Journal: Mediators of Inflammation

Article Title: Resolution of PMA-Induced Skin Inflammation Involves Interaction of IFN- γ and ALOX15

doi: 10.1155/2013/930124

Figure Lengend Snippet: Neutralization of endogenous IFN- γ decreased the infiltration of inflammatory cells and the production of inflammatory cytokines in the skin. Mice were sacrificed 3 hours, 4 days, or 7 days after the third PMA treatment. (a) Skin sections were stained with anti-CD11b, anti-Gr1, or anti-FSP1 (red). Nucleoli were stained with DAPI (blue). Scale Bar, 50 μ m. (b) The expression levels of IL-6, TNF- α , MCP-1, IL-1 α , and IL-1 β of the skin from control group or anti-IFN- γ group were detected at indicated time points. * P < 0.05, compared to control group.

Article Snippet: For immunofluorescence analysis, sections were stained with rat anti-mouse CD11b (1 : 100; BD Pharmingen, USA), rat anti-mouse granulocyte differentiation antigen 1 (Gr1, 1 : 100; BD Pharmingen, USA), rabbit anti-mouse fibroblast-specific protein-1 (FSP1, 1 : 100; BD Pharmingen, USA), and subsequently rhodamine-labeled secondary antibody and counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma, USA).

Techniques: Neutralization, Staining, Expressing

Journal: PLoS ONE

Article Title: Cadherin-9 Is a Novel Cell Surface Marker for the Heterogeneous Pool of Renal Fibroblasts

doi: 10.1371/journal.pone.0000657

Figure Lengend Snippet: Sources of the used antibodies

Article Snippet: anti FSP1/S100A4 Ab-8 , Dianova (Hamburg, Germany).

Techniques: Transduction

Journal: PLoS ONE

Article Title: Cadherin-9 Is a Novel Cell Surface Marker for the Heterogeneous Pool of Renal Fibroblasts

doi: 10.1371/journal.pone.0000657

Figure Lengend Snippet: (A) Immunohistochemical staining of two consecutive kidney cryostat sections with antisera against cadherin-9 and FSP1, a well-known renal fibroblast marker, showed that cadherin-9 positive cells also express FSP1. Staining of FSP1, however, could be also observed in the cadherin-9 negative glomerulum and in some tubular cells. The inserts in the left panel are enlarged in the right panel. (B) Cadherin-9 is not expressed by endothelial cells, as demonstrated by double immunofluorescence staining with the endothelial cell marker CD31. But cadherin-9 expression partially overlaps with CD45 as well as with α-smooth muscle actin. Cells which express only α-smooth muscle actin or CD45 are marked with an arrowhead, whereas cells which express only cadherin-9 with an arrow. Co-localization of α-smooth muscle actin or CD45 with cadherin-9 are indicated by double arrows. The different magnifications in (A) and (B) are shown by different bars.

Article Snippet: anti FSP1/S100A4 Ab-8 , Dianova (Hamburg, Germany).

Techniques: Immunohistochemical staining, Staining, Marker, Double Immunofluorescence Staining, Expressing

Journal: PLoS ONE

Article Title: Cadherin-9 Is a Novel Cell Surface Marker for the Heterogeneous Pool of Renal Fibroblasts

doi: 10.1371/journal.pone.0000657

Figure Lengend Snippet: Together with FSP1, α-smooth muscle actin (α sm actin) and CD45, cadherin-9 is a novel cell surface marker, which can be used to differentiate the depicted renal fibroblast subpopulations from other renal cell types such as epithelial and endothelial cells, smooth muscle cells, monocytes or macrophages.

Article Snippet: anti FSP1/S100A4 Ab-8 , Dianova (Hamburg, Germany).

Techniques: Marker

Journal: Redox Biology

Article Title: Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

doi: 10.1016/j.redox.2024.103388

Figure Lengend Snippet: The primers.

Article Snippet: Primary antibodies against caspase-4/11, FSP1, GCH1 and GPx4 were obtained from Zenbio (Chengdu, China).

