Mbs142428

SJRHB012_ZC, SJRB011_YC, IC-pPDX-29_XC and RMS-ZH003_XC cells were plated at 6000 cells/well in 384-well plates coated with Matrigel. The medium was changed after one day, and cells were incubated with either asparaginase 1 u/mL (MBS142428, Biozol, Eching, Germany), imiquimod 50 uM (14956, Cayman Chemical) or both. DMSO-treated cells served as controls. After 72 h, cell viability was determined using the CellTiter-Glo 3D viability assay (Promega, G9681, Madison, WI, USA).

Cells were seeded at 750 (mouse) or 1000 (human RD) cells per well in 96-well plates with 100 µL medium. After 24 h, cells were exposed to asparaginase (MBS142428, Biozol), phenformin (14997, Cayman Chemical, Ann Arbor, MI, USA), metformin (13118, Cayman Chemical) and imiquimod (14956, Cayman Chemical) at defined concentrations. Chemical treatments were repeated every 48 h for up to 6 days in total. To determine cell growth, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M2003, Sigma-Aldrich) was solved in PBS to a final concentration of 12 mM. In brief, 10 µL of MTT solution was added per well and incubated for 4 h. Then, 100 µL dissolving solution (10% SDS (L3771, Sigma-Aldrich) with 0.01M HCL) was added to each well and incubated for 12–16 h. Optical densities at 570 nm were measured with a Tecan Sunrise absorbance microplate reader.