Figure 4. SarSP stimulates intracellular signaling pathways involved in synthesis of pro–IL-1β and TNF-α.

(A) Quantification by qRT-PCR of transcripts for pro–IL-1β, NLRP3, and pro–caspase-1 in primary cultures of keratinocytes isolated from WT or TNF-α^KO mice. (B) Analysis by qRT-PCR of the intracellular signaling pathways involved in the increase of pro–IL-1β and TNF-α transcripts in COCA cells treated with SarSP (10^–7 M) alone or with intracellular pathway inhibitors or their vehicles (Veh). (C) FACS analysis of Ca²⁺ flux induced by SarSP (10^–7 M) in COCA cells. A representative experiment of 3 is shown. (D) Quantification by ELISA-based assay of NFATc1 and NF-κB nuclear translocation in COCA cells treated with SarSP (10^–7 M) alone or with the mouse NK1R-specific antagonist RP67,580 (10^–5 M) or vehicle. (E) Quantification by ELISA-based assay of NF-κB nuclear translocation in COCA cells treated with SarSP (10^–7 M) alone or with intracellular (BAPTA-AM) or extracellular (EGTA) Ca²⁺ chelators. In A, each circle represents a keratinocyte primary culture from 1 mouse (n = 3). In B, D, and E, each circle represents a single culture of COCA cells (B, n = 3; D, n = 4; E, n = 3). Means ± 1 SD were analyzed by 1-way ANOVA and post hoc Student-Newman-Keuls test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.