Figure 4. Identification of the effector pathway downstream of mevalonate.

(a) Cardiomyocyte differentiation in EBs treated with DMSO (control) or 10 μM ATV, zaragozic acid (ZA), GGTI-2133 (GGTI) or FTI-277 (FTI) during days 3–6. Results were analysed as in Fig. 1c. (b) Real-time PCR of T expression in EBs treated with DMSO or 10 μM ATV, ZA, GGTI or FTI during days 3–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.001. *Zaragonic acid increased T and Lhx1 expression, which might be due to upregulation of HMGCR levels on day 3 and 4 (Supplementary Figure 3e). (c) Real-time PCR of T levels in EBs treated with DMSO or 10 μM ATV, with/without 12.5 μM farnesol (FOH), during days 3–5 and collected on day 5. Results were analysed as in Fig. 1b. (d) Representative 2D-PAGE images of farnesylated proteins detected with biotin-phosphine from ES cells and EBs that were left untreated (left) or treated with 10 μM FTI-277 (right) and analysed on day 4. Results are representative of three cultures. Black arrowheads, ~20 kDa farnesylated proteins; white arrowhead, EB-specific farnesylated protein. (e) Immunostaining to detect the localisation of Flag-Myc-tagged Lamin B1 (WT) or Lamin B1 (C-S mutant) in ES cells. Nuclei were visualised by Hoechst 33342 staining. Scale bars, 5 μm. (f) Real-time PCR of the indicated genes in the Lamin B1 WT, KO and REV EBs. EBs were collected at the indicated times and analysed as in Fig. 1b. *P < 0.05.