Comparison of endocannabinoid levels in mES cells and EBs at days 7 and 14, when the number of cells in each group is normalized to 10⁷ ( = 1e⁷).

<p>The groups depict the logarithms of each value. AEA, DHEA and EEA endocannabinoid levels were detected but were lower than the limit of quantitation (<0.05 ng/1e⁷ cells) for the number of cells analyzed.</p

Expression of CB1 and CB2 in Rosa26.6 (Panel A) and E14 (Panel B) ES cells.

<p>Cells were washed with PBS, and then RNA was isolated and analyzed by RT-PCR using specific primers for CB1, CB2, GAPDH and CXCR4. Panel C: RT-PCR analysis of the <i>in vitro</i> differentiation of Rosa26.6 ES cells, using specific primers for GAPDH, Flk-1, PECAM-1 and Sca-1. EBs: Embryoid bodies. ES cells: undifferentiated control ES cells. The following primers were used: GAPDH: 292 bp S 5′-CTCACTGGCATGGCCTTCCG-3′ AS 5′-ACCACCCTGTTGCTGTAGCC-3′ CB1: 430 bp S 5′-CGTGGGCAGCCTGTTCCTCA-3′ AS 5′-CATGCGGGCTTGGTCTGG-3′ CB2: 479 bp S 5′-CCGGAAAAGAGGATGGCAATGAAT-3′ AS 5′CTGCTGAGCGCCCTGGAGAAC-3′ PECAM-1: 260 bp S 5′-GTCATGGCCATGGTCGAGTA-3′ AS 5′-CTCCTCGGCATCTTGCTGAA-3′ Flk-1: 239 bp S 5′-CACCTGGCACTCTCCACCTTC-3′ AS 5′-GATTTCATCCCACTACCGAAAG-3′</p

The expression of CB1 and CB2 receptors in Rosa26.6 and E14 ES cells as analyzed by Western blot analysis.

<p>Cells were lysed in RIPA buffer and 100 mg of total cell lysates were analyzed by SDS-PAGE, followed by Western blotting with CB1 or CB2 specific antibodies (at a dilution of 1:500). The cell lines 293T and SH-SY5Y were used as negative and positive controls, respectively, for CB1 expression. Actin was used as a control for loading. ES cells: undifferentiated ES cells; EBs: Embryoid bodies at different time points as indicated.</p

Effects of Δ⁹-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.

<p>Rosa ES cells were either untreated (as control) or treated with Δ⁹-THC, control DMSO (0.01%), control methanol (0.01%), or with the inhibitors AM251 (for the CB1 receptor) or AM630 (for the CB2 receptor), as indicated. After 48 hours, the cells were analyzed for their viability by light microscopy. This is a representative experiment out of three experiments.</p

Effects of Δ⁹-THC on the differentiation of ES cells. Rosa ES cells were either untreated or treated with Δ⁹-THC in the presence or absence of cannabinoid inhibitors (AM630 and AM251), as indicated.

<p>After 14 days, the number of EBs was counted. Data represent the mean value of 3 independent experiments (mean±SD). * P values with asterisk (*, P<0.05) show significant differences from ES cells.</p