Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quantitative ELISAs for serum soluble LHCGR and hCG-LHCGR complex: potential diagnostics in first trimester pregnancy screening for stillbirth, Down’s syndrome, preterm delivery and preeclampsia

doi: 10.1186/1477-7827-10-113

Figure Lengend Snippet: Epitope mapping of LHR29 monoclonal antibody. a ) Three recombinant proteins containing 229, 291 or 318 amino acid residues of the LHCGR extracellular domain and C-terminal FLAG-epitope were expressed in CHO cells as described ; b ), recombinant as well as mock transfected extracts (no DNA) were resolved in SDS-PAGE, blotted and probed with anti-FLAG, LHR29 and PG732 mono- and polyclonal antibodies respectively; c ), two monoclonal antibodies (LHR74 and LHR29) and one polyclonal (PG732) were used in western blot analysis of proteins extracted from placental extracts at 12 wks of gestation.

Article Snippet: Purified LHR29 and LHR74 antibodies were initially provided by Dr Hugues Loosfelt (INSERM, France) and subsequently the antibody producing clones were obtained from ATCC (Clone ID CRL-2685 and CRL-2686).

Techniques: Recombinant, FLAG-tag, Transfection, SDS Page, Bioprocessing, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Glioma-associated mesenchymal stem cells-mediated PD-L1 expression is attenuated by Ad5-Ki67/IL-15 in GBM treatment

doi: 10.1186/s13287-022-02968-z

Figure Lengend Snippet: Identification of MSCs derived from human glioma tissues. A and B Adherent growth patterns and fibroblastic morphology of GA-MSCs cultured in MSC media (× 100, scale bars = 1000 µm). C Immunofluorescence showed that GA-MSCs expressed MSC specific markers CD44 and CD105 (× 400, scale bars = 500 µm)

Article Snippet: The cells were incubated with primary antibodies against CD44 and CD105 (1:100, Proteintech, China), then overnight at 4 °C.

Techniques: Derivative Assay, Cell Culture, Immunofluorescence

Journal: Frontiers in Endocrinology

Article Title: Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

doi: 10.3389/fendo.2022.853563

Figure Lengend Snippet: Decreased responsiveness of LHCGR to its ligand in endometriosis. (A) Average number of ovulated oocytes following superovulation protocols with a different dose of hCG (n = 6, each group). * P < 0.05 (two-way ANOVA). (B) The percentage of luteinized unruptured follicle for all CLs tracked per ovary. * P < 0.05, ** P < 0.001 (two-way ANOVA). (C) Western blotting of LHCGR protein expression in GCs of EMs and sham mice at different time points after hCG administration (0, 2, 4, and 8 h). GAPDH was used as a sample loading control. * P < 0.05, ** P < 0.001 (Student’s t -test). (D, E) Immunohistochemical H-score and representative images of immunohistochemical staining for LHCGR in the GCs from EMs and sham mice after PMSG-priming (48 h). Scale bar, 100 μm. ** P < 0.001 (Student’s t -test).

Article Snippet: The sections were then treated with bovine serum albumin (BSA) blocking, followed by incubation with primary antibody for LHCGR (19968-1-AP; 1:200 dilution; Proteintech), C/EBPα (18311-1-AP; 1:200 dilution; Proteintech), COX-2 (ab15191; 1:200 dilution; Abcam) and corresponding secondary antibody.

Techniques: Western Blot, Expressing, Control, Immunohistochemical staining, Staining

Journal: Frontiers in Endocrinology

Article Title: Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

doi: 10.3389/fendo.2022.853563

Figure Lengend Snippet: COX-2 involved in the hCG-induced ovulation is downregulated in EMs granulosa cells. (A) qRT-qPCR analysis of expression of ovulation-related genes VEGFA, PGF, COX-1, COX-2, AREG, EREG, MMP-2, and MMP-9 in human GCs treated with nontargeting negative control siRNA (NC) or LHCGR siRNA.** P < 0.001 (Student’s t -test). (B) qRT-PCR analysis of ovulation-related gene expression in GCs of EMs and sham mice after PMSG-priming (48 h). * P < 0.05, ** P < 0.001 (Student’s t -test). (C) Human GCs were treated with10 IU/ml hCG for 0, 12, 24, and 36 h, the protein levels of COX-2 were examined by Western blot. (D) The mRNA levels of COX-2 in hCG-treated human GCs at different time points were analyzed by qRT-PCR. * P < 0.05, ** P < 0.001 (ANOVA). (E, F) Human GCs were transfected with 50 nM siRNA against LHCGR for 24 h and then treated with 10 IU/ml hCG for another 24 h. The mRNA and protein levels of COX-2 were analyzed. * P < 0.05 (ANOVA). (G) Western blotting of COX-2 during ovulation in GCs from EMs and sham mice.* P < 0.05 (Student’s t -test). (H) Immunohistochemical H-score and representative images of immunohistochemical staining for COX-2 in the GCs from EMs and sham mice after PMSG-priming (48 h). Scale bar, 100 μm. ** P < 0.001 (Student’s t -test).

Article Snippet: The sections were then treated with bovine serum albumin (BSA) blocking, followed by incubation with primary antibody for LHCGR (19968-1-AP; 1:200 dilution; Proteintech), C/EBPα (18311-1-AP; 1:200 dilution; Proteintech), COX-2 (ab15191; 1:200 dilution; Abcam) and corresponding secondary antibody.

