Active Insulin Like Growth Factor 2 (IGF2)

Figure. The binding activity of IGF2 with EMMPRIN.
Insulin-like growth factor 2 (IGF2) is one of three protein hormones that share structural similarity to insulin. It has growth-regulating, insulin-like and mitogenic activities. IGF2 exerts its effects by binding to the IGF-1 receptor and to the short isoform of the insulin receptor. IGF2 may also bind to the IGF2 receptor (also called the cation-independent mannose 6-phosphate receptor), which acts as a signalling antagonist; that is, to prevent IGF2 responses. Besides, Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) has been identified as an interactor of IGF2, thus a binding ELISA assay was conducted to detect the interaction of recombinant human IGF2 and recombinant human EMMPRIN. Briefly, IGF2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to EMMPRIN-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IGF2 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 IGF2 and EMMPRIN was shown in Figure 1, and this effect was in a dose dependent manner.

Figure. Cell proliferation of MCF-7 cells after stimulated with IGF2.
To test the effect of IGF2 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 IGF2. 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 IGF2 for 96h observed by inverted microscope was shown in Figure 2. Cell viability was assessed by CCK-8 (Cell Counting Kit-8) assay after incubation with recombinant IGF2 for 96h. The result was shown in Figure 3. It was obvious that IGF2 significantly increased cell viability of MCF-7 cells.
(A) MCF-7 cells cultured in DMEM, stimulated with 1ng/mL IGF2 for 96h;
(B) Unstimulated MCF7 cells cultured in DMEM for 96h.

Figure. Cell proliferation of MCF-7 cells after stimulated with IGF2.