengend Snippet: miR-222-3p directly suppresses CDK19 expression by binding to its 3’-UTR and inhibits OC cell proliferation. A A Venn diagram was intersected to look for the downstream candidate genes targeted by miR-222-3p. B Expression levels of seven candidate genes were detected after transfection with miR-222-3p mimic in OC cells. C qRT-PCR analyses were used to detect CDK19 mRNA levels after transfection with miR-222-3p mimic or inhibitor in OVCAR3 cells or SKOV3 cells, respectively, the results were determined by an unpaired two-tailed t -test. D CDK19 was significantly up-regulated in OC tissues ( p < 0.0001). E Kaplan–Meier curves for overall survival probability in OC patients with low and high CDK19 expression. F Schematic description of the imaginary double strand formed by miR-222-3p with the 3′-UTR of CDK19. G Effectiveness of the CDK19 and ctrl vector with GFP fluorescence ( left ), the quantized figure is the Mean gray value ( middle ) and Integrated density ( right ). H Relative luciferase activities in HEK-293T cells co-transfected with a miR-222-3p mimic/or inhibitor and CDK19 WT/or MUT. I The proliferation ability of OVCAR3 after CDK19 transfection was assessed by the colony formation assay, the results were determined by an unpaired two-tailed t -test. J, K The colony formation assay ( J ) and EdU assay ( K ) revealed inhibition of proliferation when OVCAR3 cells were transfected with miR-222-3p mimic. Recovery assays showed that miR-222-3p suppressed the proliferation of OVCAR3 cells due to its inhibitory effect on CDK19. The results of J and K are presented as mean ± SD and determined by unpaired two-way ANOVA. All the experiments were performed in triplicate

Article Snippet: The expression levels of CDK19 in ovarian tumors/normal ovarian tissues and the mice’ section of cancerous tissue were evaluated by IHC using an anti-CDK19 antibody (Proteintech, 13761–1-AP, 1:1000).

Techniques: Expressing, Binding Assay, Transfection, Quantitative RT-PCR, Two Tailed Test, Plasmid Preparation, Fluorescence, Luciferase, Colony Assay, EdU Assay, Inhibition

Journal: Cellular and Molecular Life Sciences

Article Title: Hotair promotes the migration and proliferation in ovarian cancer by miR-222-3p/CDK19 axis

doi: 10.1007/s00018-022-04250-0

Figure Lengend Snippet: Hotair regulates CDK19 expression via miR-222-3p. A EdU assay was used to detect the rescue effect of OE-Hotair on OVCAR3 cells proliferation abilities ( left ). Scale bar , 100 µm. The number of cells was counted ( right ). B Transwell assay was used to detect the rescue effect of OE-Hotair on OVCAR3 cells’ migration abilities. Scale bar , 100 µm ( left ). The number of cells was counted ( right ). C–E Pearson’s correlation scatter plots show the fold changes of CDK19 mRNA, miR-222-3p miRNA, and Hotair mRNA levels in OC tissues ( n = 54). F After the transfection of the OE-Hotair plasmid, the protein expression of CDK19 was detected by Western blotting. G, H Western blotting of CDK19 in OVCAR3 and HO-8910 PM cells with Hotair and/or miR-222-3p mimics. I A working model describing the interaction between Hotair/miR-222-3p/CDK19 during cancer development. Hotair regulated CDK19 by sponging miR-222-3p expression and subsequent targeting of genes. Results are presented as mean ± SD

Article Snippet: The expression levels of CDK19 in ovarian tumors/normal ovarian tissues and the mice’ section of cancerous tissue were evaluated by IHC using an anti-CDK19 antibody (Proteintech, 13761–1-AP, 1:1000).

Techniques: Expressing, EdU Assay, Transwell Assay, Migration, Transfection, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of CDK8 gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Genome Wide, CRISPR, shRNA, In Vitro, Cell Counting, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Correlation between CDK8 gene dependency and sensitivity to the CDK8 inhibitor BI-1347 in 669 cancer cell lines (y-axis showing CDK8 dependency score; x-axis showing PRISM LFC value of BI-1347 treatment). Linear regression lines and Pearson correlation coefficients (R) are shown for all cell lines (black), aRMS (red), and eRMS (blue). B. Western blot analysis showing CDK8 inhibition by small molecules as assessed by STAT1 phosphorylation at serine 727. C, D. Dose response curves of inactive BI-1347 analog (BI-1374) (C) and three pharmacologic CDK8 inhibitors (D), BI-1347, SEL-120-34A, and JH-XII-178, at day 7 of treatment in Rh30, Rh4 and RHJT cell lines. E, F. Live cell proliferation assessed by Incucyte for Rh30 (E) and Rh4 (F) cells after treatment with vehicle DMSO (black), the CDK8 inhibitor BI-1347 (red) and its inactive analog BI-1374 (gray). Data were normalized to DMSO. Data represent means ± SEM (n=6, *: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. Line graph reveals mean subcutaneous tumor volume (mm ) formed by Rh30 cells after treatment with the CDK8 inhibitor SEL-120-34A. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Western Blot, Inhibition, Phospho-proteomics

