human ovarian cancer granulosa cell line (kgn) cells  (Procell Inc)

Journal: Frontiers in Immunology

Article Title: The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment

doi: 10.3389/fimmu.2021.685724

Figure Lengend Snippet: NF-κB pathway was activated with treatment of LPS in the KGN cells. (A) After treatment with LPS (200 ng/mL) for 4 h or 6 h, the relative expression of p65 was measured by RT-qPCR (4 h: P = 0.0257, 6 h: P = 0.0074). (B) The phosphorylation levels of p65 in KGN cells with the treatment of LPS (200 ng/mL) for 6 h and ATP (4 mM) for 50 min. (C) After treatment with LPS (200 ng/mL) for 4 h or 6 h, the relative expression of TLR4 was measured by RT-qPCR (4 h: P = 0.0096, 6 h: P = 0.0086). (D) The localization of p65 in KGN cells with LPS (200 ng/mL) stimulation for 3 h by immunofluorescent assays (p65, red; DAPI, blue; scale bar, 20 μm). *P < 0.05, **P < 0.01 and ***P < 0.001. *P < 0.05 was considered statistically significant.

Article Snippet: Human ovarian cancer granulosa cell line KGN cells (Procell CL-0603) ( ) were provided by Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment

doi: 10.3389/fimmu.2021.685724

Figure Lengend Snippet: NLRP3 inflammasomes were activated in KGN cells with LPS stimulation. (A) The mRNA level of IL-1β in primary human GCs treated with LPS (200 ng/mL) for 4 h was measured by RT-qPCR assays (P < 0.0001). (B) With LPS stimulation (200 ng/mL) in KGN cells, IL-1β mRNA levels were detected by RT-qPCR assays (4 h: P = 0.0004, 6 h: P = 0.0271). (C) The mRNA level of NLRP3 in KGN cells stimulated with LPS (200 ng/mL) (4 h: P = 0.0024, 6 h: P = 0.0131). (D) NLRP3 protein levels in KGN cells with LPS treatment (200 ng/mL) for 2, 4, 6, 12, and 24 h. (E) The protein levels of NLRP3, ASC, pro-Caspase-1, and Caspase-1 in KGN cells were treated with LPS (200 ng/mL) for 12 h and ATP (4 mM) for 50 min. (F) Immunofluorescent staining for co-localization of NLRP3 with ASC in KGN cells with LPS treatment (200 ng/mL) for 3 h and ATP (4 mM) for 50 min. (NLRP3, green; ASC, red; DAPI, blue; scale bar, 20μm). (G) Immunofluorescent staining for co-localization of NLRP3 with mitochondria in the KGN cells with LPS stimulation (200 ng/mL) for 3 h and ATP (4 mM) for 50 min (NLRP3, green; MitoTracker indicated mitochondria, red; DAPI, blue; scale bar, 100μm). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. *P < 0.05 was considered statistically significant.

Article Snippet: Human ovarian cancer granulosa cell line KGN cells (Procell CL-0603) ( ) were provided by Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Quantitative RT-PCR, Staining

Journal: Frontiers in Immunology

Article Title: The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment

doi: 10.3389/fimmu.2021.685724

Figure Lengend Snippet: NF-κB pathway and NLRP3 inflammasomes in KGN cells were activated by stimulation of with follicular fluid from PCOS patients. (A) With treatment of follicular fluid in KGN cells of PCOS patients and controls for 3 h, the phosphorylation levels of p65 were measured by western blotting assays. (B) The localization of p65 in KGN cells with follicular fluid treatment of PCOS patients and controls for 3 h by immunofluorescent assays. (p65, red; DAPI, blue; scale bar, 50 μm). (C) The KGN cells were treated with follicular fluid of PCOS patients and controls for 3 h, and IL-1β mRNA levels were measured by RT-qPCR (P = 0.0097). (D) The mRNA level of NLRP3 was detected in KGN cells with treatment of follicular fluid of PCOS patients and controls for 3 h (P = 0.0011). (E) NLRP3 inflammasome-related proteins (NLRP3, ASC, pro-Caspase-1, and Caspase-1) were measured by western blotting assays. (F) With treatment of follicular fluid of PCOS patients and controls for 3 h, the localization of NLRP3 in KGN cells is measured by immunofluorescent assays (NLRP3, green; DAPI, blue; scale bar, 50 μm). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. *P < 0.05 was considered statistically significant.

Article Snippet: Human ovarian cancer granulosa cell line KGN cells (Procell CL-0603) ( ) were provided by Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Phospho-proteomics, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment

doi: 10.3389/fimmu.2021.685724

Figure Lengend Snippet: Follicular fluid from PCOS patients and LPS impaired mitochondria structure and function, caused oxidative stress, and arrested cellular proliferation. (A) Immunofluorescence imaging of mitochondria in GCs from PCOS patients and controls (MitoTracker, red; DAPI, blue; scale bar, 20 μm). (B) Immunofluorescence imaging of mitochondrial morphology in KGN cells with LPS stimulation (200 ng/mL) for 3 h and ATP (4 mM) for 50 min (MitoTracker indicated mitochondria, red; DAPI, blue; scale bar, 20 μm). (C) Immunofluorescence imaging of mitochondrial morphology in KGN cells incubated with follicular fluid for 3 h (MitoTracker indicated mitochondria, red; DAPI, blue; scale bar, 20 μm). (D) After DHE staining, flow cytometry assays were used to detect ROS levels in KGN cells with LPS (200 ng/mL) treatment for 3 h and ATP (4 mM) for 50 min. (E) After DHE staining, flow cytometry assays were used to detect ROS levels in KGN cells with follicular fluid stimulation for 3 h. (F) Immunofluorescence imaging of EdU to indicate the KGN cells proliferation with LPS (200 ng/mL) treatment for 3 h and ATP (4 mM) for 50 min (EdU, green; DAPI, blue; scale bar, 50 μm). (G) Immunofluorescence imaging of EdU to indicate the KGN cells proliferation with follicular fluid treatment for 3 h (EdU, green; DAPI, blue; scale bar, 50 μm). (H) With EdU staining, flow cytometry assays were used to detect fluorescence in KGN cells with LPS (200 ng/mL) stimulation for 3 h and ATP (4 mM) for 50 min treatment. (I) After EdU staining, flow cytometry assays were used to detect fluorescence in KGN cells with treatment of follicular fluid for 3 h. **P < 0.01. *P < 0.05 was considered statistically significant.

Article Snippet: Human ovarian cancer granulosa cell line KGN cells (Procell CL-0603) ( ) were provided by Procell Life Science&Technology Co., Ltd (Wuhan, China).

Techniques: Immunofluorescence, Imaging, Incubation, Staining, Flow Cytometry, Fluorescence