Figure 4. S100A6 regulates calcium entry and barrier function in PMVECs.
Global calcium entry following ISOC activation (R+TG) was measured using the ratiometric dye Fura2. Here, cells were pretreated with rolipram (R) for 15 minutes prior to mounting on microscope stage. Thapsigargin (TG) was added when cells were in low extracellular calcium to reveal calcium release and then extracellular buffer was supplemented to 2 mM final calcium concentration to reveal calcium entry. A. Doxycycline-inducible S100A6 over-expressing PMVECs revealed decreased calcium entry following R+TG, as compared to non-doxycycline-treated cells. B. Comparison of peak calcium entry observed. Peak calcium entry was decreased by 25% in doxycycline-treated cells compared to non-doxycycline-treated cells. n = 3 independent experiments. C. ISOC was measured using patch-clamp electrophysiology. Here, cells were pretreated with rolipram (R) for 15 minutes prior to obtaining a Giga ohm seal and whole-cell patch configuration. Thapsigargin (TG) was applied through the patch pipette. Doxycycline-inducible S100A6 over-expressed PMVECs revealed decreased ISOC following R+TG, as compared to non-doxycycline-treated cells. n = 4 measurements per condition performed over two different recording periods. D. Endothelial barrier integrity was assessed using ECIS. Here, cells were pretreated with rolipram (R) followed by thapsigargin (TG) (black arrow) to activate ISOC and changes in resistance were measured. Doxycycline-inducible S100A6 over-expressing PMVECs revealed decreased barrier disruption following ISOC activation as compared to non-doxycycline-treated cells. Non-doxycycline-treated cells revealed decreased resistance following ISOC activation, but this decrease was attenuated in doxycycline-treated S100A6 over-expressed cells. E. Comparison of peak resistance decrease observed in D. The peak resistance decrease was less in doxycycline-treated cells compared to non-doxycycline-treated cells. n = 3 independent experiments.