Figure 9.

Proteomic analysis of the effects of CDK8 and CDK19 kinase inhibition. Tandem Mass Tag (TMT) based proteomic analysis was carried out for the effects of CDK8 and CDK19 kinase inhibition across three TMT-11plex batches: dKO-8 vs dKO-8M, dKO-19 versus dKO-19M and parental 293 cells treated with DMSO (Ctrl), Senexin B (3 h) or Senexin B (72 h). (A) Comparison of the effects of CDK8 kinase domain mutation (dKO-8M versus dKO-8) on the RNA and protein levels for the genes differentially expressed at the protein level. Red dots: genes whose RNA expression levels differ <1.3-fold. Blue dots: genes whose RNA expression levels differ ≥1.3-fold. (B) Comparison of the effects of CDK19 kinase domain mutation (dKO-19M versus dKO-19) on the RNA and protein levels as in (A). (C) Comparison of the effects of CDK8 and CDK19 kinase domain mutations on the expression of proteins affected ≥1.3-fold at the RNA level. (D) Comparison of the effects of CDK8 and CDK19 kinase domain mutations on the expression of proteins affected <1.3-fold at the RNA level. (E) Comparison of the effects of 72-h treatment with Senexin B on the RNA and protein levels as in (A). (F) Comparison of the effects of 3- and 72-h Senexin B treatment for the proteins affected <1.3-fold at the RNA level. Slope and Pearson correlation coefficients (r) in were calculated by linear regression and correlation analysis for (A–F). (G) Heatmap of the effects of Senexin B treatment (3 or 72 h) and CDK8 or CDK19 kinase domain mutations on the protein and RNA levels for the genes regulated by CDK8/19 kinase activity at the post-transcriptional level and detected in all the protein batch comparisons. (H) The same heatmap for all the components of the kinase module and the core Mediator complex (grey: protein not detected). (I) Expression of the indicated proteins in parental 293 cells, untreated or treated with 1 μM Senexin B, Senexin C or 15w for 24 h and in untreated 293-dKO cells.