Fig 1: C3AR1 is a putative receptor for AQEE30 in osteoblasts.a, The expression of C3ar1 mRNA in MC3T3-E1 cells before (pre-OB) and after (OB) differentiation into osteoblasts for 7 days in osteogenic culture medium. The mRNA levels were analyzed by real-time PCR (n = 3). b, MC3T3-E1 cells were generated as described in a, and total cell lysates were analyzed by western blotting with an anti-C3AR1 antibody. c, MC3T3-E1 cell lysates at the indicated doses were seeded onto ELISA plates coated with 5 μM AQEE30 for 2 h. The wells were further incubated with an anti-C3AR1 antibody and quantitatively analyzed by incubation with an HRP-conjugated secondary antibody. The absorbance was measured at 450 nm on a microplate reader (n = 4). d, The indicated biotin-labeled AQEE30 peptides were incubated with His-tagged C3AR1 expressed in HEK293T cells and then immunoprecipitated with streptavidin agarose beads. The precipitates were analyzed by immunoblotting with an anti-His antibody. The input represents 5% of the total cell lysates used for immunoprecipitation. e, MC3T3-E1 cells were treated with the indicated doses of AQEE30 in the presence or absence of SB290157, and cell viability was measured (n = 4). f, MC3T3-E1 cells were treated with AQEE30 in the presence or absence of SB290157 and analyzed in a bone nodule formation assay. Mineralization was determined with Alizarin red S staining (top), and the absorbance was measured at 570 nm (bottom, n = 6). g,h, MC3T3-E1 cells were transfected with scrambled siRNA and C3ar1 siRNA. The knockdown efficiency of C3ar1 was analyzed by real-time PCR (g, n = 3) and western blotting (h). Actin was used as a loading control. i, MC3T3-E1 cells were transfected with scrambled and C3ar1 siRNAs in the presence or absence of AQEE30 and subjected to a bone nodule formation assay (n = 4). j, After MC3T3-E1 cells were treated as described in i, a cAMP assay was performed (n = 3). k, MC3T3-E1 cells were generated as described in e, and western blotting was carried out with the indicated antibodies against p21, phospho-Rb (pRb) and Rb; their relative band densities, normalized to those of α-tubulin and Rb, respectively, are shown on the right (n = 3). The data represent the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control groups or between the groups. NS, not significant.
Fig 2: Effects of mini-PEGylated AQEE30 on bone formation in vitro.a, The structures of mini-PEG2 (PEG2) and mini-PEGylated AQEE30 (PEG2-AQEE30). b–d, MC3T3-E1 cells were treated with 5 μM AQEE30 or PEG2-AQEE30 for 48 h, after which cell viability (b, n = 4), BrdU (c, n = 5) and mineralization (d, n = 4) assays were performed. Mineralized samples were stained with Alizarin red S (upper) and measured at 570 nm. e, MC3T3-E1 lysates (30 μg) were seeded onto ELISA plates coated with 5 μM AQEE30 or PEG2-AQEE30 for 2 h. The wells were further incubated with an anti-C3AR1 antibody, and the absorbance was measured at 450 nm using a microplate reader (n = 3). f, MC3T3-E1 cells were treated with 1 μM AQEE30 or PEG2-AQEE30 for 30 min. Whole-cell lysates were analyzed in a cAMP assay (n = 3). The data represent the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control.
Supplier Page from Sino Biological, Inc. for Human C3a Receptor Gene ORF cDNA clone expression plasmid, C-His tag