LYN-1604

Kinase experiment:

Cells or tumor tissues are treated with LYN-1604 for the indicated times. Both adherent and floating cells are collected and western blot analysis is carried out. Briefly, the cell pellets ae resuspended with lysis buffer consisting of 50 mM HEPES, pH 7.4; 1% Triton-X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM PMSF, 10 mg/L aprotinin and 10 mg/Lleupeptin, and lysed at 4°C for 1 h. After 12?000 rpm centrifugation for 15 min, the protein content of the supernatant is determined by the Bio-Rad DC protein assay[1].

Cell experiment:

Cells are dispensed in 96-well plates at a density of 5×104 cells per mL. After 24 h of incubation, cells are treated with different concentrations of LYN-1604 for the indicated time periods. Cell viability is measured by the MTT assay[1].

Animal experiment:

24 female nude mice (BALB/c, 6 to 8 weeks, 20 to 22 g) are used and injected subcutaneously with MDA-MB-231 cells (5×106 cells per mouse). When the tumors reach 100 mm3 in volume (V=L×W2/2), the mice are divided into four groups. Three groups are treated with different doses of LYN-1604 once a day by intragastric administration for 14 days (low dose, 25 mg/kg; median dose, 50 mg/kg; high dose, 100 mg/kg), whereas the control group is treated with vehicle control. During the treatment, the tumor volumes and body weight are measured every day until the end of the study. At the end of treatment, all mice are sacrificed. The spleen, liver, kidney and tumor tissue are harvested, weighed, and photographed, then immediately frozen in liquid nitrogen or fixed in formalin[1].

References:

[1]. Zhang L, et al. Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo. Chem Sci. 2017 Apr 1;8(4):2687-2701.