Supplemental Figures 1-3

Figure S1. Optimization of FLICA assay for detection of active caspase-1 in PMVECs. A) Optimal washing parameters for the FLICA reagent assay were determined under non-infection conditions. PMVECs were serum starved for 60 min prior to loading with FAM-YVAD-FMK (FLICA) (1X). PMVECs were harvested at 180 min post-loading, washed in apoptosis wash buffer at the indicated number of times prior to fixation (0.6% formalin), and analyzed by flow cytometry. Non-FLICA loaded PMVECs were used to set the analysis gate. B) Optimal substrate loading parameters for the FLICA assay were determined under non-infection conditions. PMVECs were serum starved for 60 min prior to loading with FLICA at varying concentrations. PMVECs were harvested at 180 min post-loading, washed 3 times prior to fixation, and analyzed by flow cytometry. Non-FLICA loaded PMVECs were used to set the analysis gate. C) Optimal substrate loading parameters for the FLICA assay were determined during PA103 infection. PMVECs were serum starved for 60 min prior to loading with FLICA and infection with PA103 (MOI 10:1). PMVECs were harvested at 180 min post-infection, washed 3 times, fixed, and analyzed by flow cytometry. D) To verify that infection alone did not stimulate cryptic auto-fluorescence, PMVECs were treated with saline or infected with PA103 (MOI 10:1) in the absence of FLICA, followed by washing, fixation and flow cytometric analysis. Auto-fluorescence of infected PMVECs in the FITC channel did not differ from non-infected PMVECs. E) To determine whether caspase-1 activation required live bacteria, strain PA103 was heated to 60 °C for 1 hour (label as HIA) prior to infection and FLICA measurement. A loss of viability was verified by bacteria plating. F) PMVECs were serum starved for 60 min prior to loading with FLICA and infection with PA103 (MOI 40:1). Saline treated PMVEC monolayers served as a control. PMVECs were harvested at 180 min post-infection and whole cell lysates prepared for western blotting. Blots were probed with antibody against the FAM moiety on the FLICA reagent (α-FAM), antibody against caspase-1 (α-casp-1), or antibody against β-actin.Figure S2. 8-br-cAMP increases PA103-induced caspase-1 activation in PMVECs in a dose-dependent manner. PMVECs were serum starved for 60 min. The 8-br-cAMP (panel A) and 8-br-cGMP (panel B) compounds were added to the culture medium 30 min prior to addition of FLICA reagent and PA103 (MOI 10:1). Saline-treated PMVEC monolayers served as negative controls. PMVECs were harvested at 180 min post-infection and analyzed for activated caspase-1 by flow cytometry. For all conditions n=3. For 8-br-cAMP in panel A, the (*) indicates a significant difference, PFigure S3. Analysis of cAMP FRET signals in PMVECs treated with cAMP elevating compounds. Representative data from Fig. 4 are depicted here showing temporal and spatial cAMP signals in different planes.