Fig 1: Potential MSCs recruitment by NAB in vivo. (A) H&E staining and Alizarin red staining. Representative H&E staining (20×) showed that hematomas, accompanied by lots of trabecular bone growth could be observed in the NAB group at 2 weeks, while the random DNA group was not that obvious; At 4 weeks, the trabecular bone growth the NAB group gradually matured. Absorption and shaping occurred. Attaching growth of bone could be observed on the inner side of the cortical bone as the white circle highlighted, while the control group remained in the trabecular bone growth stage. Alizarin red staining (40×) images of bone defect region show distinct calcium contents between NAB and random DNA group at either 2 weeks or 4 weeks. (B) Serology assessment. Serum markers of osteogenetic processes such as OPN, BGP, and ALP were analyzed at the 2 or 4 weeks after the initiation regiment. Each bar represents means with SD of six replicates. *P<0.05; **P<0.01.
Fig 2: Evaluation of the mRNA transcription and protein expression of OPN and MCP-1 in HK-2 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis of expression levels of OPN (left panel) and MCP-1 (right panel) in HK-2 cells; values are corrected for GAPDH. Protein expression of (B) OPN (left panel) and MCP-1 (right panel) in HK-2 cells. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control group; #P<0.05 vs. sodium oxalate group. Statistical analyses were performed by one-way analysis of variance. OPN, osteopontin; MCP-1, monocyte chemoattractant protein 1.
Fig 3: Immunohistochemical distribution of the expression of OPN and MCP-1 in rat kidneys harvested following an 8-week treatment period. The left column comprises representative images showing the expression of OPN and MCP-1 in control rats, the middle column comprises representative images showing the expression of OPN and MCP-1 in EG-treated rats and the right column comprises representative images showing the expression of OPN and MCP-1 in EG + metformin-co-treated rats. OPN, osteopontin; MCP-1, monocyte chemoattractant protein 1; EG, ethylene glycol.
Fig 4: Production of OPN and MCP-1 in the cell culture supernatants of HK-2 cells and MDCK cells. Expression of (A) OPN (upper-left panel) and (B) MCP-1 (upper-right panel) in HK-2 cell culture supernatants. Expression of (C) OPN (lower-left panel) and (D) MCP-1 (lower-right panel) in MDCK cell culture supernatants. The data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control group; #P<0.05 vs. sodium oxalate group. Statistical analyses were performed by one-way analysis of variance. OPN, osteopontin; MCP-1, monocyte chemoattractant protein 1.
Fig 5: Evaluation of the mRNA transcription and protein expression of OPN and MCP-1 in rat kidneys. (A) Reverse transcription-quantitative polymerase chain reaction analysis of expression levels of OPN (left panel) and MCP-1 (right panel) in rat kidneys; values were corrected for GAPDH. Protein expression of (B) OPN (left panel) and MCP-1 (right panel) in rat kidneys. The data are presented as the mean ± standard deviation of six independent experiments. *P<0.05 vs. control group; #P<0.05 vs. sodium oxalate group. Statistical analyses were performed by one-way analysis of variance. OPN, osteopontin; MCP-1, monocyte chemoattractant protein 1; EG, ethylene glycol.
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