Figure 7. TTP destabilizes Areg mRNA in neoplastic epidermal cells.

(A) Primary keratinocytes isolated from Zfp36^fl/fl or Zfp36^ΔEP newborns were stimulated with TPA (100 ng/mL) for 2 hours prior to actinomycin D (10 μg/μl) and SB202190 (1 μM) treatment for the indicated time. Half-life of Areg mRNA mRNA was quantified by qPCR and normalized by mRNA levels at t₀ (mean ± SEM, n = 3–4, representative out of 2 experiments). (B) Schematic representation of the dual-reporter plasmids generated to assess the role of the ARE elements: 3’UTR of Areg mRNA with (AU+) or without (AU-) ARE was inserted behind the sequence coding for Renilla luciferase (R-Luc). Fold change between the Rluc/Fluc ratio of AU+ and AU- luciferase signals from dual-reporter plasmids cotransfected with TTP-Flag (or BOIP-Flag as control) in HEK293T cells (mean ± SEM, representative out of 7 experiments). (C) Electrophoretic mobility supershift assay. Extracts from HEK293T cells transiently transfected with TTP-Flag or BOIP-Flag were incubated with a ³²P-labeled probe of Areg ARE or a CTRL probe corresponding to pBS polylinker and no antibody, an α-V5, or an α-Flag antibody before migration.(D) ³²P-labeled Areg probe mixed with increasing molar excess of unlabeled Areg probe or pBS control probe was used to performed EMSA competition experiment. (C and D) Results are representative of 3 independent experiments. (E) Zfp36^ΔEP mice were treated with DMBA/TPA, and 45 minutes before each application of TPA, mice were treated with the EGFR inhibitor AG1478 (500 μg/mL) or vehicle. Mice were monitored for 12 weeks at weekly intervals for tumor development (n = 8–13, representative of 2 experiments). Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was assessed by 2-way ANOVA (B) and 2-tailed Mann-Whitney test (E). TTP, tristetraprolin; DMBA, 7,12-dimethylbenz[a]anthracene; TPA, 12-0-tetradecanoylphorbol-13-acetate; ARE, AU-rich element; pBS, pBluscript polylinker region.