Fig. 3: CDK9-targeting PROTACs induce selective degradation of CDK9 in CTCL cells.

a RNA-seq analysis of Hut78 cells treated with Flavopiridol or knocked down CDK9 expression with shCDK9. The Venn diagram showed the genes differentially expressed in each group. b MOLT-4 and HH cells were treated with THAL-SNS-032 and western blots were conducted. c HH cells were treated with DMSO or candidate PROTACs. Cell viability was measured using the CCK8. d Chemical structure of 23 (GT-02897). e Cell viability of Hut78 cells treated with DMSO or 19, 23, 24, 25, 26, and 27. Viability was counted by trypan blue. P value was calculated by two-tailed Student’s t-test. f Western blot analysis of indicated proteins in Hut78 cells treated with DMSO or 23, 24, 25, and 26. g, Western blot analysis of CDK9 in Hut78 cells upon THAL-SNS-032 or 23 (GT-02897) treatment. h 293T cells were treated with DMSO or 23 (GT-02897). Viability was counted by trypan blue. i Hut78 were treated with 23 (GT-02897) in the presence or absence of MG132 followed by Western blot analysis. j Hut78 cells were pre-exposed to SNS-032 or PHA-767491 followed by treatment with 23 (GT-02897). Then CDK9 protein was analyzed by Western blot. k Quantitative proteomic analysis and volcano plot of proteins that were differently regulated in Hut78 cells upon 23 (GT-02897) treatment. CDK9 was labeled. P value was derived by Wilcoxon rank-sum test. l Hut78 cells were treated with 23 (GT-02897) and Q-PCR analysis was performed for CDK9 (55 kDa) and CDK9 (42 kDa). P value was calculated by two-tailed Student’s t-test. m The regulated proteins in (k) were annotated and analyzed for the most significantly enriched pathways using Metascape. n–r Western blot analysis of indicated proteins in Hut78 cells upon 23 (GT-02897) treatment. s Hut78 cells were treated with different doses of 23 (GT-02897) for 10 h and DC₅₀ was calculated. Results in (e, h, s) represent biologically independent experiments of n = 3. Results in (l), n = 4 for CDK9(55 kDa) and n = 3 for CDK9(42 kDa). Data are presented as mean ± SEM. Source data are provided as a Source Data file. b, f, g, i, j, n–r n = 3, independent experiments, a representative example is shown. The samples in (f) derive from the same experiment but different gels for CDK9, β-actin, another for CDK7, another for CDK2 and another for CDK6 were processed in parallel. Band intensities were analyzed and compared using ImageJ. Relative densitometric values are provided below the blot images.