Additional file 1 of IRF-1 expressed in the inner cell mass of the porcine early blastocyst enhances the pluripotency of induced pluripotent stem cells
Creators: name: Shi, Bingbo
name: Gao, Dengfeng
name: Zhong, Liang
name: Zhi, Minglei
name: Weng, Xiaogang
name: Xu, Junjun
name: Li, Junhong
name: Du, Xuguang
name: Xin, Yanli
name: Gao, Jie
name: Zhu, Qianqian
name: Cao, Suying
name: Liu, Zhonghua
name: Han, Jianyong
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Screening for potential transcription factors related to porcine pluripotency, related to Fig. 1. (A) GO terms and KEGG pathways enriched from upregulated genes in ICM.(B) Analysis of ChIP-Seq data of H3K4me3. Marks indicate promoter regions of selected transcript factors. ChIP-Seq data of H3K4me3 in pEPSCs was obtained by Liu et al. [19]. The peaks from -4 k to -5 k upstream from the transcriptional start site of IRF-1 were peaks associated with other genes not IRF-1. (C) GFP fluorescence in PEFs transfected with reporter plasmid. Scale bar, 500 μm.(D) Distribution of red fluorescence in porcine reconstructed blastocysts by SCNT. GFP fluorescence represents successful integration of the reporter system. Red fluorescence represents the promoter’s activity. Scale bar, 100 μm.(E) qRT-PCR analysis of pluripotency-associated genes in BCL3 overexpressing cells and controls. Figure S2. Pluripotency characterization of DOX-hLIF-2i piPSCs, related to Fig. 2. (A) Immunofluorescence assay of SSEA-4, TRA-1-60, TRA-1-80. Scale bar, 20 μm. (B) EBs of DOX-hLIF-2i piPSCs obtained at day 6 of differentiation. Scale bar, 100 μm. (C) Fluorescence detection of OCT4-tdTomato in DOX-hLIF-2i piPSCs. Scale bar of the top figure, 100 μm. Scale bar of the bottom figure, 50 μm. (D) Cell morphology and AP staining of DOX-hLIF-2i piPSCs with DOX and without DOX. Scale bar, 200 μm. (E) RT-PCR analysis of endogenous expression of OCT4, SOX2, KLF4 and cMYC and exogenous OKSM. EF1A was used as internal control. 1#, 2# represent two lines of DOX-hLIF-2i piPSCs. Figure S3. The effect of IRF-1 overexpression on DOX-hLIF-2i piPSCS morphology, related to Fig. 3. (A) DAPI staining of IRF-1-overexpressing and negative control piPSCs in Fig. 3a. Scale bars from left to right, 200 μm, 50 μm. (B) RT-PCR analysis of endogenous expression of OCT4, SOX2, KLF4 and cMYC and exogenous OKSM. EF1A was used as internal control. OE: IRF-1 overexpressing piPSCs, WT: DOX-hLIF-2i piPSCs. Figure S4. Detection of heterogeneity stability of IRF-1 in DOX-hLIF-2i piPSCs, related to Fig. 4. (A) Fluorescence detection of GFP positive and negative cells after passage. Scale bars from left to the right, 100 μm, 200 μm. Figure S5. The effect of treatment with IL7 or Stattic treatment on pluripotency of DOX-hLIF-2i piPSCs, related to Fig. 5. (A) Cell morphology and AP staining of DOX-hLIF-2i piPSCs after treatment with IL7. Scale bars, 200 μm. (B) qRT-PCR analysis of pluripotency associated genes in piPSCS treated with IL7. *, P &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Figure S6. Further Characterization of OSKMI piPSCs, related to Fig. 6. (A) RT-PCR analysis of endogenous expression of OCT4, SOX2, KLF4 and cMYC and exogenous OKSM. EF1A was used as internal control. 4#, 10# represent different lines of OKSMI piPSCs. The control group represents OKSM piPSCs. (B) Immunofluorescence of SSEA-4, TRA-1-60, TRA-1-80 in OSKMI piPSCs. Scale bar, 20 μm.(C) Immunofluorescence assay of 3-germ-layer cells in EBs. Scale bar, 50 μm.