Fig 2: Lack of Dose Dependent Nuclear to Cytoplasmic Translocation of FUBP1. Hela cells were treated with 0, 100, 200 or 300 µM H2O2 for 10 minutes and cultured in freshly changed DMEM containing 0.5% FBS for another 1 hour. Cells were harvested for total cell lysates or fractionated into cytosolic and nuclear extracts for Western blot as described in Fig. 8.

Fig 3: Time Dependent FUBP1 Binding to Nrf2 mRNA. HeLa cells were treated with 100 µM H2O2 for 10 min and harvested at indicated time point for in vitro binding using Nrf2 5'UTR as a probe (A), or for immunoprecipitation using antibodies against FUBP1 for detection of associated Nrf2 mRNA (B). The proteins bound to Nrf2 5'UTR probe were pull down for Western blot to detect FUBP1, with FUBP1 levels in total cell lysates serving as an input (A). The immunoprecipitates of FUBP1 were used for real time RT-PCR to detect Nrf2 mRNA, and the abundance of Nrf2 mRNA was calculated with ?CT by comparing the copy number from the immunoprecipitates of FUBP1 group or IgG group to that of the average of IgG group at 0 time point. The results are from one experiment representative of three independent experiments. * indicates statistically significant difference between control versus the H2O2 treated group (p < 0.05).

Fig 4: IL-27 induces decidual COX-2+ macrophage differentiation in vitro.(A and B) Expression of IL-27RA in DSCs (n = 9), trophoblast cells (Tros; n = 9), DLCs (n = 9), and decidual macrophages (dMfs) (n = 9) was analyzed by FCM. Statistical graph of (A) was shown in (B). (C and D) Expression of p-STAT3 and STAT3 in dMfs (n = 9) after coculture with ESCs pretreated with FBP (0.5 mM for 24 hours) or transfected with IL-27 overexpression plasmids (IL-27over) for 48 hours was detected by FCM, and the ratios of p-STAT3 to STAT3 were calculated. (E and F) Expression of COX-2 in peripheral blood monocytes (pMos) (n = 12), eMfs (n = 12), and dMfs (n = 12) was analyzed by FCM. Statistical graph of (E) was shown in (F). (G) Expression of COX-2 in dMfs (n = 9) cocultured with Ctrl or FBP (0.5 mM for 24 hours)–treated ESCs was detected by FCM. (H) Expression of COX-2 in dMfs (n = 9) cocultured with FBP (0.5 mM for 24 hours)–pretreated ESCs or IL-27over ESCs for 48 hours was detected by FCM. Data were presented as means ± SEM or median and quartile and analyzed by t test, one-way ANOVA test, or Kruskal-Wallis test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Abnormal FBP-IL-27 regulatory axis in DSCs leads to pregnancy loss.(A) Heatmap of the differential proteins (differential fold > 3) of supernatants between FBP (0.5 mM)–ESC (n = 3) and Ctrl-ESC (n = 3) by the proteomic microarray. (B) The IL-27 levels in ESCs (n = 9) treated with FBP were detected by FCM. (C) The IL-27 levels in supernatants of control ESCs (FBP1-NC, n = 9) or FBP1-overexpressing ESCs (FBP1over, n = 9) treated with DMSO (1‰) or progesterone (P; 10-8 µM) were detected by ELISA. (D) Expression of IL-27 between control endometrium (n = 6) and decidua tissues (n = 6) was detected by immunohistochemistry. (E to G) Photograph (red arrow: absorption point) of uterus, absorption rates, or CRL of embryos from WT pregnant mice (mated with male Il27ra-/- mice, n = 8) or Il27ra-/- pregnant mice (mated with male WT mice, n = 8). (H) Transcription levels of decidualization-related genes in uterus from (E) were analyzed by RT-PCR. (I) Depth of CK7+ trophoblast infiltration into uterus from (E) was observed by hematoxylin and eosin staining or immunofluorescence assays. DAPI, 4',6-diamidino-2-phenylindole. Data were presented as means ± SEM and analyzed by t test or one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.