Fig. 5: CDK9 suppresses RARα protein expression and physically interacts with RARα.

a Quantitative proteomic analysis and volcano plot of Hut78 cell proteins upon 23 (GT-02897) treatment, with P values derived from Wilcoxon rank-sum test. b Western blotting of indicated proteins in Hut78 under 23 (GT-02897) treatment. Western blot analysis of indicated proteins in HH and Hut78 (c) or 293T (d) cells after CDK9 depletion via lentiviruses. e A doxycycline (Dox)-inducible knockdown system was applied in HH and Hut78 cells, followed by Western blotting post Dox treatment. f Volcano plot revealing differentially expressed genes in Hut78 post CDK9 depletion, with P values derived by Wilcoxon rank-sum test. g Q-PCR assessment of CDK9 and RARA levels in Hut78 cells after CDK9 depletion. h Western blot analysis of proteins in Hut78 after CDK9 depletion, with or without MG132 treatment. i 293T cells were co-transfected with RARα and CDK9 constructs, followed by co-immunoprecipitation (Co-IP) and Western blot analysis. j GST-tagged CDK9 was incubated with HA-RARα from the WCL of 293T cells transfected with 3 × HA-tagged RARα, followed by GST pulldown and Western blot analysis. WCL: whole cell lysate. k Schematic diagram of full-length CDK9 protein (top). GST pulldown assay was performed using GST-tagged CDK9 and WCL of 293T cells transfected with 3 × HA-tagged RARα. l Lentiviral infection of Hut78 with shNC or shCDK9-1, followed by transfection of EV, 3 × Flag-tagged WT, T186A, and S347A CDK9, and Western blotting of indicated proteins. m Western blotting of indicated proteins in Hut78 post CDK9 depletion or Flavopiridol treatment. n Q-PCR analysis of RARA in Hut78 cells treated with Flavopiridol. o Schematic diagram of full-length RARα protein (top). Co-transfection of 3×Flag-tagged CDK9 with full-length or mutant RARα constructs in 293T, followed by Co-IP and immunoblotting (bottom). Results in (g, n) represent biologically independent experiments of n = 3, with P values calculated by two-tailed Student’s t-test. Data are presented as mean ± SEM, with source data provided as a Source Data file. b–e, h–m, o n = 3, independent experiments, a representative example is shown. The samples derive from the same experiment each but different gels for CDK9, β-actin and another for RARα (b, d, e, h), for RARα, β-actin and another for CDK9 (c), for HA-RARα, β-actin and another for Flag-CDK9 (i), for RARα, β-actin and another for Flag-CDK9 (l) were processed in parallel. Band intensities in (b–e, h, l, m) were analyzed and compared using Image J. Relative densitometric values are provided below the blot images.