Figure. 2. ELANE is the major anti-cancer protein in human PMN media.

(A) Effect of passing PMN media through a 0.22μm filter on protein levels and MDA-MB-231 cell viability (calcein-AM). n=3/group.

(B) Proteomics analysis of PMN media pre- and post-filtration. Significantly down-regulated proteins were identified by the G-test (blue circle, p<0.05 with Bonferonni correction). See also Table S2.

(C) Effect of immunodepleting ECP or ELANE from PMN media on MDA-MB-231 cell viability (calcein-AM). Depletion of ECP or ELANE was confirmed by western blotting (Input = pre-depletion, FT = post-depletion). n=4/group.

(D) Dose-response effect of purified human ELANE or ECP on MDA-MB-231 cell or HMDM viability (calcein-AM). n=3/group.

(E) Effect of purified human ELANE or ECP on MDA-MB-231 cell or HMDM viability (calcein-AM) alone or in combination. n=3/group.

(F) Effect of serine protease inhibitors (1mM PMSF or 42nM A1AT) on catalytic activity and MDA-MB-231 cancer cell viability (calcein-AM) of purified ELANE or PMN media. n=4/group. Veh = ethanol.

(G) PMN media, PMSF-inactivated PMN media, or HSA were injected intratumorally into E0771 tumors and tumor volume was measured. n=8–9/group.

(H) Linear regression analysis of MDA-MB-231 cell killing by PMN media vs. ELANE catalytic activity in PMN media from 9 healthy donors. n=6/donor.

(I) Effect of ELANE (3μg/mL, 6h) on cancer and non-cancer cell viability (calcein-AM). n=3/group.

*, p<0.05 Student’s t-test, data are mean ± SEM.