U0126 etoh

NT2D1 embryonal carcinoma cells (American Type Culture Collection (ATCC) were used in this study and cultured as reported in [4 (link),5 (link),8 (link)]. Cells, cultured in DMEM (Sigma Aldrich, cat. D6546, St. Louis, MO, USA) complemented with 10% foetal bovine serum (FBS Gibco, cat. 10270, Gland Island, NY, USA), were treated, when indicated, with 40 ng/mL of HGF (human recombinant HGF, R&D Systems, cat. 294-HG, Minneapolis, MN, USA), 5 µM of MAPK-MEK1/2 selective inhibitor U0126 (U0126-Et-OH, Selleckchem, cat. S1102, USA and Canada only) or both molecules. The U0126 toxicity was evaluated by cell viability FACS analysis, using propidium iodide. We tested different concentrations (2.5, 5, 10, 15 µM) as suggested in the literature. To obtain an increase in G1 phase cell percentage, cells were cultured for 15 h without serum (starvation). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

The human lung squamous cell carcinoma cell line H520 and the human lung adenocarcinoma cell lines H157, A549, H1299 and Anip973 were cultured in RPMI1640 (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (Gibco) and incubated with 5% CO₂ at 37 °C. Actinomycin D (Sigma-Aldrich, St. Louis, MO, US) was used to block cyclin D1 transcription at a dose of 5 μg/mL. The mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126-EtOH (Selleck Chemicals, Houston, TX, US) was used at a dose of 0.5 μmol/L. Epidermal growth factor (EGF) (PeproTech, Rocky Hill, NJ, US) was used at a dose of 50 ng/mL to stimulate the EGF receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway.

MPTP (1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) and MPP⁺ (1‐methyl‐4‐phenylpyridine) were purchased from Sigma‐Aldrich (Cat#M0896; D048). Human PDGF‐BB was obtained from Genscript (Cat#Z02892). STI‐571 [4‐[(4‐methylpiperazin‐1‐yl) methyl]‐N‐ [4‐methyl‐3‐ [(4‐pyridin‐3‐ylpyrimidin‐2‐yl) amino] phenyl] benzamide], LY294002 [2‐(4‐morpholinyl) ‐8‐phenyl‐1(4H)‐benzopyran‐4‐one], and U0126‐EtOH [1,4‐diamino‐2, 3‐dicyano‐1, 4‐bis (o‐aminophenylmercapto) butadiene] were purchased from Selleckchem (Cat#152459‐95‐5; 154447‐36‐6, and 1173097‐76‐1). Primary antibodies for phospho‐Akt (Cat#p2717), Akt (Cat#9272s), phospho‐ERK (Cat#4374s), ERK (Cat#9107s), phospho‐CREB (Cat#9198s), and CREB (Cat#9197s) were from Cell signaling technology (CST). Anti‐tyrosine hydroxylase (Cat#25859‐1‐AP) and anti‐β‐actin (Cat#60008) primary antibodies were from Proteintech Group. Anti‐mouse IgG (Cat#7076P2) and anti‐rabbit IgG (Cat#7074P2) secondary antibodies were from CST. Anti‐mouse NeuN (Cat#ab104224) were purchased from Abcam. Alexa Fluor 568‐conjugated goat anti‐rabbit secondary antibody (Cat#A11005) and Alexa Fluor 488‐conjugated goat anti‐mouse secondary antibody (Cat#A11001) were from Thermo Fisher Scientific.

DC_AC50, a CCS inhibitor, was provided by the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences. U0126-EtOH (catalog number: S1102) was purchased from Selleck. Antibody against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:1000 times dilution) (catalog number: 4370S), p44/42 MAPK (Erk1/2) (1:1000 times dilution) (catalog number: 4695S), phpspho-MEK1/2 (Ser217/221) (1:1000 times dilution) (catalog number: 9154S), MEK1/2 (1:1000 times dilution) (catalog number: 8727S), β-actin (1:1000 times dilution) (catalog number: 8457S), mouse IgG (1:3000 times dilution) (catalog number: 7076S), and rabbit IgG (1:3000 times dilution) (catalog number: 7074S) were from cell signaling technology. Anti-Superoxide Dismutase 4 (1:500 times dilution) (catalog number: ab167170) was from Abcam. Anti-Flag tag (1:1000 times dilution) (catalog number: 66008) was from proteintech. CCS shRNA was purchased from Open Biosystems, Huntsville, AL. The sequence of targeted CCS shRNA was as follows: 5′-CCGGCTGATTATTGATGAGGGAGAACTCGAGTTCTCCCTCATCAATA ATCAGTTTTTG-3′. Lipofectamine RNA iMAX was purchased from Invitrogen. The sequences of targeted CCS siRNA were as follows: sense: 5′-GUCUUGGUACACACCACUCUA-3′; Antisense: 5′-UAGAGUGGUGUGUACCAAGAC-3′.

The Library of Pharmacologically Active Compounds (Sigma-Aldrich) was purchased from the SickKids Proteomics, Analytics, Robotics & Chemical Biology Centre (SPARC BioCentre). The APExBIO DiscoveryProbe Kinase Inhibitor Library was a gift from Jim Dowling. The OICR Kinase Inhibitor Library was a gift from Rima Al-Awar and David Uehling at the Ontario Institute of Cancer Research. The GSK Published Kinase Inhibitor Set (PKIS) molecules was obtained from GlaxoSmithKline. MEK Inhibitors trametinib, pimasertib, U0126-EtOH and BI-847325 and EGFR inhibitor gefitinib were purchased for further testing from Selleck Chemicals.

Antibodies against PDGFRα (#5241), Caspase-3 (#9665), PARP (#9542), Caspase-9 (#9508), Bax (#2772), Bcl-2 (#2872), PI3K (#4257), AKT (#4691), phospho-AKT (#4060), mTOR (#2983), phospho-mTOR (#5536), p38-MAPK (#8690), phospho-p38MAPK (#4511), MEK (#9126), phospho-MEK (#9154), ERK1/2 (#4695), phospho-ERK1/2 (#4370), GAPDH (#2118) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody to LC3 (#AL221) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Antibody to Atg-5 (#ab108327) was obtained from Abcam (Cambridge, MA, USA). Z-VAD-FMK, chloroquine, 3-Methyladenine, U0126-EtOH were from Selleck Chemicals (Houston, TX, USA).

P5091, SB203580, SP600125, and U0126-EtOH were purchased form Selleck. HBX-19818 and GNE-6776 were purchased from MCE. CFSE, LPS, CCK-8 reagent, NMP, Tween-80 and PEG400 were purchased from Biosharp (Hefei, China). Clodronate liposomes were purchased from Biolegend. PD-1 antibody was purchased from Invitrogen.