Nystatin

After culturing gel-encapsulated cells for 1 day, DMSO or chemical inhibitors (latrunculin A, blebbistatin, or nystatin, purchased from Cayman Chemical) were added and incubated for 2 hours at 37°C. Cells were then rapidly retrieved on ice as described in characterization of NF-κB activation kinetics, but they were not fixed. Retrieved cells were incubated with biotinylated TNFα (R&D Systems) at saturating concentration (1 μg/ml) for 1 hour, followed by avidin–fluorescein isothiocyanate (FITC) for 30 min on ice. Flow cytometry analysis was then performed. The sample incubated with avidin-FITC alone was used as a negative control.

For inhibitor studies, SiNWs grown from 100-nm seed Au NPs were sonicated (7 min) into M200 medium with growth supplement (Life Technologies) and then allowed to settle overnight. After settling, HUVECs were introduced at ~15% initial confluence, and drugs were administered after 8 hours of incubation. All drugs were obtained from Sigma-Aldrich and were administered at the following concentrations (dissolved in dimethyl sulfoxide): chlorpromazine, 2.5 μg/ml; nystatin, 50 ng/ml; dynasore, 80 μM; Cyto D, 5 ng/ml; A5, 4 and 16 nM; Lova, 10 μM (NC9907790, Cayman Chemical). Throughout this process, the SiNW-cell overlap was monitored by taking alternating DF and PC micrographs every ~2 hours at random substrate locations (selected before viewing to avoid sampling bias).