Fig. 4: IGF1 treatment of human embryos promotes ICM proliferation.

a Representative western blots for selected proteins related to MAPK/PI3K/AKT/mTOR signalling in mouse and human embryos. n = 6 human blastocysts at 6 days post fertilisation (dpf) per experiment, and 5 or 10 mouse blastocysts. Given the limited human material, after WB transfer the higher and lower molecular weight regions of the blot were cut and probed separately. The cut junction can be seen in the upper edge of pS6. Full-length western blots are shown in Supplementary Fig. 11. Data represents n = 5 replicate experiments. b Schematic of human embryo treatment schedule. c Immunofluorescence analysis for NANOG (EPI, green) and SOX17 (PE, red) with DAPI (blue) nuclear stain at 6.5 dpf in control embryos or embryos treated with 17 nM IGF1 from 2 dpf. Scale bar: 100 μm. d Immunofluorescence analysis for NANOG (green), KLF17 (red) and DAPI (blue) at 6.5 dpf following 17 nM IGF1 treatment from 2 dpf. Scale bar: 100 μm. e Quantification of total cell number (DAPI stained nuclei) and percentages of NANOG- or SOX17-expressing cells, and ICM (combined NANOG and SOX17 totals) in control or 17 nM IGF1-treated embryos. Data points represent the percentage per individual embryo; horizontal line indicates the mean. n = 5 control, 4 treated embryos. One-tailed t-test. *P < 0.05, **P < 0.01. f Immunofluorescence analysis for NANOG (red) and GATA6 (PE, green), with DAPI (blue) at 6.5 dpf in control embryos or embryos treated with 1 µg/ml FGF2 and 1 µg/ml heparin, or 100 ng/ml FGF2 from 2 dpf. Scale bar: 100 μm. g Immunofluorescence analysis for NANOG (green) and SOX17 (red) with DAPI (blue) nuclear stain at 6.5 dpf in control embryos or embryos treated with 100 ng/ml FGF2 from 5 dpf. h Quantification of total cell number (DAPI) and percentages of NANOG- or SOX17-expressing cells, and ICM in control embryos or embryos treated with 100 ng/ml FGF2 from 5 dpf. Data points represent the percentage per individual embryo; horizontal line indicates the mean. n = 10 control, 9 treated embryos. One-tailed t-test. *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.