(A) Top panel: scheme of the plasmid constructs used for the generation of the L3 and L3R transgenic mouse lines. Lower panel: scheme of the cross between the transgenic mouse lines and isolation of pMEFs from E13.5 mouse embryos. (B) PCR and RT-PCR analysis of mouse embryos showing Leu3 expression specifically in embryos expressing L3 transgene. (C) RT-PCR analysis of pMEFs showing Leu3 expression in the cell lines isolated from L3 transgenic embryos and lack of GFP expression in L3R pMEFs when α-ΙΡΜ is not added in the culture. RT-PCR for GAPDH was used as a control. L3: Leu3 transgene/L3R: Leu3 reporter transgene/E1-5: embryo 1/M1-5: pMEF cell line 1 (scale bar 200μm).

Two cell stage embryos were harvested from F1 pregnant females and cultured for two days ex vivo in the presence of increasing concentrations of α-IPM (0, 5, 10 or 20 mM) until they reach the blastocyst stage. Thirty one embryos were used for every experimental group. (A) Bright field photographs of embryos cultured for two days under different concentrations of α-ΙΡΜ. In the presence of α-ΙΡΜ in concentrations of 5 and 10 mM, two cell stage embryos develop normally to blastocyst stage compared to the control. However, early mouse embryos cultured in the presence of 20 mM α-ΙΡΜ arrest at the 2-cell stage due to osmolarity changes. (scale bar 50μm) (B) Assessment of α-IPM toxicity in early mouse embryos ex vivo.

(A) Incorporation of ¹⁴C-α-IPM into fibroblast cells. 10T1/2 were grown to confluency of 80–90% before they were incubated in the presence of a constant amount α-IPM (2 mM) and various amounts of ¹⁴C-α-IPM (10–40 nM). After 48 hours, the cells were lysed in the presence of digitonin and the radioactivity incorporated into the cell was counted. The average percent of ¹⁴C-α-IPM incorporated in the cells for each α-IPM concentration is presented as the mean ± standard deviation of the mean (SD) (Table S1). (B) and (C). Ex vivo analysis of “Leu3p-α-ΙΡΜ” inducible gene expression system in pMEFs. (B) Detection of GFP expression with western blot in primary fibroblasts in the presence or absence of α-ΙΡΜ. β-actin expression was used as a positive control. (C) Immunohistochemical detection of GFP expression in primary fibroblasts derived from the mating of L3 and L3R transgenic lines. GFP expression is detected only upon α-ΙΡΜ addition in the double transgenic fibroblasts. Results from GFP immunoreactivity analysis are in accordance with the results obtained from western blot. (D) Kinetics of α-ΙΡΜ. Titration of [α-ΙΡΜ] for maximum inducibility in primary mouse fibroblasts (pMEFs). WT, L3R and double transgenic pMEFS were cultured in the presence of increasing concentrations of α-ΙΡΜ (0, 0.078, 0.156, 0.312, 0.625., 1.25, 2.5, 5, 10, 15, 20 Mm) and induced GFP was quantitated. Following data analysis performed using the GraphPad PRISM 5 software (GraphPad, Inc., USA), the EC₅₀ was calculated to be 0.8837 mM. The data are derived from three independent experiments for each experimental group (WT, L3R, L3/L3R) and for each different concentration of the inducer (0, 0.078, 0.156, 0.312, 0.625., 1.25, 2.5, 5, 10, 15, 20 Mm) and the absolute values are presented (Table S2) as the mean ± standard deviation of the mean (SD). (E) α-ΙΡΜ “ON” kinetics. Double transgenic pMEFS were cultured in the presence of 5 and 20 mΜ α-ΙΡΜ for different time points. The time required for 50% of inducible GFP expression is t₅₀”ON” equal to 49±0.9 min after 5 mM α-IPM addition and to 43+3 min after 20 mM α-ΙΡΜ addition. (F) α-ΙΡΜ “OFF” kinetics. Double transgenic pMEFs were cultured in the presence of 5 and 20 mM α-ΙΡΜ for 24 hrs, then α-ΙΡΜ was removed from the media and cells were left in culture for a period up to 48 hrs. After α-IPM removal from the media, the time required for 50% reduction of GFP expression is t₅₀OFF⁵ equal to 3.64±0.94 h, when the initial [α-IPM] concentration was 5 mM and t₅₀OFF²⁰ equal to 2.18±0.43 h, when the initial [α-IPM] concentration was 20 mM (scale bar: 50 μm). The data are derived from three independent experiments for each experimental group.