DOI: 10.5061/dryad.kkwh70sb6
title: Eif3d safeguards the homeostasis of Key signaling pathways in Human primed pluripotency

description: Although pluripotent stem cell (PSC) properties, such as differentiation
 and infinite proliferation, have been well documented within the
 frameworks of transcription factor networks, epigenomes, and signal
 transduction, they remain unclear and fragmented. Directing attention
 toward translational regulation as a bridge between these events can yield
 additional insights into previously unexplained mechanisms. Our functional
 CRISPR interference screen-based approach revealed that EIF3D, a
 translation initiation factor, is crucial for maintaining primed
 pluripotency. Loss of EIF3D disrupted the balance of
 pluripotency-associated signaling pathways, thereby compromising primed
 pluripotency. Moreover, EIF3D ensured robust proliferation by controlling
 the translation of various p53 regulators, which maintain low p53 activity
 in the undifferentiated state. In this way, EIF3D-mediated translation
 contributes to tuning the homeostasis of the primed pluripotency networks,
 ensuring the maintenance of an undifferentiated state with high
 proliferative potential. This study provides further insights into the
 translation network in maintaining pluripotency.
description: These are raw data, uncropped images, numerical data, and
 statistical results from the manuscript "EIF3D Safeguards the
 Homeostasis of Key Signaling Pathways in Human Primed Pluripotency"
 by Okubo et al.
description: # Eif3d safeguards the homeostasis of Key signaling pathways in Human
 primed pluripotency
 [https://doi.org/10.5061/dryad.kkwh70sb6](https://doi.org/10.5061/dryad.kkwh70sb6) This data set contains the original data used in the paper "Eif3d safeguards the homeostasis of key signaling pathways in Human primed pluripotency" published in Science Advances. The .fcs files are the raw data for flow cytometry analysis, the .tif files are the raw image data for immune-stained cells, the .pdf files are the raw data for Western blotting, and the .xls file contains the numerical data and statistical test results used in the paper. ## Description of the dataset ### 20221114\_3D1-1\_control.fcs * The flow cytometry data of EIF3D knockdown human iPS cel clone 1-1 stained for DNA. ### 20221114\_3D1-1.fcs * The flow cytometry data of EIF3D knockdown human iPS cell clone 1-1 stained for EdU (5-ethynyl-2�f-deoxyuridine) and DNA. ### 20221114\_3D1-1(EdU-).fcs * The flow cytometry data of EIF3D knockdown human iPS cell clone 1-1 without EdU incorporation stained for EdU and DNA. ### 20221114\_3D1-4.fcs * The flow cytometry data of EIF3D knockdown human iPS cell clone 1-4 stained for EdU and DNA. ### 20221114\_3D4-4.fcs * The flow cytometry data of EIF3D knockdown human iPS cell clone 4-4 stained for EdU and DNA. ### 20221114\_C1\_control.fcs * The flow cytometry data of Control human iPS cel clone 1 stained for DNA. ### 20221114\_C1.fcs * The flow cytometry data of Control human iPS cel clone 1 stained for EdU and DNA. ### 20221114\_C1(EdU-).fcs * The flow cytometry data of Control human iPS cel clone 1 without EdU incorporation stained for EdU and DNA. ### 20221114\_C3.fcs * The flow cytometry data of Control human iPS cel clone 3 stained for EdU and DNA. ### 20221114\_C3.fcs * The flow cytometry data of Control human iPS cel clone 3 stained for EdU and DNA. ### Control\_EN\_Hoechst.tif * The image of endoderm differentiated Control human iPS cell line 1 stained for DNA. Related to the files Control_EN_PH.tif and Control_EN_SOX17.tif. ### Control\_EN\_PH.tif * The phase contrast image of endoderm differentiated Control human iPS cell line 1. Related to the files Control_EN_Hoechst.tif and Control_EN_SOX17.tif. ### Control\_EN\_SOX17.tif * The image of endoderm differentiated Control human iPS cell line 1 stained for SOX17. Related to the files Control_EN_Hoechst.tif and