Fig. 2

ABCB5⁺ MSCs restore ER Ca²⁺ in hypoxic HUVECs. A–D HUVECs transduced with the ratiometric ER-Ca²⁺ sensor-D1ER, were treated with 80 μM CoCl₂, or 120 μM DFO or 3 μM TG for 4 h in medium. Thereafter, HUEVCs were either co-cultured with ABCB5⁺ MSCs (CoCl₂-CO; DFO-CO; TG-CO) or cultured in ABCB5⁺ MSC-CM (CoCl₂-CM; DFO-CM; TG-CM) for 24 h. The ratio of CFP (Ex 430 nm/Em 474 nm) intensity by YFP (Ex 514 nm/Em 527 nm) intensity represented the dynamic changes in ER Ca²⁺ concentration and were measured using a Tecan multimode plate reader at every two hours. 100 μM of H₂O₂ was used as positive control. Changes in ER-Ca²⁺ in HUVECs under A CoCl₂, CoCl₂-CM and CoCl₂-CO treatments; B DFO, DFO-CM and DFO-CO treatments; C TG, TG-CM and TG-CO treatments; and D H₂O₂ treatments. E Representative images of HUVECs under various treatments taken by SP5 microscopy system (scale bar is 200 μm). F–H D1ER expressing HUVECs were grown on 13 mm Ø glass coverslips treated with 80 μM CoCl₂, or 120 μM DFO or 3 μM TG in medium for 4 h. Subsequently, HUEVCs were either co-cultured with ABCB5⁺ MSCs (CoCl₂-CO; DFO-CO; TG-CO) or cultured in ABCB5⁺ MSC-CM (CoCl₂-CM; DFO-CM; TG-CM) for 24 h and fixed with 4% PFA. Changes in ER-Ca²⁺ in HUVECs under F CoCl₂, CoCl₂-CM and CoCl₂-CO treatments; G DFO, DFO-CM and DFO-CO treatments; H TG, TG-CM and TG-CO treatments. Data is represented as mean ± SD, n = 3 in each group, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001