Gefitinib

Glioma cells (5 × 10³) were plated on 96‐well plates in triplicates and treated with various concentrations (0.1–10 μM) of gefitinib (InvivoGen, USA). After 48 h MTT was added, cells were lysed and the absorbance was measured as described above. Half‐maximal inhibitory concentration (IC₅₀) was determined for each cell line using the following formula: IC₅₀ = (50%–Low Inh%)/(High Inh%–Low Inh%) × (High Conc–Low Conc) + Low Conc, where: Low Inh%/High Inh% = % inhibition directly below/above 50% inhibition; Low Conc/High Conc = Corresponding concentrations of the test compound.

LY294002 (Cell Signaling, #9901) and Bapta-AM (Selleckchem, #S7534) were used at a 50 μM concentration. PP2 (Cayman Chemical, #13198–1), U0126 (Cell signaling, #9903), PF-573228 (Sigma, #PZ0117), Gefitinib (Invivogen, #tlrl-gef) were used at 10 μM concentration. Thapsigargin (Cayman Chemical, #10522) was used at 5 μM concentration. Wotrmannin (Cell Signaling, #9951) and MK-2206 2HCl (Selleckchem, #S1078) were used at 1 μM concentration.
Crystal violet was purchased from Sigma (#3886).
Antibodies for AKT (#4691), p-AKT-S473 (#4060), p-NDRG1-T346 (#5482), NDRG1 (#9485), p-P70S6K-T389 (#9234), P70S6K (#2708), RICTOR (#2114), p-PRAS40-T246 (#2997), PRAS40 (#2691), TSC2 (#4308), p-TSC2-T1462 (#3617), 4EBP1 (#9644), p-4EBP1-T37/46 (#2855), ITGB1 (#9699), ILK (#3856), SRC (#2123), YES (#3201), were from Cell Signaling. Antibody against β-Actin (#A3854) was from Sigma.

RPMI 1640 (Lonza) supplemented with 10% fetal‐bovine‐serum, 100 μg/mL normocin, 50 μg/mL penicillin‐streptomycin, 1% non‐essential aminoacids, 1% MEM‐vitamins and 1 mM sodium pyruvate (cRPMI). PBP MV130 (Bactek)composed of heat‐inactivated bacteria (60% S. pneumoniae, 15% S. aureus, 15% S. epidermidis, 4% K. pneumoniae, 3% M. catarrhalis and 3% H. influenzae), PBP MV140 (Uromune) composed of heat‐inactivated bacteria (Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae and Enterococcus faecalis, 25% each) and control (all the excipients without bacteria) were from Inmunotek S.L. Type B TLR9‐ligand (ODN 2006), inhibitors for MyD88 (Pepinh‐MYD), RIPK2 (Gefitinib) from InvivoGen and neutralizing anti‐IL‐10 (clone JES3‐9D7, Biolegend) were used.

CFPAC-1 cells were seeded on glass coverslips in 12-well plates containing 1 ml of culture medium per well. At 30% confluency, cell media was replaced by low serum medium containing 2% FBS. After 8 days of compound treatment, cells were fixed with 4% Formaldehyde (Merck), permeabilized with 0.1% Triton X100 (Merck) in DPBS, blocked with 1% BSA (Applichem) in DPBS, and incubated with 1:1000 dilution of anti-acetylated tubulin antibody or 1:500 dilution of anti-IFT88 antibody (Cat. No. 13967–1-AP, Proteintech) for 1 h, followed by incubation with 1:1000 dilution of fluorescent secondary antibody for 1 h. Nuclei were counterstained with DAPI (Vector Laboratories, Vectashield (Cat. No. H-1500). Images of primary cilia were captured by acquiring Z-stacks using either a Bio-Rad Radiance or Nikon C2 Eclipse Ti-E confocal laser scanning microscope by 40X or 60X oil immersion lenses. All drugs selected for confocal reconfirmation experiments were purchased from Sigma, except for Almotriptan Malate and Sirolimus which were obtained from Selleckchem. Gefitinib was from Invivogen, Cefprozil Monohydrate from Abcam and Imexon from MicroSource Discovery Systems Inc.

J774A.1 macrophages were seeded in 96-well plates and incubated overnight at 37°C and 5% CO₂ to allow adherence. Cells were treated with PF 06650833 (100 nM) (TOCRIS, Abingdon, United Kingdom), a specific inhibitor of IRAK4, or Gefitinib (10 μM) (InvivoGen, San Diego, CA, United States), an inhibitor of RIP2 for 1 h before being stimulated by bacteria at a concentration of 10⁶ CFU/ml (ratio bacteria/macrophages 10:1) for 24 h (Seganish, 2016 (link)). The release of TNF-α and IL-6 was evaluated using enzyme-linked immunosorbent assays (ELISA).

Esomeprazole, Pargyline, Leflunomide, Clofibrate, Ciprofloxacin, Ketorolac, Paroxetine, Dobutamine, Tranilast, Loperamide, Xylazine, Capsaicin, Papaverine, Oxymetazoline, Dexamethasone, Docetaxel, Topotecan, Paclitaxel were purchased from Sigma. Almotriptan Malate and Sirolimus were purchased from Selleckchem, Gefitinib from Invivogen, Cefprozil Monohydrate from Abcam and Imexon from MicroSource Discovery Systems Inc.; ATP, Apyrase, Suramin and Mefloquine were purchased from Sigma; Latrunculin B was purchased from Abcam.