Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: YAP1 interacts with EGFR signaling pathway to drive granulosa cell proliferation. A) Knockdown of Yap1 in mouse granulosa cells reduced mRNA levels of Egfr, EGF-like ligands (Hbegf and Areg), and cyclin D1 (Ccnd1). Relative mRNA levels were determined using RT-PCR. The experiment was repeated ≥3 times, and representative images are presented. B) Expression of HBEGF in hGCs, KGN cells, and HGrC1 cells transfected with empty vectors [MX as control (CTL)], vectors expressing wild-type YAP1 (YAP), or constitutively active YAP1 (YAPS127A). Relative mRNA levels were quantified using real-time PCR. Each bar represents the mean ± sem (n = 4). C, D) HBEGF treatment (50 ng/ml, 3 d) stimulated cell proliferation (C), but suppressed estradiol (E2) and progesterone (P4) production (D) in cultured granulosa cells. Each bar represents the mean ± sem (n = 4). E) HBEGF (50 ng/ml) induced rapid dephosphorylation of LATS1 (S909) and YAP1 (S127) and phosphorylation of ERK1/2 in cultured granulosa cells. β-Actin protein was used as loading CTL. The relative levels of the total proteins and phosphoproteins were determined by Western blot. Ctgf, connective tissue growth factor; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; p, phosphorylated. Experiments were repeated ≥3 times, and representative images are presented. *P < 0.05, **P < 0.01, ***P < 0.001, compared with corresponding CTL groups (MX groups).

Article Snippet: HGrC1 [an immortalized human granulosa cell (hGC) line] and KGN (an ovarian granulosa cell–like tumor cell line) cells were obtained from the Riken Cell Bank (Riken BioResource Research Center, Tsukuba, Japan).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction, Cell Culture, De-Phosphorylation Assay, Phospho-proteomics, Western Blot

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: YAP1 stimulates proliferation but suppresses differentiation of granulosa cells. A) Representative images showing spheroids formed in KGN-MX, KGN-YAP, and KGN-YAPS127A cells in a 3D culture system. Scale bars, 200 μm. B) Numbers of spheroids with different diameters formed by KGN-MX, KGN-YAP, and KGN-YAPS127A cells in the 3D culture system in A. Each bar represents the mean ± sem (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with corresponding control (CTL). C) Production of E2 and progesterone in KGN-MX, KGN-YAP, and KGN-YAPS127A cells in the 3D culture system. *P < 0.05, **P < 0.01, ***P < 0.001 compared with MX control. D) Knockdown of Yap1 with Yap1-specific siRNA (siYap) in cultured mouse granulosa cells up-regulated the expression of granulosa cell differentiation–associated genes. The relative mRNA levels of Star, Fshr, Sult1e1, Ptgfr, cyp11A1, cyp19a1, and Gapdh were determined by RT-PCR. E) Forskolin (FSK) induced expression of differentiation-associated genes in cultured primary mouse granulosa cells. The relative mRNA levels of Yap1, Star, Fshr, Sult1e1, Ptgfr, cyp11a1, and cyp19a1 in mouse granulosa cells treated with 10 μM FSK for the indicated time points were determined by quantitative PCR. *P < 0.05, **P < 0.01, ***P < 0.001 compared with CTL. F) FSK (10 μM) induced phosphorylation of YAP1 in cultured HGrC1 cells. Please note that phosphorylation results in inactivation of YAP1 protein. The relative levels of the total proteins and phosphoproteins were determined by Western blot. All experiments were repeated ≥3 times, and representative images were presented. G) FSK (10 μM) suppressed the expression of Hbegf mRNA in cultured primary mouse granulosa cells in a time-dependent manner. Each bar represents the mean ± sem (n = 4). *P < 0.05, **P < 0.01 compared with CTL. H) FSK (10 μM, 72 h) suppressed HBEGF mRNA expression in cultured primary hGCs. CREB, cAMP responsive element binding protein; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; p, phosphorylated. Each bar represents the mean ± sem (n = 4). **P < 0.01 compared with CTL.

Article Snippet: HGrC1 [an immortalized human granulosa cell (hGC) line] and KGN (an ovarian granulosa cell–like tumor cell line) cells were obtained from the Riken Cell Bank (Riken BioResource Research Center, Tsukuba, Japan).

Techniques: Control, Knockdown, Cell Culture, Expressing, Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Binding Assay

Journal: Journal of Ovarian Research

Article Title: Integrating network pharmacology and experimental verification to explore the pharmacological mechanisms of asparagus against polycystic ovary syndrome

doi: 10.1186/s13048-023-01210-5

Figure Lengend Snippet: ASP reverses DHEA induced decrease of PRKCA in PCOS granulosa cells. A - C qRT-PCR and Western blot were performed to detect the expression of PRKCA in KGN cells. β-actin was used as a normalization standard. D Estrus status of mice in the DHEA and control groups (n = 6 per group). E ELISA assay for T and E2 in mouse serum. F Histological staining of ovaries from DHEA and control mice. G qRT-PCR was performed to detect the expression of PRKCA in PCOS mouse models

Article Snippet: KGN cells, a steroidogenic human granulosa cell-like tumor cell line was purchased from iCell Bioscience Inc (China) and identified by short tandem repeat (STR) profiling.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining