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. 2020 Sep 28;130(11):5765–5781. doi: 10.1172/JCI134217

Figure 2. STAT3 mediates CDK4/6-dependent IκBζ induction in keratinocytes.

Figure 2

(A) Luciferase assay of the NFKBIZ promoter in HEK293T cells after transient expression of CDK4, CDK6, STAT3, or p65, alone or in combination. The plasmid amounts for STAT3 (200 ng) and p65 (70 ng) were adjusted to achieve similar luciferase activity in the absence of CDK4/6 expression. Overexpression of the HA-tagged CDK4 and CDK6 proteins was detected using a HA-antibody. (B) Primary human keratinocytes with a transient overexpression of hyperactive STAT3 (STAT3C) were treated for 1 hour with 100 ng/mL IL-36α and abemaciclib (Abe). NFKBIZ mRNA levels normalized to RPL37A. Immunoblot analysis of STAT3C overexpression and CDK4/6 inhibition. (C) IκBζ target gene expression in STAT3C-overexpressing primary keratinocytes. Treatment as in B. (D) Luciferase activity assay of the NFKBIZ promoter in HEK293T cells overexpressing STAT3 alone or in combination with WT CDK6 (wt), hyperactive CDK6 (S178P), or a kinase-dead CDK6 mutant (CDK6 DN). For all analyses, n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.