Control_EN_PH.tif. ### Control\_ME\_HAND1.tif * The image of mesoderm differentiated Control human iPS cell line 1 stained for HAND1. Related to the files Control_ME_Hoechst.tif and Control_ME_PH.tif. ### Control\_ME\_Hoechst.tif * The image of mesoderm differentiated Control human iPS cell line 1 stained for DNA. Related to the files Control_ME_HAND1.tif and Control_ME_PH.tif. ### Control\_ME\_PH.tif * The phase contrast image of mesoderm differentiated Control human iPS cell line 1. Related to the files Control_ME_HAND1.tif and Control_ME_Hoechst.tif. ### Control\_NANOG\_DNA.tif * The image of Control human iPS cell line 1 stained for DNA. Related to the file Control_NANOG_ICC.tif. ### Control\_NANOG\_ICC.tif * The image of Control human iPS cell line 1 stained for NANOG. Related to the file Control_NANOG_DNA.tif. ### Control\_NE\_Hoechst.tif * The image of neuroectoderm differentiated Control human iPS cell line 1 stained for DNA. Related to the files Control_NE_PAX6.tif and Control_NE_PH.tif. ### Control\_NE\_PAX6.tif * The image of neuroectoderm differentiated Control human iPS cell line 1 stained for PAX6. Related to the files Control_NE_Hoechst.tif and Control_NE_PH.tif. ### Control\_NE\_PH.tif * The phase contrast image of neuroectoderm differentiated Control human iPS cell line 1. Related to the files Control_NE_Hoechst.tif and Control_NE_PAX6.tif. ### Control\_OCT4\_DNA.tif * The image of Control human iPS cell line 1 stained for DNA. Related to the file Control_OCT4_ICC.tif. ### Control\_OCT4\_ICC.tif * The image of Control human iPS cell line 1 stained for OCT4. Related to the file Control_OCT4_DNA.tif. ### Control\_p21\_DNA.tif * The image of Control human iPS cell line 1 stained for p21. Related to the file Control_p21_ICC.tif. ### Control\_p21\_ICC.tif * The image of Control human iPS cell line 1 stained for p21. Related to the file Control_p21_DNA.tif. ### Control\_p53\_DNA.tif * The image of Control human iPS cell line 1 stained for p53. Related to the file Control_p53_ICC.tif. ### Control\_p53\_ICC.tif * The image of Control human iPS cell line 1 stained for p53. Related to the file Control_p53_DNA.tif. ### Control\_PSC\_Hoechst.tif * The image of undifferentiated Control human iPS cell line 1 stained for DNA. Related to the files Control\_ PSC_NANOG.tif and Control_PSC_PH.tif. ### Control\_PSC\_NANOG.tif * The image of undifferentiated Control human iPS cell line 1 stained for NANOG. Related to the files Control_PSC_Hoechst.tif and Control_PSC_PH.tif. ### Control\_PSC\_PH.tif * The phase contrast image of undifferentiated Control human iPS cell line 1. Related to the files Control_PSC_Hoechst.tif and Control_PSC_NANOG.tif ### Control\_SOX2\_DNA.tif * The image of Control human iPS cell line 1 stained for DNA. Related to the file Control_SOX2_ICC.tif. ### Control\_SOX2\_ICC.tif * The image of Control human iPS cell line 1 stained for SOX2. Related to the file Control_SOX2_DNA.tif. ### sgEIF3D\_EN\_Hoechst.tif * The image of endoderm differentiated EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the files sgEIF3D_EN_PH.tif and sgEIF3D_EN_SOX17.tif. ### sgEIF3D\_EN\_PH.tif * The phase contrast image of endoderm differentiated EIF3D knockdown human iPS cell line 1-1. Related to the files sgEIF3D_EN_Hoechst.tif and sgEIF3D_EN_SOX17.tif. ### sgEIF3D\_EN\_SOX17.tif * The image of endoderm differentiated EIF3D knockdown human iPS cell line 1-1 stained for SOX17. Related to the files sgEIF3D_EN_Hoechst.tif and sgEIF3D_EN_PH.tif. ### sgEIF3D\_ME\_HAND1.tif * The image of mesoderm differentiated EIF3D knockdown human iPS cell line 1-1 stained for HAND1. Related to the files sgEIF3D_ME_Hoechst.tif and sgEIF3D_ME_PH.tif. ### sgEIF3D\_ME\_Hoechst.tif * The image of mesoderm differentiated EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the files sgEIF3D_ME_HAND1.tif and sgEIF3D_ME_PH.tif. ### sgEIF3D\_ME\_PH.tif * The phase contrast image of mesoderm differentiated EIF3D knockdown human iPS cell line 1-1. Related to the files sgEIF3D_ME_HAND1.tif and sgEIF3D_ME_Hoechst.tif. ### sgEIF3D\_NANOG\_DNA.