Enzastaurin LY317615 1% Triton-X. Cells were then washed three times with PBS and incubated with an appropriate fluorescein isothiocyanate-labeled secondary antibody (Jackson ImmunoResearch). Specimens were analyzed on an Olympus fluorescence microscope. For teratomas from ES cells, ES-Hepa hybrid cells and Hepa1�C6 tumors, ~1 �� 106 cells of each clone, were subcutaneously injected into the inguinal region of immunodeficient nude mice. The mice were monitored for tumor growth for 6 weeks. Tumor volume (V) was calculated in all experiments according to V = ab2/2, where a and b designate the long and short diameters of the tumor, respectively. At 4 weeks, teratomas or tumors were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.
In Vitro Differentiation For embryonic body formation, ES and ES-Hepa hybrid cells were harvested by treatment with 0.25% trypsin. The cells were cultured in bacterial-grade Petri dishes in Dulbecco’s modified Eagle’s medium/F-12 containing 20% knock-out serum replacement (Invitrogen), 2 mm l-glutamine, 1 �� 10?4 nonessential amino acids, 1 �� 10?4 M2-mercaptoethanol (Invitrogen), and 1% penicillin/streptomycin. The medium was changed every other day. Embryonic bodies were harvested at days 3, 5, 7, and 9 for gene expression analysis. For induction of ES and ES-Hepa hybrid cells to differentiate by monolayer culture, ES and ES-Hepa hybrid cells were dispersed into a single cell suspension with 0.25% trypsin, and plated the cells on the 0.2% gelatin-coated cell culture dish.
The cells were differentiated in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen) containing 15% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin/glutamine and changed the medium every 1 or 2 days. RNA Extraction, cDNA Synthesis, Quantitative PCR, and RT-PCR For real-time quantification of gene expression, cells were lysed with buffer RLT (Qiagen 79216), and Cilengitide RNA was extracted with RNeasy microcolumns (RNeasy lysis buffer, Qiagen), according to the manufacturer’s instructions. Random hexamer-primed first-strand cDNA was prepared with the SuperScript III reverse transcriptase kit (Invitrogen, cat. no. 18080-051) according to the manufacturer’s instructions. The raw quantification data for the transcripts were normalized to the endogenous GAPDH gene (ABI PRISM 7700 Sequence Detection System User Bulletin 2, Applied Biosystems). PCR primers are listed as follows.