Fig. 3

17-AAG inhibits the expression of IL-33, the secretion of IL-6, T-helper cell-associated cytokines, and reactive oxygen species in cultures of activated human keratinocytes, CD4 + T lymphocytes, and eosinophils, respectively. TNF-α/IFN-γ- stimulated human keratinocytes (HaCaT) cells were cultured in presence of DMSO 0.1% (Vehicle) or various doses of 17-AAG. (a) Cell proliferation ELISA results as BrdU incorporation (%) after 6 h of incubation. (b) Relative expression of IL-33 and TSLP, by qPCR. (c) IL-6 levels in culture supernatant, by ELISA. (d) Phosphorylated STAT-1, STAT-3, and STAT-6 protein levels, corrected for β-Actin, were analyzed in HaCaT cell lysates following 1-hour or 24-hour activation using Western blot. Representative bands are presented. (e) NF-κB phosphorylation measured by ELISA. (f) FLG relative expression analyzed by qPCR. (g) IL-5, IL-17 A, and IL-22 secretion in the culture supernatant of anti-CD3/CD28 antibody-stimulated (1 µg/mL) CD4⁺ T cells derived from healthy donors, assessed by ELISA. (h) Luminol-enhanced ROS release in eosinophils derived from healthy donors, activated with fMLP (2 µM) or IL-33 (100 ng/mL). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, no significance; BrdU, BromodeoxyUridine; RLU, Relative Light Unit; AUC, Area Under Curve; ROS, Reactive Oxygen Species; OD, optical density.