Figure 3. BPIFA1 from normal but not asthmatic HBECs is secreted basolaterally and blocks Ca²⁺ influx in ASMCs.

(a) Representative immunoblots (left panel) and densitometry (right panel) showing BPIFA1 from healthy and asthmatic HBECs (n=6 per group). (b) Representative immunoblots (left panel) and densitometry (right panel) showing BPIFA1 levels in vehicle and IL-13 exposed HBECs (n=3 per group). (c) Mean changes in fura-2 fluorescence in ASMC with time, as an indicator of cytosolic Ca²⁺. Serosal media from normal and asthmatic HBECs were co-incubated with human ASMCs for 1 h. (d) BPIFA1 inhibits TG-induced cytosolic Ca²⁺ increases in a dose-dependent manner. ASMCs were incubated with BPIFA1 and changes in the fura-2 emission ratio over time were recorded. ΔF/F₀ represents the average peak fluorescent intensity changes of three independent experiments. (e) Mean change in the fura-2 emission ratio over time following TG and the addition of extracellular Ca²⁺. (f) Typical Ca²⁺ oscillations in ASMCs±BPIFA1 in response to 5 μM Mch. (g) Summary of Ca²⁺ oscillation frequency data following 5 μM Mch. (h) Summary of peak fluorescence ratio changes when ASMCs were pre-incubated with BPIFA1 mutants or peptides (n=3 per group). Data in a–e,g and h are mean±s.e.m. The data were analysed using Student's t-test in a and b, Mann-Whitney test in g and one-way ANOVA followed by Tukey post hoc analysis in h. *P<0.05, **P<0.01 and ***P<0.0001 different to control.