Fig 1: C3AR1 overexpression reduces migration and invasion of osteosarcoma cells. (A–D) Assessment of wound distance of Saos-2 and U-2OS cells with C3AR1 overexpression. (E–G) Number of invasive Saos-2 and U-2OS cells with C3AR1 overexpression. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 2: C3AR1 mRNA expression associates with distinct kinds of immune cells in osteosarcoma. There were significant correlations between C3AR1 expression and (A) dendritic cells, (B) macrophages M1, (C) macrophages M2, (D) neutrophils, (E) T cells CD4 memory activated, (F) T cells CD8, (G) T cells CD4 naïve and (H) macrophages M0.
Fig 3: C3 alternative pathway components expression in human VSMCs. (A) mRNA quantification by real-time PCR using specific primers for human C3 in human VSMCs treated with or without agLDL (100 µg/mL). (B) Protein levels of C3 and C3-derived products in the supernatant of hVSMCs treated with or without agLDL (100 µg/mL). Human serum (Serum) was used as a positive control for C3 (n = 3 independent experiments). (C) Western blot analysis for C3a receptor (C3aR) and aMß2 (C11b/CD18) integrin (receptor for C3b/iC3b) in lysates of hVSMCs incubated with or without agLDL (100 µg/mL). Band intensity is given in arbitrary units as mean ± SEM and statistical significance (p < 0.05, Mann–Whitney test) is indicated (n = 4 independent experiments).
Fig 4: Complement C3aR and C5aR antagonist inhibit inflammation and NF-?B signaling in HRPE cells challenged with complement C3a and C5a. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM), and the release of (A) TNF-a, (B) IL-1ß, (C) IL-6, (D) PGE2 and (E) IL-10 was determined by ELISA. HRPE cells were treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM), and the release of (F) TNF-a, (G) IL-1ß, (H) IL-6, (I) PGE2 and (J) IL-10 was determined by ELISA. (K) The phosphorylation of NF-?B and expression of NF-?B in HRPE cells treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM) were determined by western blot. (L) The phosphorylation of NF-?B and expression of NF-?B in HRPE cells treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM) were determined by western blot. ***P<0.001 relative to control; ###P<0.001 relative to C3a or C5a treatment. p-NF-?B, phosphorylated NF-?B; C5aR, C5a receptor; HRPE, human retinal pigment epithelium; CCX, CCX168; SB, SB290157.
Fig 5: Association between C3AR1 mRNA expression and prognosis and clinicopathological characteristics among osteosarcoma. (A) Kaplan-Meier of overall survival between high (red) and low (blue) C3AR1 mRNA expression osteosarcoma groups. (B–G) The association between C3AR1 mRNA expression and age (B), gender (C), race (D), specific tumor site (E), metastasis (F) and primary tumor sites (G).
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