Techniques:

Journal: Redox Biology

Article Title: Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

doi: 10.1016/j.redox.2024.103388

Figure Lengend Snippet: CSE triggered lipid peroxidation and pyroptosis in Beas-2b cells. (A) The LDH release rate was measured by the LDH cytotoxicity detection kits. (B) The GSH/GSSG ratio in treated cells were measured with the corresponding kit. (C) The levels of MDA in treated cells were measured with the corresponding kit. (D–F) ELISAs showed that CSE increased the levels of IL-1α, IL-1β and IL-18 in culture medium in a dose-dependent manner. (G) Lipid peroxidation in cells was examined by Liperfluo staining and observed by confocal microscopy (400 × ); ROS production was examined with a DCFH-DA probe and observed with a fluorescence microscope (200 × ). (H) The expression levels of xCT, GPx4, NLRP3, cleaved caspase-1/4, ASC, and cleaved IL-1β were detected by western blotting and analyzed by ImageJ. (I) The expression levels of FSP1 and GCH1 were tested by western blotting and analyzed by Image J. (J) The number of living cells and dead cells were detected by Calcein/PI staining (200 × ). (K) TEM showed the pyroptotic body formation, cell membrane and mitochondria. Green arrows indicate normal mitochondria, blue arrows indicate normal cell membranes, red arrows indicate abnormal mitochondria, and white arrows indicate damaged cell membranes (0.5 μm). NS p＞0.05, ∗p＜0.05,∗∗p＜0.01 compared with the control group.

Article Snippet: Primary antibodies against caspase-4/11, FSP1, GCH1 and GPx4 were obtained from Zenbio (Chengdu, China).

Techniques: Staining, Confocal Microscopy, Fluorescence, Microscopy, Expressing, Western Blot, Membrane, Control

Journal: Redox Biology

Article Title: Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

doi: 10.1016/j.redox.2024.103388

Figure Lengend Snippet: Fer-1 alleviated lipid peroxidation induced by CSE in Beas-2b cells. (A) CCK-8 assays indicated that Fer-1 reversed the CSE-induced death of Beas-2b cells. (B) LDH assays showed that Fer-1 suppressed LDH release caused by CSE. (C, D) The effects of Fer-1 on GSH/GSSG ratio and MDA production were determined by the corresponding kits. (E, F) The ability of Fer-1 to inhibit lipid peroxidation and ROS production was measured by Liperfluo and DCFH-DA. (G) Redox lipidomics was performed by LC-MS to differentiate different lipid peroxidation species in CSE- or Fer-1-treated cells. (H) The relative quantitative values of differential metabolites were normalized transformed and clustered. (I) The expression levels of xCT and GPx4 were measured by western blotting. (J) The mRNA levels of xCT and GPx4 were analyzed by qPCR. (K) Western blot showed Fer-1 failed to inhibit the degradation of xCT and GPx4. ∗p＜0.05,∗∗p＜0.01 compared with the control group; NS p＞0.05, #p＜0.05,##p＜0.01 compared with the CSE-treated group.

Article Snippet: Primary antibodies against caspase-4/11, FSP1, GCH1 and GPx4 were obtained from Zenbio (Chengdu, China).

Techniques: CCK-8 Assay, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Expressing, Western Blot, Control

Journal: Redox Biology

Article Title: Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

doi: 10.1016/j.redox.2024.103388

Figure Lengend Snippet: Fer-1 protected against CS-induced inflammation and lipid peroxidation in the lungs. (A) Exudative inflammatory cells in BALF were examined by Giemsa staining and analyzed by optical microscopy (200 × ). (B) Pathologic changes were assessed by H&E staining (200 × ). (C) Mucus secretion by the airway epithelium was examined by PAS staining (200 × ). (D) The level of ROS in inflammatory cells was analyzed with a DCFH-DA probe and observed by fluorescence microscopy (200 × ). (E, F) The GSH and MDA contents were measured with detection kits. (G) The production of 4-HNE was quantified by immunohistochemical staining (200 × ). (H) The expression levels of xCT and GPx4 were assessed by western blotting. ∗∗p＜0.01 compared with the control group; #p＜0.05,##p＜0.01 compared with the CS-exposed group.

Article Snippet: Primary antibodies against caspase-4/11, FSP1, GCH1 and GPx4 were obtained from Zenbio (Chengdu, China).

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

Journal: Redox Biology

Article Title: Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

doi: 10.1016/j.redox.2024.103388

Figure Lengend Snippet: xCT downregulation contributed to lipid peroxidation and pyroptosis in Beas-2b cells. (A) The knockdown effects of three xCT-siRNAs were assessed by western blotting. (B) Cell viability was determined by a CCK-8 kit. (C, D) The levels of IL-1β and IL-18 were measured by ELISAs. (E, F) The contents of GSH and MDA were measured with corresponding kits. (G, H) Western blot analysis showed that xCT knockdown decreased the expression of components of the xCT/GPx4 axis and increased the expression of pyroptosis-related proteins. (I) Immunofluorescence staining was used to assess the expression of N-GSDMD, and the results were analyzed by immunofluorescence microscopy (200 × ). ∗p＜0.05,∗∗p＜0.01 compared with the respective control group; #p＜0.05,##p＜0.01 compared with CSE-exposed NC group.

