Fig. 4: AFF1 associates with inactive CDK9 to repress target gene expression.

a qRT-PCR showing mRNA levels of differentiation activators with 3-hour CDK9 inhibitor (CDK9i) treatment with flavopiridol (Flav) or NVP2 relative to DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR showing the expression of differentiation-activating TFs in keratinocytes treated with KL alone or in combination with CDK9 inhibitors, as compared to DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). c Western blot showing knockdown (KD) efficiency of HEXIM1 shRNA’s (n = 3 biological replicates, quantified in Supplementary Fig. 4c). d Clonogenic assay of human keratinocytes expressing HEXIM1-shRNA’s or non-targeting control shRNA. e Quantification of colonies >1 mm² in HEXIM1 KD conditions relative to control (n = 3/group, ****P < 0.0001, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). f qRT-PCR comparing the mRNA levels of differentiation activators in HEXIM1 KD relative to control (n = 3 technical replicates, data are presented as mean values ± standard deviation). g–i qRT-PCR showing the relative expression of differentiation-activating TFs between DMSO control and KL treatment where control non-targeting KL is relative to non-targeting DMSO, HEXIM1 sh1 KL is relative to HEXIM1 sh1 DMSO, and HEXIM1 sh2 KL is relative to HEXIM1 sh2 DMSO (n = 3 technical replicates, data are presented as mean values ± standard deviation). j Venn diagram comparing genes differentially expressed in AFF1 KD versus HEXIM1 KD RNA-seq (Fisher’s exact test, two-Tail, P = 2 × 10⁻²²⁰). k Heatmap showing shared genes significantly changed (fold change ≥2, P < 0.05, two-tailed, Wald test) in AFF1 KD RNA-seq or HEXIM1 KD RNA-seq. l Top Gene Ontology terms of the genes differentially expressed in AFF1 and HEXIM1 KD RNA-seq (two-tailed, Fisher’s exact test). m, n Co-immunoprecipitation of HA-AFF1 western blot showing interactions between AFF1 and HEXIM1, CDK9, and phosphorylated threonine 186 CDK9 (P-CDK) in progenitors (UD) and in differentiated (DF) keratinocytes (n = 3 biological replicates). Source data are provided as a Source Data file.