1. Hypercholesterolemia inhibits re-endothelialization of arterial injuries by TRPC channel activation 
Michael A. Rosenbaum, MD, Pinaki Chaudhuri, PhD, Linda M. Graham, MD 
Journal of Vascular Surgery 
Volume 62, Issue 4, Pages e2 (October 2015)
DOI: /j.jvs
Copyright © 2015 Society for Vascular Surgery Terms and Conditions

2. Fig 1
Lysophosphatidylcholine (lysoPC) induces externalization of canonical transient receptor potential (TRPC) 6 protein in TRPC5−/− endothelial cells (ECs). A, Wild-type (WT) and TRPC5−/− ECs were incubated with or without lysoPC for 1 hour. Cell surface proteins were biotinylated, and immunoblot analysis was performed for biotinylated TRPC6 (top panel). Before incubation with streptavidin-agarose beads, an aliquot of cell lysate was removed for immunoblot analysis to determine total TRPC6 protein level (bottom panel). B, WT or TRPC5−/− ECs were incubated with or without lysoPC for 15 minutes and then exposed to anti-TRPC6 antibody followed by Alexa 488 conjugated secondary antibody. TRPC6 location was assessed by fluorescence microscopy. Nuclei were detected by counterstaining with propidium iodide. Original magnification ×40. Bar, 40 μm.
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Copyright © 2015 Society for Vascular Surgery Terms and Conditions

3. Fig 2
The antimigratory effect of lysophosphatidylcholine (lysoPC) is attenuated in canonical transient receptor potential (TRPC) 5–deficient (TRPC5−/−) endothelial cells (ECs). Migration assay was initiated in quiescent wild-type (WT), TRPC6−/− (previously reported,9 but included for comparison), and TRPC5−/− ECs; lysoPC (10 μM) was added, and migration was quantitated at 24 hours. The arrow indicates starting line of migration. Original magnification ×40. Bar, 100 μm. The bottom panel represents migration results by mean ± standard deviation (n = 3; *P < .001 compared with WT control ECs; **P < .001 compared with TRPC6−/− control [previously reported,9 but included for comparison]; and ***P < .001 compared with TRPC5−/− control).
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Copyright © 2015 Society for Vascular Surgery Terms and Conditions

4. Fig 3
Endothelial healing after carotid artery injury. A, Representative images 120 hours after carotid electrocautery injury. The area without an intact endothelial monolayer is stained with Evans blue. The arrow identifies the length of the original injury. B, Re-endothelialization results shown as the percentage of re-endothelialized area relative to the total injured area. Results are expressed as the mean ± standard error for each group: wild-type (WT) chow diet (n = 10), WT high-cholesterol (HC) diet (n = 10), TRPC6−/− chow diet (n = 8), TRPC6−/− HC diet (n = 8; *P < compared with WT HC), TRPC5−/− chow diet (n = 8), and TRPC5−/− HC diet (n = 8; **P = compared with WT HC).
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Copyright © 2015 Society for Vascular Surgery Terms and Conditions

5. Supplementary Fig 1 (online only)
Lysophosphatidylcholine (lysoPC) does not induce phosphorylation or externalization of canonical transient receptor potential (TRPC) 3 protein in wild-type (WT) endothelial cells (ECs), and the antimigratory effect of lysoPC is not attenuated in TRPC3-deficient (TRPC3−/−) ECs. A, WT ECs were incubated with or without lysoPC for 15 minutes. Tyrosine phosphorylation of TRPC3 was evaluated as an indicator of TRPC3 activation (n = 3; top panel). An aliquot of cell lysate was used for immunoblot analysis to determine total TRPC3 protein levels (bottom panel). B, WT ECs were incubated with or without lysoPC for 1 hour. Cell surface proteins were biotinylated, and immunoblot analysis was performed for biotinylated TRPC3 (n = 3; top panel). Before incubation with streptavidin-agarose beads, an aliquot of cell lysate was removed for immunoblot analysis to determine total TRPC3 protein levels (bottom panel). C, Migration assay was initiated in quiescent WT and TRPC3−/− ECs, lysoPC (10 μM) added, and migration quantitated at 24 hours. Graph represents migration results by mean ± standard deviation (n = 3; *P < .001 compared with WT control ECs; **P < .001 compared with TRPC3−/− control). D, Representative images 120 hours after carotid electrocautery injury. The area without an intact endothelial monolayer is stained with Evans blue. The arrow identifies the length of the original injury. E, Re-endothelialization results shown as the percentage of re-endothelialized area relative to the total injured area. Results are expressed as the mean ± standard error for each group: WT chow diet (n = 10), WT high-cholesterol (HC) diet (n = 10), TRPC3−/− chow diet (n = 5), and TRPC3−/− HC diet (n = 5; *P = not significant compared with WT HC).
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Copyright © 2015 Society for Vascular Surgery Terms and Conditions

6. Supplementary Fig 2 (online only)
Lysophosphatidylcholine (lysoPC) inhibits migration and induces externalization of canonical transient receptor potential (TRPC) 5 and 6 proteins in human endothelial cells (ECs). A, Migration assay was initiated in quiescent human EA.hy926 ECs, lysoPC (12.5 μM) added, and migration quantitated at 24 hours. The panel represents migration results by mean ± standard deviation (n = 3; *P < .001 compared with control ECs). B, Human EA.hy926 ECs were incubated with or without lysoPC (12.5 μM) for 1 hour. Cell surface proteins were biotinylated, and immunoblot analysis was performed for biotinylated TRPC6 (top panel) and TRPC5 (third panel). Before incubation with streptavidin-agarose beads, an aliquot of cell lysate was removed for immunoblot analysis to determine total TRPC6 (second panel) and TRPC5 (bottom panel) protein levels as appropriate.
Journal of Vascular Surgery  , e2DOI: ( /j.jvs )
Copyright © 2015 Society for Vascular Surgery Terms and Conditions