Idelalisib

The PI3K inhibitor copanlisib, TGX-221, alpelisib, idelalisib, and LY294002 were purchased from MedChemExpress (Princeton, New Jersey, USA). The PI3K inhibitor pictilisib, the ALK inhibitor crizotinib and NVP-TAE684 were obtained from ADOOQ (Irvine, California, USA). The antibodies against ALK, phospho-ERK1/2, Akt1/2, N-Myc, GAPDH, and beta-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Antibodies targeting Phospho-Akt (Ser473), Phospho-S6 Ribosomal Protein (Ser235/236), S6 Ribosomal Protein, and anti-Phospho-Histone H3 (Ser10) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Other antibodies used in this study included anti-MAPK1/2 (Milipore, NG1946), anti-β-Tubulin antibody (Abcam, Ab6046).

Mouse single-cell suspensions were incubated with fluorescently labelled antibodies for 30 min at 4 °C after blocking FC receptors using anti-CD16/32 antibody for 20 min at 4 °C. Afterwards, cells were washed in PBS + 2% FBS and data were collected on a FACS Canto II (Becton Dickinson) cytometer. A minimum of 50,000 and a maximum of 200,000 events was acquired in every measurement. Analyses were performed using FlowJo software (TreeStar). Counting of total cells was performed with CountBright™ beads. For Phosflow assays, cells were first stained with extracellular antibodies, and then fixed by adding paraformaldehyde to a final concentration of 2% for 20 min at 4 °C. After two washes, cells were permeabilized for 30 min with 90% methanol on ice. Intracellular labelling was performed at 4 °C O/N with specific phospho-Abs and their corresponding secondary antibodies. For Phosflow assays of the MEC-1 cell line, cells were first serum-starved and treated with specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK), U0126 (Promega), phosphatidylinositol 3 kinase (PI3K), wortmannin (Millipore), idelalisib (MedChemExpress) and LY294002 (Sigma), mTOR complex Rapamycin (Millipore), Btk inhibitor Ibrutinib (MedChemExpress) and Src tyrosine kinases family PP2 (Sigma). After 6 h, cells were treated as previously described.

Selective small molecule inhibitors specifically against the SYK/PI3K signaling pathway were investigated (108 (link)). For repurposing purpose, only approved or investigational compounds in phase-III clinical trials were used in the screening assay. All inhibitors (dexamethasone [D1756; Merck], entospletinib [S7523; Selleckchem], R406 [S1533; Selleckchem], alpelisib [S2814; Selleckchem], idelalisib [HY-13026; MedChemExpress], duvelisib [HY-17044; MedChemExpress], were purchased in powdered form and dissolved according to the distributor’s instructions). Macrophages were preincubated with inhibitors (or DMSO as a control) for 30 min at 37°C. After preincubation, macrophages were stimulated with 20 μg/ml polyinosinic:polycytidylic acid (poly(I:C), Sigma-Aldrich) and seeded in a density of 50,000 cells/well in pre-coated 96-well plates in 200 μl/well medium.