Fig 2: Suppression of AEP alleviated breast cancer cells-induced bone pain while overexpression of AEP suffered severe bone pain. (a) Flinches of paw in 2 min of mice injected with MDA-MB-231 cells with AEP knock-down, rescue, or overexpression (n = 8). (b) Paw withdrawal mechanical threshold of mice injected with MDA-MB-231 cells with AEP knock-down, rescue, or overexpression (n = 8). (c) Paw withdrawal thermal latency of mice injected with MDA-MB-231 cells with AEP knock-down, rescue, or overexpression (n = 8).

Fig 3: MM-specific upregulation of the cysteine protease legumain (LGMN) in chronic hypoxia: Four MM cell lines and the bone marrow stromal cell line HS-5 were cultured under hypoxic conditions (1% O2) and analyzed at day 0, 1 and 7 for protein and mRNA expression. (A) Relative LGMN mRNA levels in MM cell lines, normalized to TATA-box-binding protein (TBP) mRNA levels. * p < 0.05; ***, p < 0.001. (B) Representative Western blots for LGMN at 56 kDa (pro-LGMN) and 37 kDa (activated LGMN). ß-tubulin served as a loading control. (C) Densitometric quantifications of LGMN expression (pro-LGMN and active LGMN). Signals were normalized to ß-tubulin expression. Bar graphs represent the mean ±SEM of three independent experiments. One-way-ANOVA with Bonferroni’s post-hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (D) The extracellular concentration of LGMN in RPMI-8226, OP;-2 and U266 cells was assessed by enzyme-linked immunosorbent assay (ELISA) in the supernatant under normoxic and chronic hypoxic conditions. Extracellular concentration of LGMN [ng/mL] in RPMI 8226, OPM 2 and U266 cells. Bar graphs indicate the mean ±SEM of three technical replicates. Unpaired student’s t-test, *, p < 0.05; **, p < 0.01.

Fig 4: GSEA analyses of KEGG signaling pathways activated in GC patients with high expression of LGMN compared with the ones with low expression. (A) Focal adhesion, (B) ecm receptor interaction, (C) cell adhesion molecules cams, (D) TGF-ß signaling pathway, (E) JAK-STAT signaling pathway, and (F) gap junction were differentially enriched when LGMN overexpressed.

Fig 5: Expressions of P75 and TrkA were suppressed in DRGs and spinal cords of mice injected with AEP inhibitors while increased in mice injected with AEP proteins. (a–c) Western blot analysis of P75 and TrkA expression in DRGs of mice injected with MDA-MB-231 cells treated with AEP inhibitors, recombinant AEP proteins, or saline control. (d–f) Western blot analysis of P75 and TrkA expression in spinal cords of mice injected with MDA-MB-231 cells treated with AEP inhibitors, recombinant AEP proteins, or saline control. (g) Immunohistochemical analysis of P75 and TrkA expression in DRGs of mice injected with MDA-MB-231 cells treated with AEP inhibitors, recombinant AEP proteins, or saline control. (h) Immunohistochemical analysis of P75 and TrkA expression in spinal cords of mice injected with MDA-MB-231 cells treated with AEP inhibitors, recombinant AEP proteins, or saline control.