Powerplex 16 hs

UM-SCC-1 (SCC1), UM-SCC-6 (SCC6) and SCC25 cells were cultured in Dulbecco’s Modified Eagle’s Medium and Ham’s F12 supplemented with 10% foetal bovine serum and maintained at 37 °C and 5% CO₂. A total of 25,000 cells were plated in quintuplicate in six-well plates. Cetuximab (Lilly) was purchased from Johns Hopkins Pharmacy, and JQ1 from Selleck Chemicals. Cell lines were treated daily with cetuximab (100 nM), JQ1 (500 nM), the combination or vehicle (PBS + DMSO; mock) for 5 days. Proliferation was measured using alamarBlue assay (Thermo Scientific). AlamarBlue (10% total volume) was added to each well, and fluorescence (excitation 544 nm, emission 590 nm) was measured after 4 h of incubation at 37 °C. A media only well was used as blank. The measurements were repeated in three independent experiments. Growth rate was calculated using the formula: $GR=2^{k\left(c,t\right)/k\left(0\right)}-1$ Where k(0) = fluorescence measured for non-treated cells and k(c,t) = fluorescence for treated cells.^{20 (link)}Parental cell lines were authenticated before and after all assays using short tandem repeat (STR) analysis kit PowerPlex16HS (Promega) through the Johns Hopkins University Genetic Resources Core Facility.

All established human T-ALL cell lines were obtained from the laboratories of Drs. Thomas Look (DFCI, Boston), Jon Aster (Brigham & Women’s Hospital, Boston), and Adolfo Ferrando (Columbia University, New York) and have undergone extensive genotypic characterization including STR DNA typing (PowerPlex 16 HS, Promega) [1 (link), 3 (link), 26 (link)]. Known gene mutations in PI3K/AKT and MAPK pathways are summarized in S1 Table. Cell lines were grown in RPMI 1640 medium supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM l-glutamine, and antibiotics. Recombinant human IGF-1, IL-7, and SDF-1α/CXCL12 (Peprotech) were resuspended in PBS/1% BSA prior to addition to culture media. Phorbol 12-myristate 13-acetate (PMA, Sigma P1585) was resuspended in DMSO prior to addition to culture media.