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plots showing the number of gene body changes after 24 hrs of CDK8 inhibitor BI-1347 treatment. Significantly up-regulated genes are highlighted in red; significantly downregulated genes are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). B. Metagene plots of PRO-seq reads of genes with significant upregulation or downregulation of gene body transcription at 24 hrs of BI-1347 treatment. C. Heatmap of Log 2 transformed fold change values of PRO-seq pausing indices of genes with changes in gene body transcription at the 24 hr time point. D. Heatmap showing log 2 transformed fold change of RNA-seq read counts for indicated gene sets after DMSO and BI-1347 (10 nM) treatment for 24 or 72 hrs. E. Bubble dot plot shows Gene Set Enrichment Analysis (GSEA) of PRO-seq gene hits that are upregulated at 24 hrs. Dot size indicates number of genes in each gene set, and dot color indicates p -adjusted value. F. Immunofluorescence analysis of myogenin (green; 20X) in Rh30 (left) and Rh4 (right) cells after 7 days of DMSO or BI-1347 treatment. G. Representative images of H&E stain of xenograft tumors. Arrows indicate myofibrils. H. Volcano plots reveal the number of changes in enhancer RNA (eRNA) transcripts after 24 hrs of BI-1347 treatment. Significantly upregulated eRNAs are highlighted in red; significantly downregulated eRNAs are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5) I. MA plots showing the changes of chromatin accessibility by ATAC-seq following 4 and 24 hrs of BI-1347 treatment. Significantly increased ATAC-seq peaks are highlighted in red; significantly decreased ATAC-seq peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). J. Pie chart showing the peak annotation of up-regulated ATAC-seq peaks to gene features at 24 hrs of BI-1347 treatment. K. Histogram of PRO-seq reads around up-regulated ATAC-seq peaks at indicated time points of BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Transformation Assay, RNA Sequencing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. IGV gene tracks showing a time course analysis of BI-1347 treatment by PRO-seq signal at the PAX3::FOXO1 locus. B. Heatmaps of CUT&RUN analysis of CDK8 and PAX3::FOXO1 binding in Rh30 and Rh4 cell lines. C. MA plots showing changes of PAX3::FOXO1 binding following BI-1347 treatment at 24 and 72 hrs. Significantly increased PAX3::FOXO1 peaks are highlighted in red; significantly decreased PAX3::FOXO1 peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). D, E. Heatmaps of CDK8 (D) and PAX3::FOXO1 (E) signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. F. Heatmaps of log 2 transformed fold change of RNA-seq read counts plotted by the shared gene between 24 hrs of PAX3::FOXO1 degradation and 72 hrs of CDK8 inhibition at the indicated time points. G. IGV gene tracks showing a time course analysis for PRO-seq signal at the VGLL2 locus after BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Binding Assay, Transformation Assay, RNA Sequencing, Inhibition

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Scatter plot showing the Z-scored average log2 fold change (LFC) of gene knockout effects in Rh30 cells treated with BI-1347 versus DMSO at day 14 (y-axis) and day 21 (x-axis) from genome-wide CRISPR-Cas9 screens. Each point represents an individual gene; dot size corresponds to statistical significance, and dot color indicates classification. B. Bubble dot plot of GSEA for gene hits scoring at day 21 in the CRISPR-Cas9 BI-1347 drug modifier screen ranked by C5 gene sets. C. Box plots showing construct-level Z-score averages for individual genes in the SAGA complex from the genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by SAGA functional modules. D-F. Live cell proliferation assessed by Incucyte for BI-1347+/-sgTADA2B ( D ), BI-1347+/-sgTAF5L ( E ), and BI-1347+/-GSK699 ( F ). G. Quantitative real-time TaqMan qPCR analysis of RUNX1 , SEMA3D , and VGLL 2 expression at day 7 following treatment with vehicle control (DMSO), CDK8 inhibitors, or GSK699, and the indicated combination treatments. Expression levels were normalized to GAPDH gene expression and shown relative to DMSO control. Data represent means ± SEM (n=6).

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Gene Knockout, Genome Wide, CRISPR, Construct, Functional Assay, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Live cell proliferation assay by Incucyte for BI-1347 treatment combined with CRISPR knockout of SIX4 with two different guide RNAs. B. Western blot analysis of SIX4 protein level after treatment with three CDK8 inhibitors at indicated time points. Lamin B included as a loading control. C, D. Western blot analysis of SIX4 protein levels from cytoplasmic and nuclear fractions from Rh30 ( C ) and Rh4 ( D ) cells treated with DMSO or BI-1347 for 14 days. GAPDH and Lamin B served as nuclear and cytoplasmic loading controls, respectively. E, F. Volcano showing log 2 fold change of SIX4 ( E ) and TADA2B ( F ) genome binding sites determined by CUT&RUN analysis. Red highlights significantly up regulated sites and blue highlights significantly down regulated sites (log 2 FC 1.5, padj<0.05). G, H. Pie charts showing the annotation of upregulated CUT&RUN peaks for SIX4 ( G ) and TADA2B ( H ) sites after 24 hrs of BI-1347 treatment in Rh30 cells. I. Heatmaps of time course analysis of ATAC-seq signal, SIX4 signal, TADA2B signal, H3K27ac signal around upregulated ATAC-seq peaks at 24 hrs of BI-1347 treatment. J. IGV gene tracks showing the PRO-seq, SIX4, TADA2B, ATAC-seq, PAX3::FOXO1, CDK8, H3K4me3, and H3K27ac at the VGLL2 gene body and enhancers loci at indicated time points of BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Proliferation Assay, CRISPR, Knock-Out, Western Blot, Control, Binding Assay

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

Techniques: Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay