Fibroblast growth factor bfgf

U87MG-Luc2 (U87), primary E2 and G7 glioma cell lines were used in this study. E2 and G7 cells were derived from freshly resected human GBM specimens as previously described [49 (link)]. U87 cells were cultured in Hyclone MEM/EBSS (Fischer Scientific), 10% fetal calf serum, 1% L-Glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen) and 1% sodium pyruvate (Invitrogen). E2 and G7 cells were cultured in MEMα (Gibco) supplemented with 5% L-Glutamine. The U87 cell identity was confirmed using the short tandem repeat (STR) analysis (Identicell, Denmark).
For western blotting and flow cytometry both cell lines were cultured in stem cell enriching conditions in Advanced DMEM F12 medium (Gibco) supplemented with 1% B27 (Invitrogen), 0.5% N2 (Invitrogen), 4 μg/ml heparin, 20 ng/ml fibroblast growth factor (bFGF, Sigma), 20 ng/ml epidermal growth factor (EGF, Sigma) and 1% L-Glutamine. Stem cell enriched cultures were grown on Matrigel (BD Biosciences) coated flasks (1:50 dilution) in serum free media.

The lung cancer cell lines HCC827 and A549 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line H520 was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and was authenticated through short tandem repeat profiling by BCRC; it was free of Mycoplasma. HCC827 and A549 were used for tumorsphere formation and Western blotting, and they were reauthenticated through short tandem repeat profiling (Applied Biosystems, Massachusetts, USA). The HCC827 and H520 cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. A549 was cultured in Dulbecco’s modified Eagle medium (DMEM) with the same additives. For tumorsphere formation, the cells were cultured in low-attached 6-well plates with serum-free medium containing B27 (Invitrogen, Massachusetts, USA), 20 ng/mL of EGF (Sigma, Missouri, USA), 20 ng/mL of fibroblast growth factor (bFGF, Sigma), 5 μg/mL of bovine insulin (Sigma), and 4 μg/mL of heparin (Sigma) [24 (link)]. All cells were incubated at 37°C and 5% of CO₂. Cancer-initiating and early progenitor cells survived and proliferated, but differentiated cells died [25 (link)]. The cells were observed using an inverted microscope.