Ml349

PTE activity was analyzed by imaging flow cytometry (Amnis/EMD Millipore). In brief, 10⁶ U937 cells were pre-incubated for 10 min at room temperature followed by 10 min on ice with DPP-2 or DPP-3 fluorescent probes (provided by B.C. Dickinson) [22 (link)]. The APT2 selective inhibitor ML349 (#5344, bio-techne), the APT1 selective inhibitor ML349 (#5345, bio-techne) and the pan PTE inhibitor Palmostatin B (Sigma-Aldrich) were used as controls. TNF was added and incubated for another 20 min on ice. Activation was triggered by temperature shift to 37 °C for the indicated time points, followed by immediate cooling/fixation in 2%PFS/PBS. The plasma membrane was stained using cell mask deep red stain (1:10.000 in PBS) for 5 min on ice, followed by washing with PBS. Images were acquired using the Inspire software (200.1.388.0) and changes in fluorescence intensity were analyzed using the IDEAS software (6.0.154.0).

Palmostatin B was purchased from Merck Millipore (178501). ML348 and ML349 were purchased from Tocris Bioscience (USA). Cells were plated in 96-well plates with a density of 4000-8000 cells per well and incubated for 24 h at 37°C with 5% C02. Then cells were treated with increasing drug concentrations and their combinations. Cell viability was measured with the CellTiter-Glo Luminescent Cell Viability Assay (Promega; Wisconsin, USA) according to the manufacturer's protocol. Luminescence was measured on the SynergyHT plate reader (BioTek, Vermont, USA) using Gen5 software (Version 1.11.5). For apoptotic assays 0.1–0.2 × 10⁶ cells were plated in 12-well plates and treated with DMSO or an inhibitor. After 72 hours apoptosis was assessed using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and Propidium Iodide according to the manufacturer's instructions (Invitrogen, V13241) with the AccuriC6 Flow Cytometer using the CFlow software (Ver. 1.0.227.4). Concentrations of drugs resulting in 50% decrease in cell viability relative to DMSO treated controls (GI50) were calculated with CalcuSyn software (Biosoft, Cambridge, UK, Version 2.1).

Ceramide was analyzed by imaging flow cytometry (Amnis/EMD Millipore). In brief, cells were incubated with ML349 (50 μM, Tocris), GW4869 (20 μM, Sigma-Aldrich) or left untreated for 30 min at RT followed by 20 min cooling down on ice and centrifugation for 4 min 350 x g, 4 °C. 100 ng/mL TNF was incubated for 20 min on ice, followed by 15 min temperature shift to 37 °C. Cells were fixed in 2%PFA/PBS for 15 min on ice, 2x washing and permeabilization in 0.2%Saponin/0.1%BSA/PBS for 15 min on ice. Cells were 2x washed with 0.1%BSA/PBS followed by 30 min incubation with anti-ceramide antibody (clone 15B4, 1:100 in 0.1%BSA/PBS), 2x washing and incubation with anti-mouse-alexafluor488 antibody, diluted 1:200 in 0.1%BSA/PBS for 30 min. Images were acquired using the Inspire software (200.1.388.0) and changes in fluorescence intensity was analyzed using the IDEAS software (6.0.154.0).