Figure 4.

Knockdown of Orai1 inhibited chronic hypoxia-induced increases of basal [Ca²⁺]_i and SOCE in rat distal PASMCs. (a) Expression of Orai1, 2, 3 mRNA relative to 18 s mRNA measured by qRT-PCR in non-targeted small interfering RNA (siNT)- or siRNA targeted to Orai1 (siOrai1)-treated PASMCs. *P<0.05 versus siNT. (b) Western blot showing expression of Orai1, 2, 3 and β-actin protein in PASMCs treated with siNT or siOrai1. (c) Mean ratios of Orai1, 2 and 3 proteins relative to β-actin protein measured by western blotting in siNT- and siOrai1-treated PASMCs. *P<0.05 versus siNT. (d) Representative traces of different treatments on the time course of F340/F380 before and after restoration of extracellular Ca²⁺ in distal PASMCs perfused with Ca²⁺-free KRB solution containing 10 μM CPA, 0.5 mM EGTA and 5 μM nifedipine. (e) The effect of Orai1 silencing on hypoxia-induced changes of basal [Ca²⁺]_i in PASMCs. Basal [Ca²⁺]_i is determined as the average F340/F380 level during 0–5 min perfusion. *P<0.05 versus normoxic control. ^#P<0.05 versus hypoxic siNT control. (f) The effect of Orai1 silencing on hypoxia-induced changes of SOCE in PASMCs. SOCE is determined as the peak increase of F340/F380 level following calcium restoration between 15 and 30 min perfusion. Bar values are means±S.E.M. (n=5 experiments in 74–116 cells). *P<0.05 versus normoxic control. ^#P<0.05 versus hypoxic siNT control. (g) SOCE is determined by measuring the time course of fura-2 fluorescence intensity excited at 360 nm before and after adding 200 μM Mn²⁺ in Ca²⁺-free KRB solution containing 10 μM CPA and 5 μM nifedipine in PASMCs. Data at each time point were normalized to fluorescence at time 0 (F/F0). (h) Average quenching of fura-2 fluorescence by Mn²⁺. Data are expressed as the percentage decrease in fluorescence at time 10 min from time 0. Bar values are means±S.E.M. (n=5 experiments in 88–109 cells). *P<0.05 versus normoxic control. ^#P<0.05 versus Hypoxic siNT control. Brackets indicate ±S.E.