Article Snippet: Primary antibodies against caspase-4/11, FSP1, GCH1 and GPx4 were obtained from Zenbio (Chengdu, China).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Expressing, Immunofluorescence, Staining, Microscopy, Control

Journal: Redox Biology

Article Title: Lipid peroxidation triggered by the degradation of xCT contributes to gasdermin D-mediated pyroptosis in COPD

doi: 10.1016/j.redox.2024.103388

Figure Lengend Snippet: Cigarette smoke induced lipid peroxidation and pyroptosis through the ubiquitination and degradation of xCT. (A) Lipid peroxidation due to the suppression of the xCT/GPX4 axis facilitated pyroptosis in cigarette smoke-induced COPD. (B) The process of xCT protein ubiquitination. In an ATP-dependent manner, Ub is attached to the ubiquitin-activating enzyme E1 and then delivered to the ubiquitin-binding enzyme E2. Next, the E3 ligase acts as a bridge, identifying E2-Ub and the substrate xCT to facilitate the binding of Ub to xCT. Finally, the ubiquitin chain is identified by the 26S proteasome, leading to the degradation of xCT.

Article Snippet: Primary antibodies against caspase-4/11, FSP1, GCH1 and GPx4 were obtained from Zenbio (Chengdu, China).

Techniques: Binding Assay

Journal: Scientific reports

Article Title: Exploring Liraglutide's mechanism in reducing renal fibrosis: the Fsp1-CoQ10-NAD(P)H pathway.

doi: 10.1038/s41598-025-85658-z

Figure Lengend Snippet: Fig. 5. The ferroptosis in kidney tissue of db/db mice. (A) The ultrastructure of mitochondria observed by TEM. (B–C) The activity of GSH and GSH-Px in the kidney tissue (n = 5). (D) The expression and statistics of GPX-4 and Fsp1 proteins (n = 6). (E) The immunohistochemical staining of GPX-4 protein (scale bar = 100 μm, n = 3). (F–G) The ratio of NAD+/NADH and CoQ10 (H2)/CoQ10 (n = 4). The results are presented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. db/m group. #p < 0.05, ##p < 0.01 vs. db/db group.

Article Snippet: The blots were then incubated with primary antibodies overnight at 4 °C, including Collagen-I (GB112543, Servicebio), Collagen-III (GB11023, Servicebio), TGF-β(bs-0086R, Bioss), NOX-4 (A00403, Boster), 4-HNE (ARG23717, Arigo), MDA (ab243066, Abcam), TfR1(3-6800, Invitrogen), DMT1(abs112967, Absin), FPN1(MTP11-A, Alpha Diagnostic International), FTH(ab183781, Abcam), FTL(ab218400, Abcam), GPX-4, Fsp1, GAPDH (GB15002, Servicebio), β-actin (GB15001, Servicebio), α-Tubulin (GTX628802, GeneTex).

Techniques: Activity Assay, Expressing, Immunohistochemical staining, Staining

Journal: Scientific reports

Article Title: Exploring Liraglutide's mechanism in reducing renal fibrosis: the Fsp1-CoQ10-NAD(P)H pathway.

doi: 10.1038/s41598-025-85658-z

Figure Lengend Snippet: Fig. 6. Liraglutide inhibited HG-induced ferroptosis in HK-2 cells. (A–B) The cell viability of HK-2 cells treated with HG with 0, 10, 20, 30, and 50 mM for 24 h and 48 h respectively (n = 5, **p < 0.01 vs. Con group). (C) The cell viability of HK-2 cells treated HG (30 mM), RSL3 (5 µM), liraglutide (LIRA, 500 nM), and Fer-1 (5 µM) (n = 6). (D–E) The cell viability of HK-2 cells detected by flow cytometry (n = 3). (F) The ultrastructure of HK-2 cells mitochondria was observed by TEM (scale bar = 1.0 μm, n = 3). (G) The JC-1 staining of HK-2 cells (scale bar = 100 μm, n = 3). (H–I) The fluorescence absorption spectrum of BODIPY 581/591 C11 (scale bar = 100 μm, n = 5). (J) The expression and statistics of GPX-4 and Fsp1 proteins (n = 3). (K) The immunofluorescence double label staining of Fsp1 and 4-HNE (scale bar = 50 μm, n = 3). (L–M) The ratio of NAD+/NADH and CoQ10 (H2)/CoQ10 (n = 4). The results are presented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. Con group. #p < 0.05, ##p < 0.01 vs. HG group.

