Journal: Bioactive Materials

Article Title: Nanomaterial-assisted theranosis of bone diseases

doi: 10.1016/j.bioactmat.2022.12.014

Figure Lengend Snippet: Nanocrystal-polymer and chitosan-based nanocarriers for osteoarthritis therapy. A) Schematic representation of a KGN-NPP. (i) SEM of surface and core of KGN-NPPs. (ii) Representative safranin-O/fast green and IHC showing the medial side of the knee of mice at day 56. Femur (above), tibia (below). Arrows indicate cartilage erosion. Scale bar = 200 μm . The images reproduced with the permission from WILEY-VCH. B) Illustration of the procedures to synthesize PN-KGN. (iii) TEM of PN-KGN. (iv) In vitro release of KGN from PN-KGN at 37 °C. (v) Macroscopic views of OA development at 3, 6, and 12 weeks after OA induction. Arrows indicate the locations of cartilage degeneration (n = 6 per group) . The images reproduced with the permission from Taylor & Francis. C). Proposed reaction scheme of CHI-KGN conjugate. (vi) Safranin-O and Alcian-blue staining on day 21. (vii) In vivo fluorescence imaging and relative intensity using fluorescence dye-labeled CHI-KGN NPs (left knee) and CHI-KGN MPs (right knee) at various time points from rats in which OA was induced surgically 6 weeks before . The images reproduced with the permission from Elsevier Ltd. D) Effects of BBR-loaded CNs on protein expressions of caspase-3, Bcl-2 and Bax in rat articular cartilage from each experimental group. (viii) Effect of BBR-loaded CNs on histological changes in articular cartilage of the rat ACLT + MMx model . The images reproduced with the permission from Elsevier B.V. E) SEM of C-CP-Fe-bLf-NCs and chemical bonding analysed using FTIR analysis. (ix) Fluorescence and biodistribution of arthritic mice treated with (a) control diet and (b&c) Cy5.5 dye labeled AEC-CP-Fe-bLf-NC diet were fed orally 36 h prior to being euthanized and imaged. Arrows show the localization of NC in joint cartilage. (x) Schematic diagram illustrating the signaling mechanism modulated by AEC-CP-Fe-bLf-NCs in arthritis . The images reproduced with the permission from Elsevier Ltd.

Article Snippet: KGN coated with chitosan NPs using EDC/NHS catalysis methods (CHI-KGN) showed a distinct inhibitory effect on cartilage degeneration ( C) [ ].

Techniques: Polymer, In Vitro, Staining, In Vivo, Fluorescence, Imaging, Labeling, Control

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: Cancers

Article Title: In Vitro Systematic Drug Testing Reveals Carboplatin, Paclitaxel, and Alpelisib as a Potential Novel Combination Treatment for Adult Granulosa Cell Tumors

doi: 10.3390/cancers13030368

Figure Lengend Snippet: Patient-derived cell line characteristics.

Article Snippet: KGN, a human AGCT cell line derived from a 63-year-old patient heterozygous for FOXL2 c.402C > G, was used as an additional AGCT cell line to evaluate potential AGCT response [ ].

Techniques: Control

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

Journal: The Journal of Clinical Investigation

Article Title: Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

doi: 10.1172/JCI89857

Figure Lengend Snippet: (A and E) Confirmation of stable shRNA-mediated (A) or transient siRNA-mediated (E) gene expression silencing by Western blot in Huh7 cells. (B and G) DENV entry measured via luciferase assay 6 hours after infection. (C and H) DENV RNA replication monitored by luciferase activity every 24 hours following transfection of Huh7 cells with a Tet-inducible DNA-launched DENV replicon and induction by doxycycline for 6 hours (GND is a replication-incompetent DENV). Data are normalized to signal at 24 hours. (D and I) Infectious DENV production measured via luciferase assays by inoculation of naive cells with supernatants from stable or siRNA-transfected cells 48 hours after electroporation with DENV RNA. (F) Relative cell viability following gene expression knockdown measured by alamarBlue assays. (J and K) Cell viability (blue) and dose response of DENV infection (black) to more selective AAK1 (J) and GAK (K) inhibitors (structures shown) measured by luciferase assays 48 hours after infection. Data are plotted relative to vehicle control. Shown are representative experiments from at least 2 conducted. Individual experiments in B–D and F–K had 8–10 biological replicates; shown are means ± SD; ***P < 0.001 relative to corresponding NT control by 1-way ANOVA (B, D, F, G, and I) or 2-way ANOVA (C and H), followed by Dunnett’s multiple comparisons test.

