Figure 6. SOX4 inhibits progesterone receptor (PGR) degradation by regulating E3 ligase HERC4.

(A) Venn diagram of overlapping genes between upregulated genes in SOX4 knockdown from RNA sequencing (RNA-Seq) and PGR-associated protein form IP-MS. mRNA and protein levels of HERC4 in the absence of SOX4 by qPCR (B) and western blot (C). Results were presented as means ± standard error of the mean (SEM); n = 3. (D) Protein interaction between PGR and HERC4 after overexpression of HA-PGR and Flag-HERC4 in 293T cells. (E) Protein interaction between PGR and HERC4 after overexpression of HA-PGR and Flag-HERC4 in decidualized immortalized human endometrial stromal cells (HESCs) for 2 days, D2: 2 days. (F) Localization of PGR and HERC4 after overexpression of PGR and HERC4 in decidualized HESCs for 2 days, scale bar: 100 μm. (G) PGR ubiquitination at the present of MG-132 after overexpression of HERC4 in decidualized HESCs for 2 days.(H) PGR ubiquitination at the present of MG-132 and ubiquitin with HERC4 overexpression or knockdown in 293T cells. (I) PGR ubiquitination at the present of MG-132, ubiquitin, and SOX4 with or without HERC4 in 293T cells. (J) Degeneration of PGR after HERC4 overexpression in 293T cells. (K) PGR protein levels at the present of HERC4 overexpression and in HERC4 knockdown decidualized immortalized HESCs for 2 days. (L) Protein levels of PGR, HERC4, and SOX4 after SOX4 and/or HERC4 knockdown in decidualized immortalized HESCs for 2 days. (M, N) The protein half-life of PGR after HERC4 knockdown at the present of protein synthesis inhibitor cycloheximide (CHX) in 2 days decidualized immortalized HESCs. (O, P) The protein half-life of PGR after SOX4 and/or HERC4 knockdown at the present of protein synthesis inhibitor CHX in 2 days decidualized immortalized HESCs. The value for relative protein level is band quantification of indicated protein, relative to GAPDH. The above experiments were repeated three times. n=3; *p < 0.05; **p < 0.005.

Figure 6—figure supplement 1. The SOX4 regulated ubiquitin E3 ligase expression during the endometrial stroma decidualization.

(A) Coomassie blue staining of PAGE-Gel with progesterone receptor (PGR) immunoprecipitated complex. (B, C) Expression of RNF213 and UBR3 in SOX4-knockdown human endometrial stromal cells (HESCs). Results were presented as means ± standard error of the mean (SEM); n = 3; *p < 0.05. (D) PGR expression after HERC4 overexpression in HESCs as detected by immunofluorescence, scale bar: 100 μm. (E) Knockdown efficiency of HERC4 by siHERC4. The above experiments were repeated three times. (F) Protein levels of IGFBP1 in the indicated treatment. n=3; **p < 0.005.