Journal: Cells

Article Title: Kisspeptin Alleviates Human Hepatic Fibrogenesis by Inhibiting TGFβ Signaling in Hepatic Stellate Cells

doi: 10.3390/cells13191651

Figure Lengend Snippet: KPA treatment upregulates KISS1 and KISS1R expression in hPCLS. Human PCLS generated from patients with fibrotic liver biopsies were treated with KPA (3 nM, 100 nM), vehicle (PBS), or ALK5i (10 mM) for 48 or 72 h. Changes in ( A – C ) KISS1 and KISS1R ( D – F ) gene expression were determined by qPCR in patient livers. Data are shown from n = 3 patients. Results are expressed as mean +/− S.E.M. * p < 0.05 vs. control. ** p < 0.01 vs. control. a, p < 0.05 vs. PBS (vehicle); b, p < 0.05 vs. PBS (vehicle); and c, p < 0.05 vs. PBS (vehicle), for each time point. Two-way ANOVA was conducted, followed by multiple comparison test. ( G ) Representative confocal images of human hepatic stellate cells in MASH patient biopsies, immunostained for endogenous KISS1R (red) and desmin (green), a marker for stellate cells. Areas of colocalization (yellow) are shown in overlay; magnified images and trichrome staining of liver fibrotic section are shown on the right.

Article Snippet: Protein expression was determined using the following anti-human antibodies: anti-rabbit KISS1 (Protein Tech, Rosemont IL, USA catalog # 18375-1-AP; 1:1000), anti-rabbit GPR54 (Abcam, Cambridge, MA, USA catalog #ab137483; 1:1000), anti-mouse fibronectin (R&D Systems, Minneapolis, MN USA catalog # MAB19182; 1:1000).

Techniques: Expressing, Generated, Gene Expression, Control, Comparison, Marker, Staining

Journal: Cells

Article Title: Kisspeptin Alleviates Human Hepatic Fibrogenesis by Inhibiting TGFβ Signaling in Hepatic Stellate Cells

doi: 10.3390/cells13191651

Figure Lengend Snippet: KPA treatment decreases activation of human hepatic stellate LX-2 cells. Representative Western blots showing expression of endogenous ( A ) KISS1 protein ( n = 5 biological replicates) and ( B ) KISS1R protein expression ( n = 4 biological replicates). MDA-MB-231, SKBR3, and KISS1R-SKBR3 were used as reference for expression ( n = 4). ( C ) Secreted kisspeptin protein in culture media measured by ELISA (N-4 biological replicates). ( D – G ) Changes in fibrogenic gene expression in response to KPA (3 nM, 48 h) +/− TGFb (5 ng/mL, 48 h). ( n = 4–6 biological replicates) ( H ) Western blot analysis and ( I , J ) densitometric analyses of blots, showing changes in fibrogenic protein in response to KPA (3 nM, 72 h) +/− TGFb (5 ng/mL, 72 h). ( n = 4 biological replicates) Results are expressed as mean +/− S.E.M. * p < 0.05 vs. control. ** p < 0.01 vs. control. *** p < 0.001 vs. control. **** p < 0.0001 vs. control. One-way ANOVA was conducted, followed by multiple comparison test.

Article Snippet: Protein expression was determined using the following anti-human antibodies: anti-rabbit KISS1 (Protein Tech, Rosemont IL, USA catalog # 18375-1-AP; 1:1000), anti-rabbit GPR54 (Abcam, Cambridge, MA, USA catalog #ab137483; 1:1000), anti-mouse fibronectin (R&D Systems, Minneapolis, MN USA catalog # MAB19182; 1:1000).

