Victorx4 reader

For immunostaining, infected Calu-3 cells were fixed with 4% PFA for 15 min. Following a washing step with PBS, cells were blocked and permeabilized overnight with 1% BSA in PBS (+0.2% Triton) and afterwards stained with a polyclonal rabbit anti-NP antibody (GeneTex, Cat. No. GTX135357, Irvine, CA, USA) for 24 h. Subsequently, cells were incubated for 1 h with a goat anti-rabbit-AlexaFlour488 (Invitrogen, Cat. No. A11008, Waltham, MA, USA) and finally stained with 4′,6-Diamidino-2-phenyl-indol –dihydrochlorid (DAPI) (Sigma Aldrich, D9542, St. Louis, MO, USA) for 10 min. For analysis, immunostaining was quantitative analyzed with a PerkinElmer VictorX4 reader (488 nm) (PerkinElmer, Waltham, MA, USA) and pictures were taken using a CTL-ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH, USA).

T-ALL cells were treated with DMSO, single compound or a combination of compounds for up to 14 days. Cells were seeded at a starting concentration of 3 × 10⁵ cells per mL and sub-cultured every 2–3 days by centrifugation, resuspension in fresh medium and addition of new compound. Cell concentration and viability were determined by FSC/SSC on a Guava EasyCyte Benchtop Flow cytometer (Merck Millipore). MRK-560 does not cause an immediate effect on cell proliferation and survival, and therefore, pretreatment was used when combined with other drugs in short-term experiments. Seven-day pretreated or untreated cells were seeded in 96-well plates (3 × 10⁵ cells/mL), and compounds (inhibitor or DMSO) were dispersed in a randomized fashion by a D300e digital dispenser (Tecan). Compound concentration was normalized to DMSO. A quantitative evaluation of proliferation was done after 48 h with ATPlite (PerkinElmer) and measured on the VICTOR X4 Reader (PerkinElmer). Synergy was analyzed using CompuSyn software.
Apoptosis assay. Apoptosis was measured after 12 days of treatment with DMSO, single compound or combination with the FITC Annexin V detection kit with PI (Biolegend). Cells were analyzed on a MACSQuant Vyb flow cytometer (Miltenyi Biotec), and data were analyzed using FlowJo Software (Becton, Dickinson and Company).