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. 2022 Mar 4;11:e72073. doi: 10.7554/eLife.72073

Figure 4. Genome wide binding of SOX4 in decidualized stromal cells.

(A) Distribution of SOX4-binding peaks as revealed from SOX4 ChIP-Seq in SOX4 overexpressed (HA-SOX4) immortalized human endometrial stromal cells (HESCs) decidualized for 2 days. (B) Distribution SOX4 binding in genebody. (C) Heatmap of SOX4-binding sites distribution. (D) Motif analysis of SOX4-binding sites. (E) Venn diagram of SOX4 directly binding peaks and SOX4 regulated genes after SOX4 knockdown. (F) SOX4-binding site in the STAT3, FOXO1, and FOSL2 with progesterone receptor (PGR) binding and chromatin accessibility in both primary stromal cell and stromal cell line. The chromatin accessibility is depicted in undecidualized and decidualized stromal cells and genome-wide PGR binding is generated from proliferated and middle secretory endometrium as revealed from previous reports. Undec: undecidualized HESCs; Dec: decidualized HESCs. (G–J) ChIP-qPCR assay of SOX4 binding on PRL, STAT3, FOXO1, and FOSL2 in HA-SOX4 overexpressed decidualized HESCs. Results were presented as means ± standard error of the mean (SEM); n = 3; *p < 0.05; **p < 0.005; ***p < 0.0001. (K) Read number of SOX4 binding in SOX4 regulated genes after SOX4 knockdown. (L) KEGG analysis of overlapped genes of SOX4 directly binding and SOX4 downregulated genes as well as nonoverlapped genes. (M) mRNA levels of FOXO1 in SOX4 knockout undecidualized and decidualized HESCs at indicated time points. Results were presented as means ± SEM; n = 3; **p < 0.005. (N) Protein levels of SOX4, FOXO1, and IGFBP1 in SOX4 knockout undecidualized and decidualized HESCs at indicated time points. Band quantification of indicated proteins, relative to loading control GAPDH. *p < 0.05; ***p < 0.0001. n = 3. (O) mRNA levels of FOXO1 after SOX4 overexpression in undecidualized or decidualized HESCs for 4 days. Results were presented as means ± SEM; n = 3; **p < 0.005. The above experiments were repeated three times.

Figure 4.

Figure 4—figure supplement 1. FOXO1 knockdown abolished the effect of SOX4 overexpression on decidualization.

Figure 4—figure supplement 1.

(A) FOXO1 expression after FOXO1 knockdown in the primary decidualized human endometrial stromal cells (HESCs). (B) Protein levels of FOXO1, IGFBP1, and SOX4 after overexpression of SOX4 in the presence of FOXO1 knockdown or not in decidualized HESCs for 4 days; n=3; *p < 0.05; **p < 0.005.
Figure 4—figure supplement 2. The upregulated genes after SOX4 knockdown with SOX4-binding sites in their regulatory regions.

Figure 4—figure supplement 2.

(A) Venn diagram of SOX4 directly binding peaks and upregulated genes after SOX4 knockdown. (B) Gene Ontology (GO) analysis of the overlapped gene list in A.