(A)MCF-7 cells cultured in DMEM, stimulted with 100ng/mL IL6 for 96h. (B)Unstimulated MCF-7 cells cultured in serum-free DMEM for 96h.

Interleukin 6 (IL-6) is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. Current data suggest that direct application of IL-6 on breast cancer cells inhibits proliferation in ER-positive (estrogen- receptor- positive) cells through the Jak/Stat3 pathway. To test the inhibitory effect of IL-6 on proliferation of ER-positive MCF-7 cell line, cells were seeded into triplicate wells of 96-well plates at a density of 5,000 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 IL-6. 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 measure the absorbance at 450nm using a microplate reader after incubating the plate for 1-4 hours at 37°C.