FIG 5 .

Sap2-induced inflammasome activation in vaginal epithelial cells. Vaginal washes of mice treated for 24 h with saline, LPS (50 µg/10 µl/mouse), or Sap2 (0.5 µg/10 µl/mouse) in the presence or absence of IC-1 were analyzed for the percentage of double-positive CD326⁺/caspase-1⁺ (A and B) and CD326⁺/IL-1β⁺ (A and C) cells, or for IL-18 production (F). Data are expressed as means ± SEM. *, P < 0.05 for LPS- or Sap2-treated versus saline treated mice and for IC-1 plus LPS- or Sap2-treated mice versus LPS- or Sap2-treated mice. For the gating strategy (A) of flow cytometry analysis, vaginal cells were gated on CD326-positive epithelial cells (based on side light scatter and CD326 staining; R1), caspase-1-positive cells (based on side light scatter and caspase-1 staining; R2), and IL-1β-positive cells (based on side light scatter and IL-1β staining; R3). Hence, the logical “AND” operator was used: for R4 results from the intersection between R1 and R2 and for R5 from the intersection between R1 and R3. For Western blotting, the A-431 cell line was untreated (NS) or treated for 2 h with LPS (10 µg/ml) plus ATP (5 mM) or Sap2 (20 µg/ml) in the presence or absence of IC-1 or Pepstatin A. After incubation, cell lysates were analyzed by Western blotting. Membranes were incubated with Abs to caspase-1 and actin. Caspase-1 results were normalized against those for actin (D). Culture supernatants were collected and tested for IL-1β production (E). Data are expressed as means ± SEM. *, P < 0.05 for LPS plus ATP or Sap2 treatment groups versus NS; #, P < 0.05 for IC-1 plus LPS plus ATP or Sap2 or for Pepstatin A plus Sap2-treated versus LPS- or Sap2-treated.