Fig 2: Comparison of mouse TE with human TE.(A) Cell lineage identification of mouse S1, S2, and S3 TEs. (B) Immunofluorescence images of GATA3 and RXRA in mouse S2 embryos. Scale bar, 100 µm. (C) Cell cycle analysis of MTE and PTE in human and mouse. (D) Immunofluorescence images of Ki67 in human and mouse S2 embryos. Scale bars, 100 µm. (E) Bar plots showing relative expression levels [log2 (TPM/10 + 1), average levels ± SEMs] of specific genes in the implantation pole of human and mouse embryo. (F) Expression pattern and functional annotations of the common DEGs in implantation poles of human and mouse embryo. (G) Immunofluorescence images of FMNL2 in human and mouse S2 embryos. (H) The left panel shows immunofluorescence images showing colocalization of Fmnl2 and F-actin at the attachment site of mouse embryos. The right panel shows the same image with top view and side view. Please note that in the side view, ICM is marked by an *, MTE is marked by an arrow, and PTE is marked by an arrowhead. Scale bar, 100 µm.

Fig 3: The variant affects the inhibitory effect of RXRA on enhancer activity. (A) The variant nucleotide (boxed) is highly conserved and overlaps a predicted conserved binding site for RXRA. (B) Western blot results confirmed the successful overexpression or inhibition of the expression of RXRA in HeLa cells. Data are expressed as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 by Student's t-test. (C) In HeLa cells, luciferase constructs with wild-type and mutant fragments were cotransfected into cells with a human RXRA expression vector and RXRA siRNA, and the corresponding luciferase activity was analyzed. The variant affected the binding of the transcription factor RXRA to the enhancer so that the inhibitory effect was relieved, and the activity of the enhancer increased. Cotransfection with RXRA siRNA partially abolished the inhibition of enhancer activity. Data are expressed as the mean ± SD of three independent experiments. ***P < 0.001 by Student's t-test. (D) An electrophoretic mobility shift assay (EMSA) showed high-affinity, sequence-specific interaction of HES1 with a double-stranded oligonucleotide containing the wild-type (wt) sequence but not the mutant sequence (mu). The shifted signal was suppressed by the addition of an unlabeled consensus high-affinity binding site for HES1. The red arrow indicates the shifted HES1 complex. The areas indicated by the black lines represent non-specific probe binding and free probe. EMSA and supershift western blot analyses confirmed RXRA protein binding. The arrows indicate the complex containing the biotin-labeled HES1 probe and RXRA protein. (E) ChIP-qPCR confirmed that RXRA was enriched in the HES1 promoter (near the HES1 promoter variant).

Fig 4: Inhibition of PPAR signaling pathway eliminates the effects of didymin pretreatment on OGD/R treated PC12 cells. (A) Western blot was performed to detect the protein expression of PPAR-? and RXRA in PC12 cells. (B) MTT assay was performed to detect the cell viability of PC12 cells. (C and D) Flow cytometry analysis was performed to detect the apoptosis of PC12 cells. (E) Western blot was performed to detect the protein expression of Bax, c-caspase-3, and Bcl-2 in PC12 cells. *p<0.05 vs. control group; †p<0.05 vs. OGD/R group; ‡p<0.05 vs. OGD/R+didymin group. PPAR, peroxisome proliferator-activated receptors; OGD/R, oxygen-glucose deprivation/reperfusion.

Fig 5: Didymin activates the PPAR signaling pathway. (A) SymMap analysis showed that didymin activated the PPAR signaling pathway. (B) Western blot was performed to detect the protein expression of PPAR-? and RXRA in PC12 cells. *p<0.05 vs. 0 µM group; †p<0.05 vs. 10 µM group; ‡p<0.05 vs. 20 µM group. (C) Western blot was performed to detect the protein expression of PPAR-? and RXRA in OGD/R treated PC12 cells. *p<0.05 vs. control group; †p<0.05 vs. OGD/R group; ‡p<0.05 vs. OGD/R+10 µM didymin group; §p<0.05 vs. OGD/R+20 µM didymin group. PPAR, peroxisome proliferator-activated receptors; OGD/R, oxygen-glucose deprivation/reperfusion.