Supplementary material - Impact of Na+ permeation on collective migration of pulmonary arterial endothelial cells (Xu et al)

The first phase started with fast closure speed which then rapidly reduced within 5 hours after scratching. The second phase maintained at slow and stable speed from 6 to 24 hours. In the absence of extracellular Na + , we found that wound closure distance reduced more than 50%, fewer cells at the leading edge protruded prominent lamellipodia, and some sustained interendothelial gaps progressively increased in size over time and fused with adjacent gaps.

When scratch-associated cellular injury was also missing, PAEC monolayer migrated much more slowly, and interendothelial gaps increased in size obviously towards the end. Pharmacological inhibition of the epithelial Na + channel (ENaC) using amiloride reduced wound closure distance by 30%. Inhibition of both the ENaC and the Na + /Ca 2+ exchanger (NCX) using benzamil further reduced wound closure distance in the second phase and caused accumulation of floating particles in the media.

Surprisingly, pharmacological inhibition of the Ca 2+ release-activated Ca 2+ (CRAC) channel protein 1 (Orai1) using GSK-7975A, the transient receptor potential channel protein 1 and 4 (TRPC1/4) using Pico145, or both Orai1 and TRPC1/4 using combined GSK-7975A and Pico145 treatment did not affect wound closure distance dramatically. GSK-7975A also inhibits smaller inward Ca 2+ currents via Orai2 and Orai3 channels, whereas Pico145 also blocks TRPC4, TRPC5, and TRPC1/5 channels.

Nevertheless, the combined treatment appeared to also cause accumulation of floating particles. Orai1 gene silencing using shRNAs led to a 25% reduction of wound closure in the first 6 hours but had no effect afterwards. However, in the absence of extracellular Na + or cellular injury, Orai1 did not affect PAEC collective migration.

Overall, the data revealed that Na + permeation into cells contributed to PAEC monolayer collective migration by increasing lamellipodial formation, causing accumulation of floating particles, and impairing intercellular adhesion.