DEV041160 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -
• Supplemental Figure S1 -

  Fig. S1. Lentiviral Nanog knockdown and rescue experiments in ESCs. (A) Effect of Nanog knockdown on clonal expansion of ESCs. NRi- and NC-shRNA ESCs were reseeded onto a feeder layer at single-cell density after Cre induction. (B) Comparison of the number of GFP-negative colonies. *P<0.01. Error bars, s.e.m. (C) Rescue of Cre-induced NRi-ESCs by the silent-mutation form of Nanog (Mut-Nanog). Nanog expression in the NRi+Mut-Nanog was detected by immunocytochemistry even after the wild-type Nanog was knocked down, as shown by the substantial reduction in GFP expression. (D) Maintenance of the undifferentiated state by rescue with Mut-Nanog. Cre-induced NRi+Mut-Nanog ESCs sustain an undifferentiated state in morphology even after knockdown of wild-type Nanog, as shown by the substantial reduction in GFP expression.

• Supplemental Figure S2 -

  Fig. S2. Generation of NRi transgenic mice. (A) Integration site of the lentiviral Nanog knockdown vector of NRi-shRNA in the R1 ESC genome. (B) Germline transmission of the lentiviral Nanog knockdown vector. Germline-transmitted progeny are GFP positive (green). (C) Transcription of NRi-shRNA in E12.5 ER-Cre/NRi-Tg embryos assessed by northern blotting. PC, positive control RNA from AdCre-infected NRi-ESCs.

• Supplemental Figure S3 -

  Fig. S3. Reduction in the number of PGCs in E11.5 ER-Cre/NRi-Tg embryos. (A) Reduction in the number of alkaline phosphatase-positive PGCs in E11.5 ER-Cre/NRi-Tg genital ridges. Tamoxifen (TM) was injected into 7.5 dpc pregnant mice. (B) Expression of Nanog in SSEA1⁺ PGCs of E11.5 ER-Cre/NRi-Tg embryos. Nanog expression is repressed in SSEA1⁺ PGCs. (C) The proportion of Nanog⁺ PGCs in SSEA1⁺ PGCs in E11.5 ER-Cre/NRi-Tg embryos. **P<0.05. Error bars, s.e.m.

• Supplemental Figure S4 -

  Fig. S4. Immunostaining for Oct4 and phosphorylated histone H3 in Nanog knockdown PGCs in vitro. (A) Expression of Oct4 in Nanog⁻ ER-Cre/NRi-Tg PGCs cultured for 12 and 36 hours in the presence of tamoxifen (TM). Circles indicate Nanog⁻ PGCs. (B) Immunohistochemistry of PGCs for phosphorylated histone H3 after 36 hours of culture with TM. (C) The proportion of phosphorylated histone H3⁺ cells among SSEA1⁺ cells after 36 hours of culture with TM. *P<0.01, **P<0.05. Error bars, s.e.m. (D) The efficiency of Cre-mediated DNA recombination in mouse embryonic fibroblasts (MEFs) from E12.5 ER-Cre/NRi-Tg embryos over time post-TM treatment.

• Supplemental Figure S5 -

  Fig. S5. Reduction in the number of spermatogonia in TNAP-Cre/NRi-Tg adult and newborn testes. (A) External morphology of testes of 6-week-old TNAP-Cre/NRi-Tg mice. (B) Comparison of the wet weight of testes. (C) A section of the testis of a 6-week-old TNAP-Cre/NRi-Tg mouse. Spermatogonia were visualized by immunostaining with TRA98 (red). (D) Cryosection of testis from TNAP-Cre/NRi-Tg newborn (P1) mouse. Spermatogonia were detected with the TRA98 antibody (red). (E) Comparison of the number of TRA98⁺ cells of TNAP-Cre/NRi-Tg and NRi-Tg newborn testes. The number of spermatogonia was counted in at least 15 sections from each testis. The areas of sections were measured using ImageJ software (NIH). **P<0.05. Error bars, s.e.m.

• Supplemental Figure S6 -

  Fig. S6. Changes in the expression level of lineage-specific genes and Nanog-binding genes. (A) No global change between Nanog high (H) and Nanog low (L) in expression of the endoderm, mesoderm and ectoderm genes. The number of genes significantly up- or downregulated in Nanog (L) is shown under each heat map (P<0.05). Gene ontology information was downloaded from the Affymetrix website (https://www.affymetrix.com/analysis/netaffx/index.affx). (B) Comparison of expression of 'Nanog-binding' genes between Nanog (L) and Nanog (H) by heat map. Significantly up- or downregulated genes are represented (P<0.05).

• Supplemental Figure S7 -

  Fig. S7. Id1, Tial1 and Suz12 expression in Nanog knockdown PGCs. (A) Nanog, Id1, Tial1 and Suz12 transcription in a single-cell library as assessed by qPCR. Fold changes were normalized to Gapdh levels using the ddCT method. Libraries were categorized as Nanog high, low and negative by expression levels. Asterisk, no detectable signal. Error bars, s.e.m. (B) A model of Nanog-mediated transcriptional regulation of Id1 in ESCs and PGCs.