Marimastat

Mouse anti-VE-cadherin antibody (sc-9989) diluted 1:50 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Cy and Alexa Fluor-conjugated secondary antibodies were acquired from Jackson Immunoresearch (Philadelphia, PA, USA) and Molecular Probes (Eugene, OR, USA), respectively, and used for immunocytochemistry. HIF2α inhibitor [51 (link)] (Axon 2034) was obtained from Axon Medchem (Hanzeplein, The Netherlands). Z-VAD-FMK was obtained from Adooq-bioscience (187389-52-2, Irvine, CA, USA) and Santa Cruz Biotechnology (sc-3067, Dallas, TX, USA). Marimastat from Santa Cruz Biotechnology (sc-202223, Dallas, TX, USA), MG-132 from Promega (G9951, Madison, WI, USA). Cytotoxgreen was obtained from Essen BioScience (Ann Arbor, MI, USA) and CellROXgreen from Molecular Probes (Eugene, OR, USA). PX-ethyl was purchased from Sigma (St. Louis, MO, USA). According to the Sigma safety data sheet, safety measures of eye shields, face shields, full-face respirator, and gloves should be taken. PX was used according to our previously reported protocol [43 (link),44 (link)] All other reagents applied in this study were used in accordance to the known literature and the supplier’s guidelines.

To attempt to inhibit the loss of ACKR1, HPMECs were first incubated with whole blood for 24 hours for ACKR1 induction. Cells were then washed with PBS and treated with MG-132 (20μM; CAS 133407-82-6), Marimastat (100μM; Santa Cruz, sc-202223), Bafilomycin A (100nM; EMD Millipore Cat# 5.08409.0001), GW4869 (2μM; Millipore Sigma CAS 6823-69-4) or DMSO in complete media for 6 hours. Cells were then lysed and processed for immunoblotting. For inhibition of protein synthesis, cells were treated with cycloheximide (100μg/ml; Sigma 01810) at the time of incubation with isolated PMNs for 6 hours. For inhibition of NF-κB signaling, HPMECs were pretreated with Bay 11-7082 (20μM; Sigma, B5556) for 30 minutes in complete media and then inhibitors were washed out and replaced with blood for 6 hours for ACKR1 induction.