Fig 1: COL4A1s induced VPC migration.A and B, overexpression of XBP1s in HVSMC induced conditioned medium promoted VPC migration. HVSMCs were infected with Ad-null or Ad-XBP1s for 24 h. Conditioned medium was then collected in the following 48 h. Transwell migration assays were performed on VPCs using the condition medium. A, representative images of migrated cells (Left panel) and VPC migration data shown as a histogram (Right panel). B, the conditioned medium was depleted with anti-COL4A1, anti-Col15A1, and anti-Col18A1 antibodies before transwell migration assays on VPCs. IgG was included as control. C and D, Exogenous COL4A1s increased VPC migration. No change observed in HUVEC and HVSMC migration. Two hundred ninety-three cells were transfected with either pS2-COL4A1s vector or pShuttle2-GFP (Mock) as control. The conditioned medium was depleted with IgG or anti-FLAG agarose beads (α-FLAG), followed by transwell migration assays on VPCs, HUVECs, and HVSMCs. C, show the representative images of migrated VPC cells. D, show histogram representation of migrated VPCs, HUVECs and HVSMCs. E, the purified COL4A1s promote VPC migration. COL4A1s was purified using anti-FLAG-agarose beads incubation with pS2-COL4A1s-transfected 293 cells conditioned medium and subsequent FLAG peptide mediated elution. This was followed by transwell migration assay on VPCs. FLAG peptide was included as control. Left panel shows the representative images, and the right panel shows migrated VPCs as histogram representation. Data are presented as mean ± SD from six independent experiments or representative images of six views from 20x microscope. ∗: p < 0.05; ∗∗: p < 0.01. COLA1, type IV collagen alpha 1; HUVEC, human umbilical vein EC; HVSMC, human smooth muscle cell line; VPC, vascular progenitor cell; VSMC, vascular smooth muscle cell; XBP1, X-box binding protein 1; XBP1s, spliced XBP1 protein.