Figure 4: Aldara, but not IMQ, activates the inflammasome in human primary keratinocytes.

(a) Keratinocytes were treated with different concentrations of Aldara for certain time periods. Whole-cell lysate (left) as well as acetone-precipitated supernatants (right) were resolved by SDS–PAGE. The resulting western blots were probed with the antibodies indicated on the left-hand side. The vehicle DMSO served as negative control. (b) The relative efficiency of cell lysis by the indicated concentrations of Aldara and DMSO 1:1,000 in a was assessed by lactate dehydrogenase (LDH) assays (left) and the amount of released IL-1β in b was measured by ELISA (right). (c) Thirty minutes before Aldara treatment (1:1,000, 4 h), keratinocytes were pretreated with a pan-caspase inhibitor (VAD, 20 μM) or a caspase-1 inhibitor (YVAD, 50 μM). The efficiency of cell lysis was assessed with LDH assays (top panel), and mature IL-1β release was measured with ELISA (middle panel). Cell lysate and acetone-precipitated supernatants were resolved by SDS–PAGE, and the western blots were probed with the antibodies indicated on the left-hand side (bottom panel). (d) Keratinocytes were transfected with scrambled siRNA (ctrl. 1), with siRNA directed against Caspase-5 (ctrl. 2) or with siRNA directed against ASC, caspase-1, NLRP3 and NLRP1 two days before Aldara treatment (1:3,000, 4 h). Secretion of mature IL-1β into the supernatant was assessed by western blotting using antibodies against pro- IL-1β and IL-1β p17. Probing with an antibody recognizing β-actin served as loading control. (e) Keratinocytes were either seeded at the indicated densities per well into a six-well plate or had been transferred into KBM-2 medium for differentiation 5 days before Aldara treatment (1:3,000, 4 h). The efficiency of cell lysis was assessed with LDH assays (top), and mature IL-1β release was measured with ELISA (bottom). (f) Cells were treated either with DMSO, Aldara, vehicle, IMQ or softcream for 4 h at the indicated concentrations. The efficiency of cell lysis was assessed with LDH assays (top), and mature IL-1β release was measured with ELISA (bottom). Error bars indicate s.d.; *P<0.05, **P<0.01 and ***P<0.001, ****P<0.0001 (unpaired Student’s t-test).