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. Author manuscript; available in PMC: 2023 Aug 1.
Published in final edited form as: J Allergy Clin Immunol. 2022 Oct 25;151(2):494–508.e6. doi: 10.1016/j.jaci.2022.09.034

FIG 5.

FIG 5.

Alternaria-induced DNA fragmentation and release involves furin-mediated activation of caspase-3. (A and B) hBE cells labeled with fluorescent caspase-3 substrate before (A) and after (B) 100 μg/mL Alternaria (original magnification 40×, scale bar = 10 μm). (C) Nuclear fluorescence detected after Alternaria (100 μg/mL) alone and after caspase-3 inhibitor AQZ-1 (25 μmol), caspase-3 siRNA, decanoyl-RVKR-CMK (100 nmol), and furin knockdown (KD) (*P < .0001). (D) Nuclear fluorescence detected after Alternaria (100 μg/mL) alone and after 5 mmol GSH or Ca2+-free conditions (*P < .0001). (E) AQZ-1 (25 μmol) or caspase-3 siRNAs inhibited ~50% of Alternaria-induced DNA release (AQZ-1: *P < .0004; CSP3 siRNA; †P < .01). Furin inhibition with TPCK (25 μmol), decanoyl-RVKR-CMK (100 nmol), or naphthofluorescein (100 nmol) blocked ~50% of Alternaria-induced DNA release (*P < .0001). (F) Furin KD blocked Alternaria-induced DNA release, similar to decanoyl-RVKR-CMK (*P < .0005, †P < .0001). (G) Comet assay showing inhibition of Alternaria-evoked DNA fragmentation after AQZ-1 (25 μmol) and caspase-3 siRNAs (*P < .0001). DNA fragmentation was blocked by decanoyl-RVKR-CMK (100 nmol), naphthofluorescein (100 nmol), and furin KD (*P < .0001). Statistics (Fig 3, C-G) involved comparisons with Alternaria alone using Brown-Forsythe and Welch ANOVA followed by Dunnett T3 posttest.