Techniques: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression, Control, Comparison

Journal: Cells

Article Title: Kisspeptin Alleviates Human Hepatic Fibrogenesis by Inhibiting TGFβ Signaling in Hepatic Stellate Cells

doi: 10.3390/cells13191651

Figure Lengend Snippet: KPA treatment decreases collagen secretion, cell migration, and proliferation of LX-2 cells. Quantification of ( A ) TGFβ-induced collagen secretion following VEH, KPA (3 nM), TGFβ (5 ng/mL), and KPA + TGFβ treatment ( n = 4 biological replicates); ( B ) cell migration following VEH and KPA (3 nM) treatment. KPA treatment decreases ( n = 4 biological replicates) ( C ) LX-2 cell proliferation following VEH and KPA (3 nM) treatment, as assessed using BrdU ( n = 11 biological replicates) and ( D ) soft agar colony formation following VEH, KPA (3 nM, 100 nM) treatment ( n = 11 biological replicates) t. Gene expression by qPCR analysis showing the effect of KPA (3 nM, 48 h) treatment on inflammatory markers ( E , F ) IL6 and TNFA , and ( G , H ) KISS1 and KISS1R ( n = 3–5 biological replicates). Results are expressed as mean +/− S.E.M. Student’s unpaired t -test or one-way ANOVA was performed, followed by multiple comparison test. * p < 0.05 vs. control. ** p < 0.01 vs. control. *** p < 0.001 vs. control. **** p < 0.0001 vs. control.

Article Snippet: Protein expression was determined using the following anti-human antibodies: anti-rabbit KISS1 (Protein Tech, Rosemont IL, USA catalog # 18375-1-AP; 1:1000), anti-rabbit GPR54 (Abcam, Cambridge, MA, USA catalog #ab137483; 1:1000), anti-mouse fibronectin (R&D Systems, Minneapolis, MN USA catalog # MAB19182; 1:1000).

Techniques: Migration, Gene Expression, Comparison, Control

Journal: Cells

Article Title: Kisspeptin Alleviates Human Hepatic Fibrogenesis by Inhibiting TGFβ Signaling in Hepatic Stellate Cells

doi: 10.3390/cells13191651

Figure Lengend Snippet: KPA treatment decreases TGFβ signaling in human HSCs. ( A ) TGFβ-induced SMAD phosphorylation ( n = 5 biological replicates) and ( B ) SNAIL expression ( n = 4 biological replicates) was observed, following KPA (3 nM, 72 h) treatment, as determined by Western blot analysis; densitometric analysis of blots are shown below. ( C ) TGFβ-induced SNAI1 mRNA expression following KPA (3 nM, 48 h) determined by qPCR ( n = 4 biological replicates). ( D ) TGFβ-induced SMAD 2/3 phosphorylation cells were pretreated with OA (5 nM) for 3 h, following TGFβ1 (5 ng/mL), and/or KPA (3 nM) for 48 as determined by Western blot analysis. OA treatment did not impact cell viability (see ) ( n = 4 biological replicates). ( E ) Schematic showing proposed signaling pathways by which KISS1R activation with kisspeptin (KP) suppresses hepatic fibrosis in hepatic stellate cells. Results are expressed as mean +/− S.E.M. * p < 0.05 vs. control. ** p < 0.01 vs. control. *** p < 0.001 vs. control. One-way ANOVA was performed, followed by multiple comparison test.

Article Snippet: Protein expression was determined using the following anti-human antibodies: anti-rabbit KISS1 (Protein Tech, Rosemont IL, USA catalog # 18375-1-AP; 1:1000), anti-rabbit GPR54 (Abcam, Cambridge, MA, USA catalog #ab137483; 1:1000), anti-mouse fibronectin (R&D Systems, Minneapolis, MN USA catalog # MAB19182; 1:1000).

Techniques: Phospho-proteomics, Expressing, Western Blot, Protein-Protein interactions, Activation Assay, Control, Comparison

Journal: Journal of Ovarian Research

Article Title: Bone marrow mesenchymal stem cells expressing Neat-1, Hotair-1, miR-21, miR-644, and miR-144 subsided cyclophosphamide-induced ovarian insufficiency by remodeling the IGF-1–kisspeptin system, ovarian apoptosis, and angiogenesis

