Figure 1. LPA induces migration of human lung-resident mesenchymal stem cells via LPA1 receptor.

(A & B) LPA induces LR-MSC migration in a dose dependent manner. Human LR-MSCs were added to the upper chamber of the Transwell and allowed to migrate through the porous membrane into the lower chamber containing medium alone or medium with LPA. Migrated cells on the lower side of the membrane were stained with Hema3 stain and counted with Olympus BX41 light microscope using a ×20 objective. n=3 separate cell lines with 3 replicates of each condition and 5 counted high power fields for each replicate. Representative image from transwell demonstrating robust migration of LR-MSCs in response to LPA are shown above. LPA induced migration was compared to that induced by 10ng/ml CCL21, 30ng/ml CXCL12, 10ng/ml EGF, 20ng/ml HGF, 0.1uM FMLP, 1ug/ml LTA, 10% FBS, and 10nM PDGF, as shown in B. n=4 individual cell lines with 3 replicates of each condition and 5 counted high power fields for each replicate. (C) LPA receptors profile of human LR-MSCs. LPA1, LPA2 and LPA3 mRNA expression by real-time quantitative PCR in human LR-MSCs. n=4 LR-MSC lines. A representative image from western blot analysis and immunofluorescent staining demonstrating LPA1 protein expression in LR-MSCs is shown below. (D) Effect of LPA receptor antagonists on LPA-induced LR-MSC migration. LR-MSCs were pre-treated with competitive antagonists of the LPA1 and LPA3 receptors (1μM VPC12249 or 1μM VPC32183) for 30 min before migration assay. Data represent mean ± SEM from 3 independent experiments. (E & F) Effect of LPA1 siRNA on LPA-induced migration of human LR-MSCs. LR-MSCs were transfected with (20nM) validated LPA1 or scrambled siRNA for 24 h and serum starved for another 24 h. Protein was collected for western blot analysis to test transfection efficiency as shown in E (n=3). Effect of LPA1 siRNA on LR-MSC migration was studied using the migration assay. Data shown in F represent mean ± SEM from 3 independent experiments. *** P < 0.0001.