D luciferin bioluminescent substrate

MT, D-luciferin bioluminescent substrate, alamarBlue Cell Viability Reagent, and Dead Cell Apoptosis Kit were purchased from ThermoFisher Scientific (Waltham, MA). MT stock solution at 1 M was prepared by dissolving it in DMSO in ultrasonic sonication bath. For in vivo assays, MT was dissolved in 20% glycofurol solutions to reduce the potential toxicity to mice. The full-length FGF19 gene was amplified from HN30 cells and cloned into pcDNA3.1 (+) expression vector (Invitrogen, USA) as previously described [7 (link)]. FGFR4-speficic inhibitor H3B-6527 was purchased from Selleckchem (Houston, TX). Antibodies that recognize ATF4, PERK, p-PERK, IRE-1, eIF2α, p-eIF2α, E-cadherin, Vimentin, Puma, Noxa, Bcl-2, and Bax were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against FGF19, FGFR4, p-FGFR4 and β-Actin were obtained from Abcam (Cambridge, MA). Western blotting, RT-PCR, wound healing assays, Annexin V FITC/PI double staining, and cell viability assays were carried out as we described previously [1 (link), 7 (link), 25 (link), 27 (link), 29 (link)]. Proteins were visualized using a SuperSignal West Pico chemiluminescence kit (Pierce) and quantified with ImageJ Fiji (version 1.2; WS Rasband, National Institute of Health, Bethesda, MD).

Antibodies that recognize ERK1/2, p-ERK1/2, RAF1, p-RAF1, AKT, p-AKT, MEK1/2, p-MEK1/2, GAPDH, COXIV, VDAC1, cleaved caspase-3, calnexin, PCNA, E-cadherin and vimentin were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against β-actin, Myc, and Flag were obtained from Sigma-Aldrich (St Louis, MO). Antibodies that recognize cyclin D1 and CDK4 were purchased from Santa Cruz Biotechnology (Dallas, TX). ATAD3A antibody was purchased from Novus Biologicals (Abingdon, UK). CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was obtained from Promega (Madison, WI). Texas-red phalloidin, D-luciferin bioluminescent substrate, and alamarBlue Cell Viability Reagent were purchased from ThermoFisher Scientific (Waltham, MA). The RAS inhibitor salirasib and the ERK1/2 inhibitor SCH772984 were obtained from SelleckChem (Houston, TX), and 4-nitroquinoline-1-oxide (4NQO) was purchased from Sigma-Aldrich (St. Louis, MO).