Cyquant nf cell proliferation assay

To measure cell growth, CPCs were subjected to a CyQUANT NF Cell Proliferation Assay (Invitrogen, C35011). CPCs were plated on a transparent 48 well plate (8,000 cells/well), cultured overnight with Growth Media and then maintained in serum free Media for 24 hours. CPCs isolated from FVB/N and used for most studies were stimulated with S1P (3µM), thrombin (0.5U/ml), lysophosphatidic acid (LPA, 10µM) or serum (20%) for 24 hours. CPCs from S1PR₂ - and S1PR₃- KOs in a C57BL/6J background were stimulated with serum (20%) for 24, 48 and 72 hours. For inhibition of Rho, cells were pretreated with C3 toxin (1µg/ml) for 6 hours prior to the addition of agonist. For knockdown of S1P receptors, cells were transfected as described in 2.4. Following stimulation, the plates were assayed according to the manufacturers’ protocol and the fluorescence intensity determined using a Tecan Spectrophotometer as previously described [48 (link)].

hPASMC and hPAEC were seeded at a density of 25 cell/mm² and 50 cell/mm², respectively, in 24-well plates containing gels with discrete stiffness of 0.1, 0.4, 1.6, 6.4, and 25.6 kPa and cultured at 37°C and 5% CO₂. For hypoxia experiments, hPASMC were placed in an airtight Modular Incubator Chamber (Billups-Rothenberg), flushed continuously for 20 minutes with a premixed gas of 1% O₂, 5% CO₂, 94% N₂ (hypoxia) or 95% air with 5% CO₂ (normoxia) and incubated at 37°C for 48 hours. In specific experiments, hPASMC were treated with iloprost (3 and 10 μmol/l) or vehicle (DMSO) 4 hours following plating and at 24 hours. After 48 hours, cell density was determined using the CyQuant NF Cell Proliferation Assay (Invitrogen). For each stiffness condition, fold change was expressed as the ratio of adherent cell number at 48 versus 4 hours. In separate experiments, hPASMC and hPAEC were plated on discrete stiffness gels, and after 48 hours, apoptosis was assessed using the Apo-ONE Homogeneous Caspase-3/7 Assay (Promega). Percent apoptosis was normalized to maximum apoptosis at 0.1 kPa.

Cells were transfected using the Nucleofector Kit V from Amaxa Biosystems (Lonza, Allendale, NJ). Gene expression was silenced with siRNAs from Santa Cruz Biotechnology (Dallas, TX) that were provided as pools of 3 target specific siRNAs designed to knock down expression of specific gene of interest, including Jab1 siRNA (Cat#sc-35717), Trx siRNA (Cat#sc-106984), and control siRNA oligonucleotides (Cat#sc-37007). The HA/Flag-Trx plasmids and His-Jab1 were also transfected into U937 and THP-1 cells. Cell proliferation was determined using the CyQUANT NF cell proliferation assay (Invitrogen, Carlsbad, CA).