Fig. 7: P-bodies are essential for D1-N3 complex to repress the translation of SOX4.

A Fluorescence observation of D1-RFP (magenta) and DDX6-GFP (cyan) in hESCs. Scale bar: 5 μm. Representative images from three independent experiments. B Immunofluorescence of N3-GFP (cyan) and DCP1A (magenta) in hESCs. Scale bar: 10 μm. Representative images from three independent experiments. C Immunofluorescence of DDX6 (yellow) and D1-RFP (magenta) and N3-GFP (Cyan) in hESCs. Scale bar: 2 μm. Representative images from three independent experiments. D Fluorescence observation of nls-MCP-RFP (magenta) and DDX6-GFP (cyan) in D1-N3-dOE hESCs with Dox. Scale bar: 2 μm. Representative images from three independent experiments. E Examination of DDX6-GFP (cyan) in hESCs without and with CHX treatment for 20 h. DAPI (gray) is counterstained to indicate nuclei. Scale bar: 20 μm. Representative images from three independent experiments. F Western blot showing the protein level of SOX4 for Dox-inducible expression of D1-N3 in the presence of CHX. Dox and CHX were added simultaneously. +Dox indicates over-expression of both D1 and N3. β-Actin serves as loading control. Representative images from three independent experiments. Source data are provided as a Source Data file. G Western blot showing the protein level of SOX4 in D1-N3 over-expressed hESCs in the absence or presence of CHX. Dox and CHX were added simultaneously. β-Actin serves as loading control. Representative images from three independent experiments. Source data are provided as a Source Data file. H Co-immunoprecipitation assay showing the physical interaction of D1 with N3, 4E-T, and CNOT1 with and without RNase treatment. Representative images from three independent experiments. Source data are provided as a Source Data file. I Schematic illustration of the Rluc-BoxB tethering reporter system. J Tethering assay showing the normalized Rluc activity in λN-RFP, N3, and λN-N3 groups. Renilla luciferase (Rluc) activity is normalized to firefly luciferase (Fluc) activity. Rluc-SOX4 3’UTR mRNA level is quantified by RT-qPCR and normalized to Fluc mRNA. The translation efficiency is calculated as the ratio of normalized Rluc activity to the normalized Rluc-SOX4 3’UTR mRNA level. Mean with SD from n = 3 experiments. Unpaired t test; two-tailed P value. Source data are provided as a Source Data file. K A proposed model showing that D1 and N3 coordinately suppress the translation of SOX4 via 4E-T in processing bodies for restricting the entry of germ cell lineage. See also Supplementary Fig. 7.