T2577

The kd of TP73-AS1 was induced by addition of doxycycline for 10 days. The cells were plated in 12-well plates and TMZ (Sigma-Aldrich, T2577) or DMSO was added. Seven to ten days post treatment, the cells were washed and trichloroacetic acid (TCA) was added for an hour at 4 °C. The plates were washed and air dried. Crystal Violet (C0075, Sigma-Aldrich) was added for half an hour and additional rinsing was conducted. After dehydration, 10% acetic acid was added and the optical density was measured at 570 nm. The results were normalized to scramble gRNA.

SK‐MEL‐28 multicellular spheroids were generated using the “hanging drop” method.
³⁸ (link),
³⁹ (link) Briefly, cells were cultured and added in suspension at 2–2.5 × 10⁴ cells/mL. Next, around 500 cells were placed on the inside cover of a 100‐mm culture dish as hanging drops (20 μL) and left for 48 h. The formed spheroids were transferred into a 96‐well plate,
³⁸ (link),
³⁹ (link) and culture medium was then added (in the absence (DMSO used as vehicle; control) or presence of TMZ (Sigma, #T2577) at 80 μg/mL (TMZ‐C1) or 200 μg/mL [TMZ‐C2]). Images were taken after 24 h and every other day, using a Leica inverted DMi1 microscope. The spheroid surface area was measured (12 spheroid measurements/condition) using the ICY Bioimage analysis software (https://icy.bioimageanalysis.org/). Ten spheroids (day 6) were selected per each condition for further ELISA measurements.

The drugs used in the study were dissolved in their respective solvents as reported in the product information. Specifically, temozolomide (Sigma-Aldrich, T2577) and enzastaurin (Sigma-Aldrich, SML0762) were dissolved in DMSO (Sigma-Aldrich, D8418), while imatinib (Sigma-Aldrich, SML1027) was dissolved in sterile milliQ water.

U87 and U343 cells were plated as monolayers in 96-well plates (4000 cells/well) and treated with indicated concentrations of CYC065 (#HY-101212, Medchemexpress, NJ, US) and THZ1 (#HY-80013, Medchemexpress, NJ, US) for 72 h. Patient-derived GBM cultures were plated in 96-well plates as gliomaspheres (3000 cells/well) and treated with indicated concentrations of CYC065, THZ1 and/or TMZ (#T2577, Sigma-Aldrich, Arklow, Ireland) and S-63845 (#S8383, Selleck Chemicals, Houston, TX, US) as indicated for 72 h. Mouse primary cortical neurons were seeded in flat-bottom 96-well plates coated with poly-L-lysine as 30,000 cells/well and treated with increasing concentrations of CYC065 and THZ1 for 96 h. Following treatment, WST-1 reagent (Sigma-Aldrich, Arklow, Ireland) was added in 1:10 final dilution, according to the manufacturer’s instructions. WST-1 salt is cleaved to a soluble formazan dye by a NAD(P)H-dependent reaction in viable cells. Plates were incubated for 2 h in a humidified incubator at 37 °C and 5% CO₂ and the absorbance of each sample was measured at 450 and 620 nm using a microplate reader (GENios, Tecan, Weymouth, UK). Background signal (620 nm) was then subtracted from the 450 nm reads. The absorbance was proportional to the number of viable cells and expressed relative to DMSO (0.1%) control-treated groups.