Techniques: Expressing, Negative Control, Quantitative RT-PCR, Gene Expression, Western Blot, Transfection, Immunohistochemical staining, Staining

Journal: Frontiers in Endocrinology

Article Title: Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

doi: 10.3389/fendo.2022.853563

Figure Lengend Snippet: C/EBPα is necessary for hCG-induced COX-2 expression in granulosa cells. (A) Western blotting of C/EBPα during ovulation in GCs from EMs and sham mice. (B) Representative images of immunohistochemical staining for C/EBPα in the GCs from EMs and sham mice after PMSG-priming (48 h). Scale bar, 100 μm. (C, D) The protein and mRNA levels of C/EBPα in hCG-treated human GCs at different time points were analyzed by Western blot and qRT-PCR, respectively. ** P < 0.001 (ANOVA). (E) hCG-treated (24 h) human GCs were analyzed by immunofluorescence to identify the subcellular localization and protein expression levels of C/EBPα (red). Nuclei were stained with DAPI (blue). Magnification: ×100. Scale bar, 50 μm. (F, G) Human GCs were transfected with negative control siRNA or LHCGR siRNA and then treated with hCG. The mRNA and protein levels of C/EBPα were analyzed. NC, negative control. * P < 0.05 (ANOVA). (H–J) Human GCs were transfected with negative control siRNA or C/EBPα siRNA. The expression of indicated genes and protein was analyzed by qRT-PCR and Western blot. ** P < 0.001 (Student’s t -test). (K) Predicted C/EBPα-binding site in the promoter region of human COX-2 . TSS, transcriptional start site; Fw primer, forward primer; Rev primer, reverse primer. (L) KGN cells were cotransfected with C/EBPα-overexpressing plasmid vectors, and luciferase reporter constructs harboring the COX-2 promoters, along with a Renilla luciferase construct for internal control. Firefly luciferase (Luc) activity was normalized to Renilla activity. Data are shown as mean ± SEM and expressed as fold increase in firefly luciferase activity compared with empty vector (PGL-basic). * P < 0.05, ** P < 0.001 (Student’s t-test). (M) KGN cells were left untreated or stimulated with hCG for 24 h. ChIP assays were performed using anti-C/EBPα antibody or isotype control antibody (IgG). qRT-PCR was used to determine C/EBPα occupancy at the potential biding site under the conditions tested. ** P < 0.001 (Student’s t-test). (N–P) Human GCs were transfected with negative control siRNA or C/EBPα siRNA and then treated with hCG. The expression of indicated genes and protein was analyzed by qRT-PCR and Western blot. ** P < 0.001 (ANOVA).

Article Snippet: The sections were then treated with bovine serum albumin (BSA) blocking, followed by incubation with primary antibody for LHCGR (19968-1-AP; 1:200 dilution; Proteintech), C/EBPα (18311-1-AP; 1:200 dilution; Proteintech), COX-2 (ab15191; 1:200 dilution; Abcam) and corresponding secondary antibody.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Quantitative RT-PCR, Immunofluorescence, Transfection, Negative Control, Binding Assay, Plasmid Preparation, Luciferase, Construct, Control, Activity Assay

Journal: Frontiers in Endocrinology

Article Title: Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

doi: 10.3389/fendo.2022.853563

Figure Lengend Snippet: Transcriptional activity of C/EBPα is regulated in a Cyclic AMP-Independent manner. (A) KGN cells were left untreated or stimulated with db-cAMP. ChIP assays were performed using anti-C/EBPα antibody or isotype control antibody (IgG). qRT-PCR was used to determine C/EBPα occupancy at the potential biding site under the conditions tested. **P <0.001 (Student’s t-test). (B) db-cAMP-treated (24 h) human GCs were analyzed by immunofluorescence to identify the subcellular localization and protein expression levels of C/EBPα (red). Nuclei were stained using DAPI (blue). Magnification: ×100. Scale bar, 50 μm. (C–E) Western blotting and qRT-PCR analysis of indicated genes and protein in human GCs after treatment with db-cAMP. * P < 0.05, ** P < 0.001 (Student’s t -test). (F–H) Human GCs were transfected with negative control siRNA or LHCGR siRNA and then treated with 1 mM db-cAMP for 24 h. The expression of indicated genes and protein was analyzed by qRT-PCR and Western blot. ns, no significance, * P < 0.05 (ANOVA). (I–K) The protein and mRNA levels of C/EBPα and COX-2 in human GCs, which were treated with 1 mM db-cAMP for 24 h following exposure to siRNAs against C/EBPα. ns, no significance, * P < 0.05, ** P < 0.001 (ANOVA).

Article Snippet: The sections were then treated with bovine serum albumin (BSA) blocking, followed by incubation with primary antibody for LHCGR (19968-1-AP; 1:200 dilution; Proteintech), C/EBPα (18311-1-AP; 1:200 dilution; Proteintech), COX-2 (ab15191; 1:200 dilution; Abcam) and corresponding secondary antibody.

Techniques: Activity Assay, Control, Quantitative RT-PCR, Immunofluorescence, Expressing, Staining, Western Blot, Transfection, Negative Control

Journal: Frontiers in Endocrinology

Article Title: Downregulation of LHCGR Attenuates COX-2 Expression and Induces Luteinized Unruptured Follicle Syndrome in Endometriosis

doi: 10.3389/fendo.2022.853563

Figure Lengend Snippet: Model of LUFs in endometriosis. Schematic depiction of the effect of LH signaling in preovulatory granulosa cells in endometriosis. During midcycle LH surge, attenuated LHCGR deactivating C/EBPα in a cAMP-dependent manner, then the transcription of COX-2 was repressed in granulosa cells. Ultimately, inducing ovulation failure and oocyte trapped in CLs.

Article Snippet: The sections were then treated with bovine serum albumin (BSA) blocking, followed by incubation with primary antibody for LHCGR (19968-1-AP; 1:200 dilution; Proteintech), C/EBPα (18311-1-AP; 1:200 dilution; Proteintech), COX-2 (ab15191; 1:200 dilution; Abcam) and corresponding secondary antibody.

Techniques: