(A) ELISA secretion assay for Flag-IL-33^Δ34-His expressing COPD airway basal cells treated with nSMase2 activator 4-methylumbelliferone (4-MU, 10 μM) and GW4869 (20 μM). 4-MU augmented secretion, which was blocked by GW4869 (n = 5). (B) Live-cell imaging for mCherry-IL33^Δ34-GFP HBE-1 cells treated with 4-MU and GW4869 for 15 minutes, demonstrating foci (white arrows) of yellow merged signal. HBE-1 cells were also fixed and permeabilized after 15 minutes and imaged for GFP only (green) and immunostained for CD9 (red), demonstrating foci of colocalization (white arrows). DAPI nuclear counterstain was also used. Scale bar: 10 μm. (C) Secretion of Flag-IL-33^full-His from COPD airway basal cells was augmented by disruption of nuclear entry (ivermectin, 1 μM) or by nSMase2 activation (4-MU, 10 μM), and both were inhibited by GW4869 (n = 5). (D) Live-cell imaging of mCherry-IL-33^full-GFP HBE-1 cells treated with DMSO or ivermectin + 4-MU, showing accumulation of cytoplasmic IL-33^full signal within 1 hour. Hoechst 33342 nuclear counterstain was also used. Scale bar: 10 μm. (E) Exosome and protein fractionation from Flag-IL33^Δ34-His–expressing HBE-1 cell supernatant (sup). Secreted IL-33 was retained above centrifugal concentrator 100 kDa filter (conc sup) and detected by anti-Flag Western blot. Lysate (lys) was used for comparison. Exosomes were resolved from free proteins by a qEV size-exclusion column, and fractions were analyzed: nonfixed sup Flag-IL-33^Δ34-His resolves into protein fractions and fixed sup protein migrates at a higher MW and resolves into both exosome and protein fractions. (F) Purified HBE-derived exosomes (10⁸ particles) incubated for 15 minutes with recombinant site–specific biotinylated IL33^Δ34 protein (1 μg) were resolved on the qEV column, showing that IL-33 coelutes in CD9-containing exosome fractions without fixation. Data are shown as the mean ± SEM. Statistical analysis: 1-way ANOVA (A and C); *P < 0.05, **P < 0.01, ***P < 0.001.