INS (Insulin) is a peptide hormone produced by beta cells of the pancreatic islets, which decreases blood glucose concentration and increases cell permeability to monosaccharides, amino acids and fatty acids. It has been reported that insulin triggers phosphorylation of a number of substrates by binding to its receptors, which was important for cell proliferation, cell cycle progression, cell division and differentiation. To detect the effect of Insulin on cell proliferation, MCF-7 cells were seeded into triplicate wells of 96-well plates at a density of 2,000 cells/well and allowed to attach overnight, then the medium was replaced with serum-free standard DMEM prior to the addition of various concentrations of INS. After incubated for 48h, 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°C.

The dose-effect curve of INS was shown in Figure 2. It was obvious that INS significantly promoted cell proliferation of MCF-7 cells. The ED50 for this effect is typically 11.71 to 57.11 ng/mL.

Insulin (INS) is a polypeptide hormone originating in the beta cells of the pancreas and serving as a principal regulator for the storage and production of carbohydrates. Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver. Besides, Insulin Receptor (INSR) has been identified as an interactor of INS, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse INS and recombinant rat INSR. Briefly, INS were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to INSR-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-INS 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 50 µL stop solution to the wells and read at 450 nm immediately. The binding activity of of recombinant mouse INS and recombinant rat INSR was shown in Figure 1, the EC50 was 8.68 ug/ml.