Annexin 5

Endothelial viability was assessed with the Lactate Dehydrogenase (LDH) assay (Invitrogen, C20300) and/or the LIVE/DEAD Viability analysis (ThermoFisher, L7013) based on SYTO10 (green, outlined both lived and dead cells) and Ethidium homodimer-2 (red, indicating dead or injured cells) staining according to instructions from manufacturers. Activation of Caspase signaling was evaluated with western blot. To stain for cell death markers, APC-conjugated Annexin V (Biolegend, 640919) was incubated at 4 °C for 15 min with the endothelial cells. PI was added right before acquisition for flow cytometry. The percentage of apoptotic or necrotic cells, as defined by Annexin V⁺ and/or Annexin V^-PI⁺ cells, were obtained using FlowJo software (FlowJo, version 10.0, Ashland, OR).

Cells grown on tissue culture dishes were washed with PBS and harvested with Accutase cell dissociation solution (Sigma-Aldrich) for 5 min at 37 °C. Cells (1 × 10⁶ cells/mL) were then re-suspended in ice-cold PBS pH 7.4/1% FBS (staining buffer) and blocked in ice-cold PBS pH 7.4/5% FBS for 10 min at 4 °C, followed by two washes in staining buffer. Cells were stained with AlexaFluor 488-conjugated Streptavidin (Invitrogen) or primary antibody for 1 h at 4 °C and washed twice with staining buffer. Primary antibodies targeted against TGFβR1/ALK-5 (1:500) is from Novus Biologicals; Integrin β1-Biotin conjugated (Ha2/5) (1:250) from BD Biosciences. Additional staining was done using AlexaFluor 488-conjugated secondary antibody for 30 min at 4 °C. For the quantification of apoptosis, adherent cells along with those in culture media were collected and washed twice with cold PBS. Cells were stained with PE-Annexin V (BD Biosciences), according to the manufacturers’ instructions, and analyzed immediately by flow cytometry. Samples were acquired using BD FACS Canto II instrument and BD FACS Diva v8.0.2 software. Data were analyzed using FlowJo v10 software, and represented as geometric mean of fluorescence is represented as Mean Fluorescence Intensity (MFI) or percentage (%) of Annexin V positive cells.