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the file sgEIF3D_NANOG_ICC.tif. ### sgEIF3D\_NANOG\_ICC.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for NANOG. Related to the file sgEIF3D_NANOG_DNA.tif. ### sgEIF3D\_NE\_Hoechst.tif * The image of neuroectoderm differentiated EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the files sgEIF3D_NE_PAX6.tif and sgEIF3D_NE_PH.tif. ### sgEIF3D\_NE\_PAX6.tif * The image of neuroectoderm differentiated EIF3D knockdown human iPS cell line 1-1 stained for PAX6. Related to the files sgEIF3D_NE_Hoechst.tif and sgEIF3D_NE_PH.tif. ### sgEIF3D\_NE\_PH.tif * The phase contrast image of neuroectoderm differentiated EIF3D knockdown human iPS cell line 1-1. Related to the files sgEIF3D_NE_Hoechst.tif and sgEIF3D_NE_PAX6.tif. ### sgEIF3D\_OCT4\_DNA.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the file sgEIF3D_OCT4_ICC.tif. ### sgEIF3D\_OCT4\_ICC.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for OCT4. Related to the file sgEIF3D_OCT4_DNA.tif. ### sgEIF3D\_p21\_DNA.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for p21. Related to the file sgEIF3D_p21_ICC.tif. ### sgEIF3D\_p21\_ICC.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for p21. Related to the file sgEIF3D_p21_DNA.tif. ### sgEIF3D\_p53\_DNA.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for p53. Related to the file sgEIF3D_p53_ICC.tif. ### sgEIF3D\_p53\_ICC.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for p53. Related to the file sgEIF3D_p53_DNA.tif. ### sgEIF3D\_PSC\_Hoechst.tif * The image of undifferentiated EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the files sgEIF3D_PSC_NANOG.tif and sgEIF3D_PSC_PH.tif. ### sgEIF3D\_PSC\_NANOG.tif * The image of undifferentiated EIF3D knockdown human iPS cell line 1-1 stained for NANOG. Related to the files sgEIF3D_PSC_Hoechst.tif and sgEIF3D_PSC_PH.tif. ### sgEIF3D\_PSC\_PH.tif * The phase contrast image of undifferentiated EIF3D knockdown human iPS cell line 1-1. Related to the files sgEIF3D_PSC_Hoechst.tif and sgEIF3D_PSC_NANOG.tif. ### sgEIF3D\_SOX2\_DNA.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for DNA. Related to the file sgEIF3D_SOX2_ICC.tif. ### sgEIF3D\_SOX2\_ICC.tif * The image of EIF3D knockdown human iPS cell line 1-1 stained for SOX2. Related to the file sgEIF3D_SOX2_DNA.tif. ### Uncropped\_Wes-1.pdf * File 1 containing unmodified western blot images. ### Uncropped\_Wes-2.pdf * File 2 containing unmodified western blot images. ### Uncropped\_Wes-3.pdf * File 3 containing unmodified western blot images. ### Uncropped\_Wes-4.pdf * File 4 containing unmodified western blot images. ### Uncropped\_Wes-5.pdf * File 5 containing unmodified western blot images. ### Uncropped\_Wes-6.pdf * File 6 containing unmodified western blot images. ### Numerical\_data\_09192024.xlsx The Excel file containing all numerical data and statistical analysis results used in the paper. * Fig. 1E: Cell number of control and EIF3D knockdown iPS cells on day 3 and 5. Three clones of each in duplicate were used for counting. * Fig. 1F: The percentages of G1, S and G2/M phases in the cell cycle of control and EIF3D knockdown iPS cells on days 3 and 5. Three clones of each were used for the analysis. * Fig. 1G&S1A: Relative expression of EIF3D, POU5F1, SOX2, NANOG, TP53, CDKN1A, ARF, INK4A, FAS, and MDM2 in control and EIF3D knockdown iPS cells from day 0 to day 5. Three clones each were used for analysis. Quantitative PCR was performed in triplicate for each sample. * Fig. 2E: Relative expression of FOXA2, FOXF1, SOX1 and NANOG in endoderm (EN), mesoderm (ME) and neuroectoderm (NE) differentiated and undifferentiated (PSC) control and EIF3D knockdown iPS cells. Three clones each were used for differentiation. Quantitative PCR was performed in