Fig 2: AQP3 provides compensation for glycolytic metabolism. (A). Glycerol content was measured in human endometrial tissues (n = 20). (B). Glycerol content was measured in siOGA-treated HEC-1A cells or siOGT-treated RL95-2 cells (n = 3). (C1). WB analysis of AQP3 protein, and the qPCR analysis (C2) of AQP3 mRNA in HEC-1A and RL95-2 cells (normalized by ß-actin; n = 3). (D). The ECAR in HEC-1A cells with empty vector, over-AQP3, over-AQP3 + glycerol (26 mM) treatments, and RL95-2 cells with empty vector, short hairpin RNA (sh)-AQP3, and sh-AQP3 + glycerol (26 mM) treatments (n = 3). (E). IHC analysis of GYK in human endometrial tissue (scale bar = 25 µm, n = 20). (F). IHC analysis of GYK expression in the uteri of mouse D1–D5 (scale bar = 25 µm, n = 3; *vs. D1). (G1). Effect of AQP3 knockdown on implantation rate with a photograph showing typical mouse uterus at D7 of pregnancy. (G2). The implanted embryo ratio in each horn was analyzed (n = 7). (H). 2-NBDG fluorescent glucose uptake assays in over-AQP3 HEC-1A and sh-AQP3 RL95-2 cells (n = 3). (I). WB analysis of AQP3, c-Myc, and GLUT1 in HEC-1A and RL95-2 cells transfected with over-AQP3 or sh-AQP3 plasmids. (n = 3; *vs. NC). Error bars represent the mean ± standard deviation. Student’s t-test, One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: QYYY promoted HIF-1a, PMK2, metabolic markers, renal inflammation and fibrosis in vitro. Cells were divided into following groups: Model group (Hypoxia + 10% blank serum); QYYY group (Hypoxia + 5% QYYY-containing serum + 5% blank serum) and valsartan group (Hypoxia + 10% Valsartan-containing serum). The control group was maintained in a humidified atmosphere of 5% CO2 at 37°C till end of study. (A) Effects of anoxia time on the cell viability of HEK293T were examined by using MTT. (B) Effects of anoxia time on the protein expression of HIF-1a and PKM2 of hypoxic HEK293T cells were examined by using western blotting. (C) Effects of QYYY on protein expression of HIF-1a, PKM2, GLUT1, LDH-A, and IL-6 in hypoxic HEK293T cells were examined by using western blotting. (D) Effects of QYYY on immunofluorescence staining of HIF-1a and PKM2 in hypoxic HEK293T cells. (E) Effects of QYYY on mRNA levels of HIF-1a, PKM2, CTGF, TGF-ß1, and TNF-a in hypoxic HEK293T cells were examined by using qPCR. (F) Effects of QYYY on changes of ATP, pyruvate and lactate using kits. (G) Effects of QYYY on nuclear and cytoplasmic protein expression of HIF-1a and PKM2 in hypoxic HEK293T cells were examined by using western blotting. * p < 0.05 and ** p < 0.01 as compared with the control group. # p < 0.05 and ## p < 0.01 as compared with the model group.

Fig 4: GLUT1 and HBP are up-regulated during the “window of implantation”. (A). GLUT1, GFAT in human endometrial tissues were analyzed by IHC (n = 20; scale bar = 25 µm). (B). HEC-1A and RL95-2 cells treated with glucose (5.5 mM, 15 mM, 25 mM, 5.5 mM glucose + 19.5 mM mannitol as high osmotic control) for 48 h. The levels of O-GlcNAcylation, GLUT1, OGA, and OGT were analyzed by WB (n = 3; *vs.5.5 mM Glc + 19.5 mM Man). (C). HEC-1A and RL95-2 cells were exposed to 5.5 mM, 25 mM glucose, with or without DON (50 mM) for 48 h. WB was used to analyze O-GlcNAcylation levels (n = 3). (D). HEC-1A and RL95-2 cells were treated at different glucosamine concentrations (0.01, 0.1, 0.5 mM) for 48 h. WB was used to analyze O-GlcNAcylation levels (n = 3; *vs.0 mM GlcNH2). (E). The qPCR analysis of Hoxa10 mRNA in the uteri of mouse D1–D5 (normalized by Gapdh; n = 3; *vs.D1). (G). IHC analysis of GLUT1, GFAT in the uteri of mouse D1–D5 (Scale bar = 25 µm; n = 3). Error bars represent the mean ± standard deviation. Student’s t-test, One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Effects of over-expression and knockdown of HIF-1a on relative markers in HEK293T cells. Cells were divided into following groups: conrtol, negative control (NC), HIF-1a-overexpression group (OE) and HIF-1a-knockdown group (KD). (A) Effects of over-expression of HIF-1a on mRNA levels of HIF-1a, PKM2, CTGF, TGF-ß1, and TNF-a were examined by using qPCR. (B) Effects of over-expression of HIF-1a on immunofluorescence staining of HIF-1a and PKM2. (C) Effects of over-expression of HIF-1a on proteins of HIF-1a, PKM2, GLUT1, LDH-A, and IL6 in HEK293T cells were examined by using western blotting. (D) Effects of knockdown of HIF-1a on proteins of HIF-1a, PKM2, GLUT1, LDH-A, and IL6 in HEK293T cells were examined by using western blotting. (E) Effects of knockdown of HIF-1a on mRNA levels of HIF-1a, PKM2, CTGF, TGF-ß1, and TNF-a in HEK293T cells were examined by using qPCR. * p < 0.05 and ** p < 0.01.