Human bfgf 2

Established pancreatic cancer tumorspheres [10] (link) were maintained in sphere media as described previously [19] (link), [20] (link) with modifications [50% NeuralBasal (Invitrogen, Carlsbad, CA), 1% N2 Supplement (Invitrogen, Carlsbad, CA), 2% B27 supplement (Invitrogen, Carlsbad, CA), 1% Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA), 10 ng/mL BMP4 (Sigma), 10 ng/mL LIF (Sigma), 20 ng/mL human bFGF-2 (Invitrogen, Carlsbad, CA), all in 1∶1 DMEM/F12 (Invitrogen, Carlsbad, CA)]. Tumorspheres were passaged every 6 days. For passaging, tumorspheres were dissociated with 0.05% trypsin for 2–5 min and then immediately washed twice with 40 mL PBS. Cells were then passed through a 40 μm nylon mesh cell strainer, counted and plated in fresh sphere medium in Costar ultra low-attachment 6 well plates (Corning, Lowell, MA).

Single cells were suspended in tumorsphere culture media containing 1% N2 supplement, 2% B27 supplement, 1% antibiotic-antimycotic, 20 ng/mL epidermal growth factor (Gibco, Carlsbad, CA), 20 ng/mL human bFGF-2 (Invitrogen), 10 ng/mL BMP4 (Sigma-Aldrich, St. Louis, MO), 10 ng/mL LIF (Sigma-Aldrich) and plated in six-well Ultra-Low Attachment Plates (Corning, Corning, NY).
siRNA transfection siRNA were purchased from Dharmacon (Lafayette, CO) and were transfected at 25 nM using Lipofectamine RNAiMAX Reagent (Invitrogen). siRNA sequences can be found in the Supplementary Material and methods.