Figure 6: Blocking of IL-20 by anti-IL-20 mAb 7E promoted osteoblast maturation and upregulated osteoblastogenic factors in MC3T3-E1 cells. | Scientific Reports

Figure 6: Blocking of IL-20 by anti-IL-20 mAb 7E promoted osteoblast maturation and upregulated osteoblastogenic factors in MC3T3-E1 cells.

From: Anti-IL-20 monoclonal antibody promotes bone fracture healing through regulating IL-20-mediated osteoblastogenesis

Figure 6

(a) MC3T3-E1 cells were incubated with osteoblast differentiation medium. Their ability to differentiate into osteoblasts was characterized using ALP staining with an assay kit 14 days post-incubation. *P < 0.05 versus untreated controls (Ctrl). (b) MC3T3-E1 cells were treated with IL-20 (200 ng/ml), 7E (2 μg/ml), or IL-20+7E for 4 h. Sclerostin and OPG expression were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; GAPDH was the housekeeping gene. *P < 0.05 versus untreated controls. #P < 0.05 versus the IL-20-treated group. (c) MC3T3-E1 cells were pre-incubated with BMP-2 for 2 h, and then treated with IL-20 (200 ng/ml) for another 4 h. OPG expression levels were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; GAPDH was the housekeeping gene. *P < 0.05 versus untreated controls. #P < 0.05 versus the BMP-2-treated group. All experiments were run three times, with similar results. Data are representative of 3 independent experiments. (d) MC3T3-E1 cells were treated with IL-20 (200 ng/ml), 7E (2 μg/ml), or IL-20+7E for 4 h. mRNA was isolated and the transcripts of OSX, Wnt3a, Wnt7a, and Wnt7b were analyzed using RTQ-PCR with specific primers. The quantification analysis of mRNA was normalized; GAPDH was the housekeeping gene. *P < 0.05 versus untreated controls. All experiments were run three times, with similar results. Data are from a representative experiment. (e) MC3T3-E1 cells were incubated with mIgG (2 μg/ml), 7E (2 μg/ml), IL-20 (200 ng/ml), IL-20+mIgG or IL-20+7E for 6 h, and cell lysates were collected and analyzed using Western blotting for the indicated protein. Data are representative of three independent experiments. (f) HEK293 cells transfected with human OSX promoter construct and incubated with IL-20 (200 ng/ml) for 24 h. Cell lysate were collected and analyzed using a luciferase assay. *P < 0.05 versus untreated controls. All experiments were run three times, with similar results. Data are from a representative experiment.

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