doi: 10.1186/s13048-024-01498-x

Figure Lengend Snippet: Effect of BM-MSCs transplantation on the expression of ovarian Kisspeptin system of the POI rats ( A – I ). Representative images from the anti-Kiss1 protein immunostained rat ovarian section of the CON ( A , B ), POI ( C , D ), and POI + BM-MSCs groups ( E , F ) lower and higher magnification, respectively, showing cytoplasmic immune expression of germinal epithelium (GE), interstitial stromal cells (IS), and granulosa lutein cells (GL) as intense, faint, and moderate immunoreactivity in CON, POI, and POI + BM-MSCs groups, respectively. Scale bars A , C , E = 200 μm; B , D , F = 50 μm, G . Relative expression of ovarian mRNA Kiss-r1, H. Relative expression of ovarian mRNA Kiss-1, and I . Area % of ovarian Kiss-1 positive immunostaining. The values represent the average of 6 to 8 rats in each group ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Immunohistochemistry for VEGF and Kiss-1 was done using a primary polyclonal rabbit anti-VEGF-A (ABclonal, Cat. No. # A0280) and a primary rabbit polyclonal anti-kisspeptin antibody (Bioss, Cat. No. # bs-0749R) and their related secondary antibodies.

Techniques: Transplantation Assay, Expressing, Immunostaining

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Primary and secondary antisera used for Western blot, IFL, and IHC.

Article Snippet: Kiss1R rabbit polyclonal (BS-2501R, Bioss Antibodies, USA) , 1:200 (IF) 1:50 (IHC).

Techniques: Western Blot, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of Kiss1R, Kiss1, Cyp19, GnRH , and miR expression in mediobasal hypothalamus homogenates. Representative Western blots ( n = 4) for Kiss1R, Kiss1, and Cyp19 (A) and normalization of Kiss1R (B) , Kiss1 (C) , and Cyp19 signals (D) carried out against α-tubulin. Quantitative expression ( n = 6) of GnRH (E) and miR-132-3p , Mir-145-5p , let-7a-5p , and let-7b-5p (F–I) carried out by qPCR. Data are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin / U6 and the control group used as reference sample. C, control group (placebo injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA (SA), animals first received SR141716A and 30 min later received AEA treatment. ** p < 0.01, * p < 0.05 vs. the control group.

Article Snippet: Kiss1R rabbit polyclonal (BS-2501R, Bioss Antibodies, USA) , 1:200 (IF) 1:50 (IHC).

Techniques: Expressing, Western Blot, Standard Deviation, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Expression and distribution of Kiss1R and Kiss1 proteins in the hypothalamic arcuate nucleus (ARC). Representative images ( n = 4 for each animal group). Nissl staining showing ARC localization at ×20 magnification (A) ; IHC of Kiss1R expression modulation in ARC, ×20 magnification (B) ; IF analysis of the Kiss1 expression in ARC, ×40 magnification (C) . C, control; KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA, animals first received SR141716A and 30 min later received AEA treatment. 3V, third ventricle. ** p < 0.01; * p < 0.05.

Article Snippet: Kiss1R rabbit polyclonal (BS-2501R, Bioss Antibodies, USA) , 1:200 (IF) 1:50 (IHC).

Techniques: Expressing, Staining, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of the KS by KP (A–C) and AEA (D–F) in rat testis. Representative Western blots for Kiss1R and Kiss1 (A, D) and normalization of Kiss1R (B, E) and Kiss1 signals (C, F) carried out against α-tubulin. N = 6 different animals in KP treatments and n = 4 in AEA treatments; data are expressed as protein OD/tubulin OD ± standard deviation. C, control group (placebo-injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA, animals first received SR141716A and 30 min later received AEA treatment. * p < 0.05.

Article Snippet: Kiss1R rabbit polyclonal (BS-2501R, Bioss Antibodies, USA) , 1:200 (IF) 1:50 (IHC).

Techniques: Western Blot, Standard Deviation, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Distribution of Kiss1 and Kiss1R in rat testis treated with KP and AEA. Double immunofluorescence of Kiss1 (red) and Kiss1R (green) expression in control (C), kisspeptin (KP), anandamide (AEA), and SR141716A (SR + AEA)-treated animals ( n = 4 for each group). The last column shows the representative images of DAPI staining on the same animals. Magnification = C ×40; KP, AEA, and SR+ AEA, ×63. * Elongated spermatids, ↑ spermatocytes, ^ spermatogonia, ° interstitium.

Article Snippet: Kiss1R rabbit polyclonal (BS-2501R, Bioss Antibodies, USA) , 1:200 (IF) 1:50 (IHC).

Techniques: Immunofluorescence, Expressing, Staining