Article Snippet: Blots were blocked and blotted with anti-AP1M1 (Abcam, catalog ab111135), anti-AP2M1 (Abcam, catalog ab75995), anti-GLuc (New England BioLabs, catalog E8023S), anti–phospho-AP2M1 (T156) (Cell Signaling, catalog 3843S), anti-AAK1 (Abcam, catalog ab134971), anti-GAK (MBL International, catalog MO573), and anti–β-actin (Sigma-Aldrich, catalog A3854) antibodies.

Techniques: shRNA, Expressing, Western Blot, Luciferase, Infection, Activity Assay, Transfection, Electroporation

Journal: The Journal of Clinical Investigation

Article Title: Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

doi: 10.1172/JCI89857

Figure Lengend Snippet: (A) Confirmation of siRNA-mediated gene expression silencing by quantitative PCR in Vero cells. Shown is normalized gene expression relative to GAPDH at 48 hours after transfection. (B and C) Relative cell viability measured by alamarBlue assays (B) and rVSV-GP EBOV infection of Vero cells measured by flow cytometry (C) at 20 hours after infection of AAK1- and GAK-depleted cells. ***P < 0.001 relative to NT (1-way ANOVA followed by Dunnett’s multiple comparisons test). (D) Dose response of rVSV-GP EBOV infection to 4-hour treatment with inhibitors measured by flow cytometry at 20 hours after infection in Vero cells. (E and F) Synergy/antagonism of sunitinib/erlotinib combination treatment on rVSV-GP EBOV infection (E) and cell viability (F). (G) Dose response to drug treatment in Huh7 cells under biosafety level 4 containment following 48 hours with EBOV infection (black) measured by immunostaining with an anti-GP antibody, and cell viability (blue) measured by Hoechst counterstain and quantified by a high-content imager. Data are plotted relative to vehicle control. All data shown are representative of at least 2 experiments; B–G have 3 biological replicates each. Shown in B–D and G are means ± SD.

Article Snippet: Blots were blocked and blotted with anti-AP1M1 (Abcam, catalog ab111135), anti-AP2M1 (Abcam, catalog ab75995), anti-GLuc (New England BioLabs, catalog E8023S), anti–phospho-AP2M1 (T156) (Cell Signaling, catalog 3843S), anti-AAK1 (Abcam, catalog ab134971), anti-GAK (MBL International, catalog MO573), and anti–β-actin (Sigma-Aldrich, catalog A3854) antibodies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Infection, Flow Cytometry, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

doi: 10.1172/JCI89857

Figure Lengend Snippet: Host kinases AAK1 and GAK regulate entry, assembly, and/or release of multiple RNA viruses through phosphorylation of the membrane trafficking adaptors AP1 (pink) and AP2 (purple). Sunitinib, erlotinib, and selective inhibitors of AAK1 and GAK disrupt these temporally distinct steps of the viral life cycle and act as broad-spectrum antivirals.

Article Snippet: Blots were blocked and blotted with anti-AP1M1 (Abcam, catalog ab111135), anti-AP2M1 (Abcam, catalog ab75995), anti-GLuc (New England BioLabs, catalog E8023S), anti–phospho-AP2M1 (T156) (Cell Signaling, catalog 3843S), anti-AAK1 (Abcam, catalog ab134971), anti-GAK (MBL International, catalog MO573), and anti–β-actin (Sigma-Aldrich, catalog A3854) antibodies.

Techniques: