Fig 2: (A) TTP silencing achieved by lentiviral infection (sample labeled shZFP36) was verified by Western blot, in comparison to endogenous TTP levels measured after infection with a vector expressing an aspecific scrambled short hairpin (labeled EV). Actin was used as loading control. (B) Western blot displaying the expression levels of inflammasome components. Inflammasome interactor levels vary after lentiviral infection with a short hairpin directed against TTP mRNA. Vinculin is included as loading control. (C) NLRP3, IL1β, and TTP mRNA levels have been analyzed by qRT-PCR in both cell samples. GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM).

Fig 3: (A) qRT-PCR shows a decrease in NLRP3 mRNA in HaCaT cells transfected with a TTP-expressing vector (pcDNA3.1-ZFP36), compared to control transfection (pcDNA3.1). GAPDH was used as endogenous control. Results are represented as the means of three experiments (±SEM) (**p < 0.01). (B) Luciferase reporter assay shows a decrease in luciferase activity in HEK293T cell line cotransfected with luciferase reporter vector and the TTP-expressing vector, compared to the control cotransfection with the empty vector. Measured luciferase activity was normalized over β-gal signals. Results are represented as the means of three experiments (±SEM) (*p < 0.05). (C) Human NLRP3 3′UTR displaying TTP binding site (highlighted). (D) NLRP3 mRNA levels measured by qRT-PCR in HaCaT cells transfected at time 0 with a TTP-overexpressing vector or an empty vector. After transfection (48 h), cells were treated with Act.D to block transcription. NLRP3 mRNA levels were recorded at the moment of ActD treatment (Ctr), after 1 h and after 3 h. Results are represented as the means of three experiments (±SEM). Statistical analysis was performed comparing ZFP36-transfected and empty vector-transfected cells at each timepoint (*p < 0.05). GAPDH was used as endogenous control.

Fig 4: Effects of all-trans retinoic acid (ATRA) on GADD34 mRNA stability in C2C12 myoblasts.(A) Western blotting of GADD34 in C2C12 myoblasts incubated in the presence of 1 μM ATRA or DMSO (NT) as a vehicle for 3 h (n = 3–4). (B) The mRNA expression levels of GADD34 in C2C12 myoblasts treated with the indicated concentrations of ATRA and 2.5 μg/ml actinomycin D (n = 3–4). *P < 0.05 versus NT (one-way ANOVA with a Student–Newman post hoc test). (C) A schematic illustration of human GADD34 mRNA sequence in the upper panels. pGL3-basic plasmid empty (control) or GADD34-3′UTR was transfected and incubated with the indicated concentrations of ATRA for 3 h in C2C12 myoblasts (n = 3–4). *P < 0.05 versus NT (one-way ANOVA with a Student–Newman post hoc test). (D, E) Human GADD34 mRNA 3′UTR reporter plasmid was transfected (E) with TTP siRNA (siTTP), HuR siRNA (siHuR), or control (siCont.) and incubated in the presence of (E) 1 μM ATRA or DMSO (D) and 10 μM SB203580 (p-p38 MAPK inhibitor) or FR180204 (p-ERK inhibitor) for 24 h (n = 3–4). *P < 0.05 versus NT (DMSO) (one-way ANOVA with a Student–Newman post hoc test). (F) A schematic illustration of the mutated human GADD34 mRNA sequence in the upper panels. Mut-A1 and Mut-A2 targeted the binding sites AREs for RNA-binding protein, respectively. Each human GADD34 mRNA 3′UTR reporter plasmid (WT, Mut-A1, and Mut-A2) was transfected and incubated in the presence of 1 μM ATRA or DMSO as a vehicle for 24 h (n = 3–4). Data are presented as the mean ± SEM. *P < 0.05 versus NT (DMSO) (one-way ANOVA with a Student–Newman post hoc test). (G) The mRNA expression levels of GADD34 by RNA ChIP assay in C2C12 cells treated with 1 μM ATRA and 10 μM SB203580 for 24 h (n = 3–4). N.S., not significant. Similar results were obtained from independent experiments. Source data are available for this figure.

Fig 5: Methylation profile of ZFP36 promoter in dermal fibroblasts from skin biopsies. CpG sites are represented by red vertical bars along the upstream region of ZFP36 transcription start site, which was taken as “+1” position of base counting. The “F1” subregion, between −1,000 and −500, was amplified by PCR after bisulfite conversion and then sequenced to obtain the presented methylation profile. Two fibroblast samples from healthy donors and two lesional fibroblast samples from psoriasis donors were analyzed. White circle, unmethylated CpG; black circle, methylated CpG.