(a) Representative images of EBs that were untreated (control) or treated with 10 μM ATV (n > 100/group) during days 1–6. EBs were stained with anti-β-tubulin III Ab to label neurites on day 12 and visualised by microscopy. Scale bars, 1 mm. (b) Real-time PCR of the cardiomyocyte marker Mhy7 and neural marker Map2 in EBs in (a). mRNA levels were normalised to Gapdh expression. Results are means ± SD (n = 3). (c) Percentage of foci with a ‘heartbeat’ (indicating cardiomyocyte differentiation) in EB cultures that were treated with 10 μM ATV for the indicated periods and evaluated on day 10. Results are means ± SD (n = 3). (d) Western blotting of EBs in (c) to detect protein expression on day 10. GAPDH, loading control. Results are representative of three trials examining at least three cultures/group. (e) Cardiomyocyte differentiation in EBs treated with 25, 100, 250 μM or 1 mM MVA in addition to 10 μM ATV during days 3–6. Results were analysed as in (c). (f) Representative images of the uteri of mice treated with DMSO, ATV or ATV plus MVA at E5.5 and examined at E10.5. Scale bars, 10 mm. (g) Survival ratios for embryos of mice in (f). (h) Whole-mount in situ hybridisation to detect the cardiac marker cMhc2 in 24 h post-fertilisation (hpf) zebrafish embryos that were treated with DMSO or treated with 30 or 90 μM ATV from one-cell stage. Scale bars, 200 μm. (i,j) Real-time PCR of cMhc2 and nestin in the zebrafish embryos in (h) at 24 hpf. mRNA levels were normalised to actin expression. Results are means ± SD (n = 3). All experiments were carried out in triplicate. *P = 0.052, **P < 0.05.