RNA-seq of human umbilical vein endothelial cells co-incubated with CYR61-treated mouse bone marrow-derived macrophages

We assess the transcriptional changes when human umbilical vein endothelial cells are co-incubated with naïve or CYR61-activated mouse bone marrow-derived macrophages. Overall design: We used primary human umbilical vein endothelial cells (HUVECs) from ATCC (PCS-100-010). We maintained HUVECs according to the manufacturer's recommended instructions. We isolated bone marrow from the tibias and femurs of C57BL/6J mice (2-5 months old). Bone marrow cells were incubated for 5-6 days in macrophage differentiation media: DMEM + 10% FBS + 10% CMG-conditioned media + 1% penicillin/streptomycin + 1% L-glutamine. After differentiation, macrophages were switched to mature macrophage media: DMEM + 10% FBS + 2% CMG-conditioned media + 1% penicillin/streptomycin + 1% L-glutamine. We plated HUVECs in the bottom compartments of 24-well transwell plates (0.4 µm pore size) along with the following in the top compartments: 1) cell-free (n=6 wells); 2) naïve BMDMs (n=6 wells); or 3) CYR61-treated BMDMs (0.5 µg/ml, 24 hr, n=6 wells). After co-incubation for 24 hrs, we washed HUVECs twice with PBS, extracted total RNA from HUVECs, and then prepared samples for RNA-sequencing.