Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 3

Figure 3. c-Jun directly regulates IL-23 expression in DCs

Heat map visualization of AP-1 family member expression in DCs sorted from back skin of indicated mice after 4h IMQ stimulation as quantified by qRT-PCR. Color code (red= up, blue = down-regulation) shows fold change of Ap-1 mRNA to untreated c-Junfl/fl DC control. Asterisk shows significant differences in IMQ activated to control c-Junfl/fl DCs. (n = 4-14; 2-4 independent experiments).

Graph shows the percentage of c-Jun positive skin DCs quantified by immunofluorescence performed as described in Fig EV2 (A). (n= 5, 3 experiments).

qRT-PCR detection of Il23p19, Il17a in total RNA isolated from the back skin of indicated mice treated with IMQ for 32 or 48h. (n = 10-21; 3-6 experiments).

IL-23 and IL-17A protein was quantified by Luminex multiplex assay in cutaneous lysates of indicated mice after IMQ treatment (48h). (n= 5; 2 independent experiments).

qRT-PCR detection of Il23p19 mRNA expression levels in BMDCs of indicated genotype stimulated with IMQ for 4h and/or pre-treated with JNKi (SP600125, 25µM, 1h). (n = 4-14; 2-5 independent experiments).

IL-23 protein was quantified by ELISA (1x106 BMDCs/well) and by intracellular flow cytometry
(IL-23p19+p40+ cells among live, single, CD45+, CD11c+ cells) in c-Junfl/fl and c-JunΔ/ΔMx1-Cre BMDCs stimulated with IMQ for 4h. (Left: n = 10-13; Right: n = 9-10; ≥ 3 independent experiments).

qRT-PCR detection of Il23p19 mRNA expression levels in CD11c+MHCII+ cells sorted from IMQ (8h)-treated back skin of indicated mice. (n = 5-12; 3 experiments).

Genomic location and sequence of a putative AP-1 binding site in the mouse promoter of IL-23p19. Blockade of c-Jun/AP-1 TF binding by inhibition of phosphorylation (JNKi, red), or mutation of the binding sequence (mut 1 = T -> C; green | mut 2 = TG -> AT; blue) reduces IL-23p19 promoter activity (see Fig 4J).

c-Jun chromatin immunoprecipitation of c-Junfl/fl and c-JunΔ/ΔMx1-Cre BMDCs stimulated with IMQ for 2h. (n = 4-6; 3 independent experiments).

Luciferase activity was quantified in lysates of RAW 264.7 cells transfected with pGL3 basic vector harboring wild-type (wt) or mutated (mut1 or 2) IL-23p19 promoter. Transfected cells were pre-treated with JNKi (SP600125, 25 µM, 1h) and stimulated with IMQ (6h). Results are shown as fold change to pGL-3-wt transfected, LAL (Limulus amebocyte lysate) treated RAW264.7 cells. (n = 4; 2 independent experiments).

IL-17A production by dermal γδ T cells was analyzed in IMQ treated skin (Day 2) of indicated mice by intracellular flow cytometry. Shown are representative plots (left) and a bar graph (right) of IL-17A+ cells pre-gated on live, single, CD45+, γδ TCRint cells. (n = 7-10; 3 independent experiments).

Epidermal thickness of back skin was quantified in H&E stained sections of indicated mice treated with rIL-23 (i.d., 1µg) and/or IMQ for two consecutive days. (n = 4-7; 3 independent experiments).

Data information: Data are shown as mean ± SEM. P‐values were calculated by unpaired, two-tailed
t-test (A, C) and one-way ANOVA with Tukey multiple comparison test (B, D- G, I- L). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.