Figure. The binding activity of IGF1 with IGFBP4.
Insulin-like growth factor 1 (IGF1), also called somatomedin C is a hormone similar in molecular structure to insulin. It plays an important role in childhood growth and continues to have anabolic effects in adults. A synthetic analog of IGF1, mecasermin, is used for the treatment of growth failure. IGF1 consists of 70 amino acids in a single chain with three intramolecular disulfide bridges. IGF1 has a molecular weight of 7,649 Daltons. Besides, Insulin Like Growth Factor Binding Protein 4 (IGFBP4) has been identified as an interactor of IGF1, thus a binding ELISA assay was conducted to detect the interaction of recombinant human IGF1 and recombinant human IGFBP4. Briefly, IGF1 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100µL were then transferred to IGFBP4-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-VDR 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℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of IGF1 and IGFBP4 was shown in Figure 1, and this effect was in a dose dependent manner.

To measure the effect of IGF1 on cell proliferation, breast cancer MCF-7 cells were seeded into triplicate wells of 96-well plates at a density of 5,000 cells/well and allowed to attach, replaced with serum-free overnight, then the medium was replaced with 1% serum standard DMEM prior to the addition of various concentrations of recombinant human IGF1. After incubated for 96h, cells were observed by inverted microscope and cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly, 10µL of CCK-8 solution was added to each well of the plate, then the absorbance at 450nm was measured using a microplate reader after incubating the plate for 1-4 hours at 37℃. Proliferation of MCF-7 cells after incubation with IGF1 for 72h observed by inverted microscope was shown in Figure 2. Cell viability was assessed by CCK-8 assay after incubation with recombinant IGF1 for 72h. The result was shown in Figure 3. It was obvious that IGF1 significantly increased cell viability of MCF-7 cells.
(A) MCF-7 cells cultured in DMEM, stimulated with 10ng/mL IGF1 for 72h;
(B) Unstimulated MCF7 cells cultured in DMEM for 72h.
Figure. Cell proliferation of MCF-7 cells after stimulated with IGF1.

Figure. Cell proliferation of MCF-7 cells after stimulated with IGF1.