triplicate for each sample. * Fig. 3H: Number of differentially expressed genes between control and EIF3D knockdown iPS cells on days 3 and 5 in the primed state, and on day 5 in the naive state. Three clones each were used for the assay. * Fig. 3I: Relative expression of EIF3D, POU5F1, SOX2 and NANOG in control and EIF3D knockdown iPS cells on day 7 in the naive state. Three clones each were used for the analysis. Quantitative PCR was performed in triplicate for each sample. * Fig. 4A: The intensity of puromycin incorporation in control and EIF3D knockdown iPS cells on day 3 was analyzed by Western blotting. Three clones each were used for the analysis. * Fig. 4C: Area under curve quantification for specified ribosomal fractions (60S/40S, 80S/40S and PS/40S) in control and EIF3D knockdown iPS cells on day 3. Three clones each were used for the analysis. * Fig. 5C: Luciferase reporter assays showing the effects of RBBP6) and GAPDH 5' untranslated regions on translation in control and EIF3D knockdown iPS cells. Three clones of each were used in duplicate for analysis. * Fig. 5E: Cell number of control, EIF3D knockdown, and RBBP6 knockdown iPS cells on day 5. * Fig. 5F: Relative expression of EIF3D, RBBP6, POU5F1, SOX2, NANOG, TP53, CDKN1A, and MDM2 in control, EIF3D knockdown and RBBP6 knockdown iPS cells on day 5. Quantitative PCR was performed in triplicate for each sample. * Fig. 6C: Cell number of iPS cells treated with DMSO, 5F, 6F, Nutlin-3a (N3a), N3a+5F, and N3a+6F for 5 days. Counts were performed in triplicate. * Fig. S2C: Nuclear area of control and EIF3D knockdown iPS cells on day 5. * Fig. S3A: Relative expression of EIF3D in 293T/17, HepG2 and human dermal fibroblasts expressing scramble shRNA, EIF3D shRNA-1 and EIF3D shRNA-2. Quantitative PCR was performed in triplicate for each sample. * Fig. S3C: Number of cells of 293T/17, HepG2 and human dermal fibroblasts expressing scramble shRNA, EIF3D shRNA-1 and EIF3D shRNA-2 on days 3 and 5. Counts were performed in triplicate. * Fig. S4B: Relative expression of EIF3D, POU5F1, SOX2, NANOG, TP53, and CDKN1A in control iPS cells, EIF3D knockdown iPS cells, and EIF3D knockdown iPS cells expressing wild-type (WT), S527D/S528D mutant (DD), or S527N/S528N mutant (NN) EIF3D cDNA on day 5. Quantitative PCR was performed in triplicate for each sample. * Fig. S4D: Cell number of control iPS cells, EIF3D knockdown iPS cells, and EIF3D knockdown iPS cells expressing wild-type (WT), S527D/S528D mutant (DD), or S527N/S528N mutant (NN) EIF3D cDNA on day 5. Counts were performed in triplicate. * Fig. S5B: Relative expression of EIF3D, POU5F1, SOX2, and endogenous NANOG in control iPS cells and EIF3D knockdown iPS cells with or without NANOG cDNA expression on day 5. Quantitative PCR was performed in triplicate for each sample. * Fig. S5D: Cell number of control iPS cells and EIF3D knockdown iPS cells with or without NANOG cDNA expression on day 5. * Fig. S5F: Relative expression of EIF3D, TP53, POU5F1, SOX2, NANOG, CDKN1A, and MDM2 in control iPS cells, EIF3D knockdown iPS cells, TP53 knockdown iPS cells, and TP53/EIF3D double knockdown iPS cells on day 5. Quantitative PCR was performed in triplicate for each sample. * Fig. S5H: Cell number of control iPS cells, EIF3D knockdown iPS cells, TP53 knockdown iPS cells, and TP53/EIF3D double knockdown iPS cells on day 5. * Fig. S6A: Relative expression of POU5F1, SOX2 and NANOG in control, POU5F1 knockdown, SOX2 knockdown, and NANOG knockdown iPS cells on day 5. Quantitative PCR was performed in triplicate for each sample. * Fig. S6D: Relative expression of POU5F1, SOX1, FOXA2, HAND1, FOXF1, PAX6, and SOX1 in endoderm (EN), mesoderm (ME) and neuroectoderm (NE) differentiated and undifferentiated (1B4) iPS cells. Quantitative PCR was performed in duplicate for each sample. Describe relationships between data files, missing data codes, other abbreviations used. Be as descriptive as possible.