BMP4 (Bone morphogenetic protein 4) is a member of the bone morphogenetic protein family, which is involved in bone and cartilage development, specifically tooth and limb development and fracture repair. It has been proven that HJV (Hemojuvelin) acts as a coreceptor of BMPs, including BMP4; therefore, a binding ELISA assay was constructed to detect the association of recombinant human BMP4 with recombinant human HJV. Briefly, BMP4 were diluted serially in PBS, with 0.01%BSA (pH 7.4). Duplicate samples of 100uL were then transferred to HJV-coated microtiter wells and incubated for 2h at 37°C. Wells were washed with PBST and incubated for 1h with anti-BMP4 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37°C. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of BMP4 with HJV was shown in Figure 1 and this effect was in a dose dependent manner.

To test the effect of BMP4 on cell apoptosis, HepG2 cells were seeded into triplicate wells of 96-well plates at a density of 4,000 cells/well and allowed to attach overnight, then the medium was replaced with various concentrations of recombinant human BMP4 diluted with 5% serum standard DMEM. After incubated for 72h,?cells were observed by inverted microscope and cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly, 10 μl of CCK-8 solution was added to each well of the plate, then the absorbance at 450 nm was measured using a microplate reader after incubating the plate for 1-4 hours at 37 ℃. Apoptosis of HepG2 cells after incubation with BMP4 for 72h observed by inverted microscope was shown in Figure 1. Cell viability was assessed by CCK-8 (Cell Counting Kit-8 ) assay after incubation with recombinant human BMP4 for 72h. The result was shown in Figure 2. It was obvious that BMP4 significantly decreased cell viability of HepG2 cells. The ED50 of recombinant human BMP4 is 5.1 ug/ml.

Figure 2. Inhibition of HepG2 cells proliferation after stimulated with recombinant human BMP4