rhil 17c  (R&D Systems)

Journal: JCI Insight

Article Title: Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling

doi: 10.1172/jci.insight.172764

Figure Lengend Snippet: ( A ) Silencing TCF4 ( siRNA-mediated ) in N/TERT keratinocytes, but not NFKB1 , JUN , or CEBPG , increases IL17C mRNA expression, which is further increased in the presence of TNF-α stimulation (10 ng/mL; n = 3, mean ± SEM, 2-way ANOVA with post hoc Tukey test. * P < 0.01; ** P < 0.005, *** P < 0.001, **** P < 0.0001; dashed line and the diamond demarcate P < 0.05 between 2 indicated groups via Student’s t test . ( B ) ATAC-Seq of human KCs isolated from fresh tissue biopsies identifies TCF4 binding sites in open chromatin regions of IL17C and ZC3H12A promoters. ( C ) Representative images of healthy normal and of lesional and nonlesional Ps skin demonstrates decreases in TCF4 (nuclear localization) and increases in IL-17C and ZC3H12A staining (using IHC; stained protein appears brown in color). Insets represent higher-magnification image. Scale bar: 100 μm; 20 μm (insets). ( D ) IL17C expression negatively correlates with TCF4 and positively correlates with ZC3H12A in lesional Ps and AD skin.

Article Snippet: Slides were treated with 3% H 2 O 2 (5 minutes), blocked with 10% goat serum (30 minutes), and incubated with primary antibodies against TCF4 (HPA025958-100UL, Sigma-Aldrich), IL-17C (AF1234, R&D Systems), and rabbit IgG isotype control (NI01, Sigma-Aldrich) overnight at 4°C.

Techniques: Expressing, Isolation, Binding Assay, Staining

Journal: JCI Insight

Article Title: Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling

doi: 10.1172/jci.insight.172764

Figure Lengend Snippet: ( A ) TCF4 decreases in KCs stimulated with IL-17C (200 ng/mL), IL-17A (20 ng/mL), TNF-α (10 ng/mL; 8 hours), and combinations of these cytokines as indicated. ( B ) Venn diagrams show the number of DEGs altered by IL-17C and IL-17A in keratinocytes with or without TNF-α stimulation. ( C ) GO BP terms. The chart shows functional categories enriched in keratinocytes induced by IL-17C and IL-17A. ( D ) Partial heatmap showing NFKBIZ and ZC3H12A increase in an IL-17C–IL17RA/RE–dependent manner. Full heatmap is presented in . ( E ) Top, TCF4 decreases in KCs engineered to have no IL17RE and IL17RA . Bottom, since ZC3H12A and NFKB had also been identified in the IL-17C promotor analyses , we focused on these target genes. Normalized counts of TCF4 , NFKBIZ, and ZC3H12A , following IL-17C stimulation in WT and IL17RA- , IL17RC- , and IL17RE -KO keratinocytes. ( F ) TCF4 decreases in KCs stimulated with TNF-α, IL-17A, and IL-17A + TNF-α and are dependent on IL-17RE/RA expression. n = 3, mean ± SEM, 1-way ANOVA ( A and E inset) and 2-way ANOVA ( E and F ) with post hoc Tukey test. * P <0.05, ** P < 0.002, *** P < 0.0005, **** P < 0.0001.

Article Snippet: Slides were treated with 3% H 2 O 2 (5 minutes), blocked with 10% goat serum (30 minutes), and incubated with primary antibodies against TCF4 (HPA025958-100UL, Sigma-Aldrich), IL-17C (AF1234, R&D Systems), and rabbit IgG isotype control (NI01, Sigma-Aldrich) overnight at 4°C.

Techniques: Functional Assay, Expressing

Journal: JCI Insight

Article Title: Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling

doi: 10.1172/jci.insight.172764

Figure Lengend Snippet: Skin inflammation occurs in an IL-17C–IL-17RA/RE–dependent manner and negatively correlates with Tcf4 expression and positively correlates with Zc3h12a expression. ( A ) Images of representative KC-Tie2 mice and KC-Tie2 mice deficient in Il17c , Il17ra , and Il17re . Representative images of H&E-stained skin isolated from the demarcated region of each mouse and adjacent skin stained for CD4 + T cells. ( B ) Dot plots of epidermal thickness measures for each mouse (control, n = 14; KC-Tie2, n = 22; KC-Tie2 × Il17re –/– , n = 6; KC-Tie2 × Il17c –/– , n = 16; KC-Tie2 × Il17ra –/– , n = 4) within each mouse strain. ( C ) Dot plots for individual quantification of CD4 + T cell numbers/field of view in skin of each mouse line ( n = 3–9/group [grp]). ( D and E ) qPCR measures for Il17a ( D , n = 4–10/grp) and ELISA of IL-17A protein ( E , n = 4–6/grp) expression for individual mice within each group. ( F ) IL-17C protein expression (using ELISA) in skin of psoriasis mouse models ( n = 8/grp), including imiquimod, KC-Tie2, Klk6 + , and IL-17C + . ( G ) IL-17C protein expression (using ELISA) for KC-Tie2 mice and KC-Tie2 mice deficient in Il17c , Il17ra , and Il17re ( n = 4–7/grp). ( H and I ) qPCR of Tcf4 ( H ) and Zc3h12a ( I ) gene expression in each mouse line ( n = 4–7/grp). ( J ) IHC of TCF4 protein in skin shows decreases in nuclear staining in KC-Tie2 mice compared with staining in control (WT) mice and KC-Tie2 mice deficient in Il17c , Il17ra , and Il17re . Insets represents higher-magnification image. One-way ANOVA, followed by post hoc Tukey test; * P < 0.05; ** P < 0.002; *** P <0.005, **** P < 0.0001. Scale bar: 50 μm ( A and J ) and 25 μm (inset of J ).

Article Snippet: Slides were treated with 3% H 2 O 2 (5 minutes), blocked with 10% goat serum (30 minutes), and incubated with primary antibodies against TCF4 (HPA025958-100UL, Sigma-Aldrich), IL-17C (AF1234, R&D Systems), and rabbit IgG isotype control (NI01, Sigma-Aldrich) overnight at 4°C.

Techniques: Expressing, Staining, Isolation, Control, Enzyme-linked Immunosorbent Assay