Fig 1: JAZF1 is dynamically expressed in human endometrial stromal cells.a–e RT-qPCR (a, b), Western blotting (c, e), and ELISA (d) analysis for JAZF1, IGFBP1, PRL, FOXO1 in immortalized HESCs treating with MPA, cAMP for 6 days. f IF staining of decidualized immortalized HESCs for JAZF1, counterstained with DAPI. undec: undecidualized HESCs; dec: decidualized HESCs. n = 3, scale bars, 100 µm. g IHC staining of JAZF1 in the proliferative and early secretory phase endometrium from RSA and normal group. n = 3, scale bars, 100 µm. h Expression of JAZF1 in proliferative and middle secretory phase endometrium. The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GDS2052). i Vlnplot of the expression level of JAZF1 in ESCs across the natural menstrual cycle identified by single-cell transcriptomics. The data were retrieved from microarray data deposited in the Gene Expression Omnibus (GSE111976). The data were shown as the median and quartile. Others were shown as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: JAZF1 depletion promoted cell death of decidualized HESCs by activating G0S2.a Results of RT-qPCR showing the expression of IGFBP1, PRL, BAX, BCL2 in immortalized HESCs transiently transfected with siJAZF1 and treated with G0S2 inhibitor NS-3. n = 3. b, c Protein expression of IGFBP1, BCL2, BAX, cleaved-caspase 3, cyt c, G0S2 in immortalized HESCs with transfection of siJAZF1 and added G0S2 inhibitor NS-3 during decidualization. n = 3. d RT-qPCR analysis for IGFBP1, PRL, BAX, BCL2 in siNC, siJAZF1 and siJAZF1+siG0S2 immortalized HESCs after decidualization. n = 3. e, f Western blotting analysis of IGFBP1, BAX, cleaved-caspase 3, cyt c, G0S2 in decidualized HESCs transfected with siJAZF1+siG0S2. n = 3. g, h Flow cytometry of decidualized HESCs transfected siJAZF1 + NS-3 or siJAZF1+siG0S2. n = 3. i Cell proliferation was determined by CCK8 assay. n = 3. The data were shown as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Overexpression of G0S2 activated mitochondrial apoptosis pathway and decidualization defect in decidualized hESCs.a, b G0S2 mRNA and protein levels in HESCs with infected with control lentivirus or G0S2-overexpression lentivirus. c RT-qPCR analysis for IGFBP1, PRL, BAX, BCL2 in G0S2-overexpression HESCs followed by cultured in medium supplemented with MPA and cAMP for 6 days. n = 3. d Western blotting analysis of IGFBP1, BAX, BCL2, cleaved-caspase3, cyt c in decidualized HESCs transfected with OE-NC or OE-G0S2 lentivirus. n = 3. e Flow cytometry of decidualized HESCs transfected with control lentivirus or overexpression-G0S2 lentivirus. n = 3. f Cell proliferation was determined by CCK8 assay. n = 3. The data were shown as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 4: JAZF1 maintains decidua homeostasis by restricting the transcription of G0S2 activated by Purß in decidualized hESCs.a Magnified image of Bio-Celastrol binding to Purß spot on the protein array. scale bars, 50 µm. b Co-IP suggested that JAZF1 interacted with Purß. c Results of RT-qPCR showing the expression of IGFBP1, PRL, BAX, BCL2 and G0S2 in immortalized HESCs transiently transfected with siJAZF1 and siPurß. n = 3. d Protein expression of IGFBP1, G0S2, BCL2, BAX, cleaved-caspase 3, cyt c in immortalized HESCs transfected with siJAZF1 and siPurß during decidualization. n = 3. e Flow cytometry of decidualized HESCs transfected siJAZF1+siPurß. n = 3. f IHC analysis of Purß in RSA decidua compared to normal decidua. n = 4. scale bars, 100 µm. g Human G0S2 promoter (-2001 to -1) Luciferase reporter plasmids and Renilla reporter plasmids were transiently co-transfected with Purß expression plasmids into HEK 293 T cells. Luciferase activity was measured 24 h after transfection and normalized to Renilla levels. n = 3. h Luciferase activity of G0S2 promoter in decidualized HESCs with co-transfection with OE-Purß and siJAZF1. n = 3. The data were shown as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.00001.
Fig 5: JAZF1 depletion promoted apoptosis and decidualization defect of stromal cells.a Results of RT-qPCR showing the expression of IGFBP1, PRL, BAX, BCL2 in HESCs after JAZF1 knockdown and knockout. n = 3. b ELISA detection showed the PRL level after JAZF1 knockdown. n = 3. c, d Protein expression of IGFBP1, BAX, BCL2, cleaved-caspase 3, cyt c in HESCs with transfection of siJAZF1 or KO-JAZF1 during decidualization. n = 3. e RT-qPCR analysis for IGFBP1, PRL, BAX, BCL2 in JAZF1-overexpression HESCs followed by culture in medium supplemented with MPA and cAMP for 6 days. n = 3. f, g Western blotting analysis of IGFBP1, JAZF1, BCL2, cyt c in decidualized HESCs transfected with OE-NC or OE-JAZF1 lentivirus. n = 3. h ELISA detection showed the PRL level after JAZF1 overexpression. n = 3. i, j Flow cytometry of decidualized HESCs transfected siJAZF1 and overexpression-JAZF1 lentivirus. n = 3. k Cell proliferation was determined by CCK8 assay. n = 3. The data were shown as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001.
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