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. 2023 Jun 28;51(14):7288–7313. doi: 10.1093/nar/gkad538

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Expression of CDK8 and CDK19 proteins and effects of CDK8 and CDK19 knockout and re-expression on STAT1 S727 phosphorylation in different cell lines. (A) Relative protein levels of CDK8 and CDK19, normalized to CDK8 protein level in 293 cells, in different cell lines, determined as shown in Supplementary Figure S6. Data are presented as mean ± SEM of biological triplicates. Ratios of CDK8 to CDK19 for each cell line are shown on top of the bars. (B) RNA levels of CDK8 and CDK19 in different cell lines (RPKM, Reads Per Kilobase, per Million mapped reads) from RNA-Seq in this study (293 cells) and from CCLE database (all the other cell lines). (C) Correlation of CDK8:CDK19 ratios between RNA and protein levels among different cell lines. (D) HAP1 (WT) and their CDK8 or CDK19 single- or double-knockout derivatives (8KO, 19KO, dKO) were treated with 1 μM Senexin B, 10 ng/ml recombinant IFNγ, or Senexin B/IFNγ combination for 5 hrs before immunoblotting analysis for phosphorylated STAT1 (S727 or Y701), STAT1, CDK8, CDK19, CCNC and GADPH. (E) HeLa (WT) and their CDK8 or CDK19 single-knockout derivatives (8KO or 19KO) were treated with 1 μM Senexin B, 5 ng/ml recombinant IFNγ, or Senexin B + IFNγ combination for 5 hrs and analyzed as in (D). (F) HCT116 (WT) and their CDK8 or CDK19 single-knockout derivative (8KO or 19KO) were treated with 1 μM Senexin B, 10 ng/ml recombinant IFNγ, or Senexin B + IFNγ combination for 1 hr before Immunoblotting analysis. (G) HCT116-8KO and their reconstitution derivatives (8KO-8, 8KO-K19) were treated and analyzed as in (D). (H) HCT116-8KO and their reconstitution derivatives (8KO-8M, 8KO-19M) were treated and analyzed as in (D). (I) 22Rv1 (WT) and their CDK8 and CDK19 single- or double-knockout derivatives (8KO, 19KO, dKO) were treated with 1 μM Senexin B, 20 ng/ml recombinant IFNγ, or Senexin B/IFNγ combination for 5 h and analyzed as in (D). Bar diagrams on the right in (D–I) represent mean densitometry signals from duplicate experiments.