Figure 3. SOX4 regulated genes in decidualized human endometrial stromal cells (HESCs).

mRNA levels of PRL (A) and IGFBP1 (B) in undecidualized and decidualized immortalized HESCs after SOX4 knockout at indicated time points after the E2, MPA, and cAMP treatment. Results are presented as means ± standard error of the mean (SEM); n = 3; *p < 0.05; ***p < 0.0001. (C) Protein levels of IGFBP1 in undecidualized and decidualized HESCs after SOX4 knockout at indicated time points. (D, E) mRNA levels of PRL (C) and IGFBP1 (D) after SOX4 overexpression in undecidualized or decidualized HESCs for 4 days. n = 3; *p < 0.05. (F) Protein levels of SOX4 and IGFBP1 in decidualized HESCs for 4 days with control or SOX4 overexpression. Band quantification of SOX4 and IGFBP1 protein, relative to GAPDH. (G) Differential expressed genes detected by RNA sequencing (RNA-Seq) in immortalized HESCs decidualized for 2 days with control or SOX4 knockdown as visualized by volcano plot. (H) Heatmap of top differential expressed genes from RNA-Seq after SOX4 knockdown in decidualized HESCs. (I, J) KEGG (J) and Gene Ontology (GO) (K) analysis of the differentially expressed genes in RNA-Seq. Results were presented as means ± standard error of the mean (SEM). *p < 0.05; **p < 0.005; ***p < 0.0001. The above experiments were repeated three times.

Figure 3—figure supplement 1. SOX4 knockout in the stromal cell by CRISPR–Cas9.

(A) Schematic diagram of SOX4 knockout by CRISPR/Cas9. (B, C) SOX4 knockout confirmation by immunoblot and immunofluorescence in human endometrial stromal cells (HESCs). n=3; *p < 0.05. Scale bar: 50 μm.

Figure 3—figure supplement 2. SOX4 knockdown by the shRNA derailed the decidualization in primary stromal cell.

(A) Knockdown efficiency of SOX4 by shRNA. Results were presented as means ± standard error of the mean (SEM); n = 3; ***p < 0.001. (B, C) Expression of PRL and IGFBP1 in the SOX4-knockdown primary decidualized human endometrial stromal cells (HESCs) from days 0 to 6. Results were presented as means ± SEM. n = 3; *p < 0.05; **p < 0.005. (D) Protein levels of SOX4 and IGFBP1 in the SOX4-knockdown primary decidualized HESCs from days 0 to 6.

Figure 3—figure supplement 3. SOX4 overexpression upregulated the expression of decidualization marker genes.

(A, B) Expression of PRL and IGFBP1 in primary decidualized human endometrial stromal cells (HESCs) for 4 days after SOX4 overexpression. Results were presented as means ± standard error of the mean (SEM); n = 3; *p < 0.05; **p < 0.005. (C) SOX4 expression after SOX4 overexpression in the primary decidualized HESCs for 4 days; n=3; *p < 0.05; ***p < 0.00 1.