Bgj398 s2183

6-well (3516), 96-well tissue culture (3596), 96-well round bottom (3799), T75 (430641) flasks, and 35 um filter cap FACS tubes (352235) were from Corning. DMEM (11960), Penicillin-Streptomycin (15070, 5000 U/mL), Glutamax (100X, 35050-061), Sodium Pyruvate (11360, 100 mM), and 0.25% trypsin/1 mM EDTA (25200) were from Invitrogen. Fetal Bovine Serum (SH30396.03) was from Hyclone. AO/PI (F23001) and Luna (L12002) slides were from Logos Biosystems. CD44-APC (clone G44-26; #559942) was from BD Biosciences. DMSO (D2650), resazurin (R7017), and DAPI (D8417) were from Sigma. EREG ELISA (SEB945HU) was from Cedarlane. RNeasy Mini Kit (74106) was from Qiagen. RNA 6000 Pico and Nano Kits were from Agilent. Ovation Pico WTA System Kit was from NuGEN. Human Gene ST 2.1 microarrays were from Affymetrix. Clinical-grade cisplatin (Teva Pharmaceuticals; 1mg/mL) was obtained from University of Michigan Hospital Pharmacy. BGJ398 (S2183) was from Selleckchem and dissolved in DMSO to 1.78 mM. DEAB (01705) and Aldefluor Assay Buffer (01702) were from StemCell Technologies. Aldefluor reagent, BAAA-DA, was synthesized by the University of Michigan Vahlteich Medicinal Chemistry Core.

MCF-7 cells were purchased from the American Type Culture Collection (ATCC) and MFM223 cells from Sigma-Aldrich. MDA-MB-231(SA) cells were a gift from Dr. Guise (University of Texas, USA). MDA-MB-231(SA) is a variant of MDA-MB-231 that effectively forms bone metastases, but does not otherwise markedly differ from the parental cell line [22 (link), 23 (link)]. MCF-7 cells were grown in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (iFBS, Gibco), GlutaMAX (Gibco), estrogen (10^–9 M) and insulin (4 μg/ml). MDA-MB-231(SA) cells were grown in 10% iFBS/DMEM (Sigma-Aldrich) supplemented with GlutaMAX and non-essential amino acids (Gibco). MFM223 cells were grown in 10% iFBS/DMEM. The FGFRis TKI258 (S2769), BGJ398 (S2183) and AZD4547 (S2801) were purchased from Selleck Chemicals as DMSO stocks (IC50 values are listed in Supplementary Table S1).

Antibodies were purchased from: FGFR3 (B-9), FGFR3 (P18) from Santa Cruz Biotechnology (Dallas, TX, USA); FGFR3 (OAAB11172) from Aviva Systems Biology (San Diego, CA, USA); phospho-p44/42 MAPK (ERK 1/2; T202/Y204; D13.14.4E), p44/42 MAPK (ERK 1/2, 9102) from Cell Signaling Technology (Danvers, MA, USA); EEA1 (610456) from BD Biosciences (San Jose, CA, USA); Alexa Fluor 488 donkey anti-goat (A11055), Alexa Fluor 594 donkey anti-goat (A11058), Alexa Fluor 488 donkey anti-mouse (A21202) from Invitrogen (Carlsbad, CA, USA); horseradish peroxidase (HRP) anti-mouse, HRP anti-rabbit, and Enhanced Chemiluminence (ECL) reagents were from GE Healthcare (Little Chalfont, UK). Geneticin (G418) was from Gibco (Waltham, MA, USA), and Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). PNGase F was purchased from NEB (P0704S) and Pierce Protein A/G Magnetic Beads (88802) were purchased from Thermo Fisher (Waltham, MA, USA). Inhibitors BGJ398 (S2183) and Trametinib (S2673) were purchased from Selleckchem (Houston, TX, USA).