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. 2018 May 1;197(9):1164–1176. doi: 10.1164/rccm.201703-0660OC

Figure 2.

Figure 2.

Role of C5a/C5aR1 in migration, invasion, and tumor-associated matrix metalloproteinase (MMP) activity. (A) Scratch wound healing assay after incubation of A549 cells with recombinant C5a. The percentage of the area covered by migrated cells (wound coverage) was calculated. (B) Invasion assay of A549 cells after treatment with recombinant C5a. Cells were counted in four independent fields of the bottom side of the Boyden chamber after crystal violet staining. (C) MMP activity in the serum-free conditioned medium of A549 cells after treatment with recombinant C5a. MMP activity was assessed by digestion of a fluorogenic substrate. (D) Left: Gene expression levels of C5aR1 were silenced in A549M1 lung cancer cells using two lentiviral transduced shRNAs targeting two different sequences (shC5aR1-1 and shC5aR1-2). Scrambled shRNA-transduced cells were used as control. IPO8 was used as the reference gene. Right: Flow cytometry analysis of the membrane expression of C5aR1 in shC5aR1-1 (red line), shC5aR1–2 (blue line), and scrambled shRNA (black line)-transduced cells. Incubation without primary antibody was used as negative control (gray line). (E) Scratch wound healing assay of C5aR1-silenced cells (shC5aR1-1 and shC5aR1-2) and control cells after treatment with recombinant C5a. (F) Invasion assay of C5aR1-silenced and control cells after treatment with recombinant C5a. (G) MMP activity in the serum-free conditioned medium of C5aR1-silenced and control cells after treatment with recombinant C5a. Data in this figure are presented as medians and interquartile ranges. Results in A–C and E–G correspond to three independent experiments. Differences between treatments were analyzed with the Mann-Whitney U test. Scales bars in representative images, 100 μm; applies to all. *P < 0.05; **P < 0.01; ***P < 0.001. C5a = cleaved complement component of C5; C5aR1 = receptor 1 for C5a; n.s. = non-significant; shC5aR1-1 = short hairpin–mediated silencing of C5aR1 gene expression, using shRNA-1; shC5aR1-2 = short hairpin–mediated silencing of C5aR1 gene expression, using shRNA-2.