Article Snippet: The blots were then incubated with primary antibodies overnight at 4 °C, including Collagen-I (GB112543, Servicebio), Collagen-III (GB11023, Servicebio), TGF-β(bs-0086R, Bioss), NOX-4 (A00403, Boster), 4-HNE (ARG23717, Arigo), MDA (ab243066, Abcam), TfR1(3-6800, Invitrogen), DMT1(abs112967, Absin), FPN1(MTP11-A, Alpha Diagnostic International), FTH(ab183781, Abcam), FTL(ab218400, Abcam), GPX-4, Fsp1, GAPDH (GB15002, Servicebio), β-actin (GB15001, Servicebio), α-Tubulin (GTX628802, GeneTex).

Techniques: Flow Cytometry, Staining, Fluorescence, Expressing, Immunofluorescence

Journal: Scientific reports

Article Title: Exploring Liraglutide's mechanism in reducing renal fibrosis: the Fsp1-CoQ10-NAD(P)H pathway.

doi: 10.1038/s41598-025-85658-z

Figure Lengend Snippet: Fig. 7. Liraglutide inhibited HG-induced ferroptosis via Fsp1-CoQ10. (A) The cell viability of HK-2 cells (n = 6, **p < 0.01 vs. HG group, #p < 0.05 vs. HG + iFsp1 group). (B–C) The fluorescence absorption spectrum of BODIPY 581/591 C11 (scale bar = 100 μm, n = 3, **p < 0.01 vs. HG group, ##p < 0.01 vs. HG + iFsp1 group). (D) The cell viability of HK-2 cells (n = 6, **p < 0.01 vs. HG + LIRA group). (E–F) The fluorescence absorption spectrum of BODIPY 581/591 C11 (scale bar = 100 μm, n = 3, **p < 0.01 vs. HG + LIRA group). (G) Flow cytometry results of BODIPY 581/591 C11 probe. (H) The immunofluorescence double label staining of Fsp1 and 4-HNE (scale bar = 50 μm, n = 3). (I–J) The expression and statistics of GPX-4 and Fsp1 proteins (n = 3, **p < 0.01 vs. HG + LIRA group). (K–L) The ratio of NAD+/NADH and CoQ10 (H2)/CoQ10 (n = 4, *p < 0.05 vs. HG + LIRA group). The results are presented as the mean ± SEM.

Article Snippet: The blots were then incubated with primary antibodies overnight at 4 °C, including Collagen-I (GB112543, Servicebio), Collagen-III (GB11023, Servicebio), TGF-β(bs-0086R, Bioss), NOX-4 (A00403, Boster), 4-HNE (ARG23717, Arigo), MDA (ab243066, Abcam), TfR1(3-6800, Invitrogen), DMT1(abs112967, Absin), FPN1(MTP11-A, Alpha Diagnostic International), FTH(ab183781, Abcam), FTL(ab218400, Abcam), GPX-4, Fsp1, GAPDH (GB15002, Servicebio), β-actin (GB15001, Servicebio), α-Tubulin (GTX628802, GeneTex).

Techniques: Fluorescence, Flow Cytometry, Immunofluorescence, Staining, Expressing

Journal: Scientific reports

Article Title: Exploring Liraglutide's mechanism in reducing renal fibrosis: the Fsp1-CoQ10-NAD(P)H pathway.

doi: 10.1038/s41598-025-85658-z

Figure Lengend Snippet: Fig. 8. The schematic diagram illustrates the involvement of ferroptosis in the kidneys of db/db mice. HG has been found to induce renal fibrosis and ferroptosis in diabetic mice. This is attributed to the increased levels of TfR1 and DMT1, as well as the decreased level of FPN1, which results in elevated LIP in the kidney. The excess of labile iron pool (LIP), primarily consisting of Fe2+, exacerbates the generation of lipid ROS in response to the Fenton reaction. Furthermore, the reduced expression of Fsp1 hinders the CoQ10 to its reduced form, CoQ10(H2), thereby impeding its antioxidant effects. Additionally, the imbalance in the Xc− system disrupts the exchange and synthesis of GSH, leading to the inhibition of GSH-dependent antioxidant defences and ultimately resulting in ferroptosis. Liraglutide has the potential to enhance the expression of Fsp1 and ameliorate the Xc− system, thereby mitigating the ferroptosis response in the renal tissue of db/db mice.

Article Snippet: The blots were then incubated with primary antibodies overnight at 4 °C, including Collagen-I (GB112543, Servicebio), Collagen-III (GB11023, Servicebio), TGF-β(bs-0086R, Bioss), NOX-4 (A00403, Boster), 4-HNE (ARG23717, Arigo), MDA (ab243066, Abcam), TfR1(3-6800, Invitrogen), DMT1(abs112967, Absin), FPN1(MTP11-A, Alpha Diagnostic International), FTH(ab183781, Abcam), FTL(ab218400, Abcam), GPX-4, Fsp1, GAPDH (GB15002, Servicebio), β-actin (GB15001, Servicebio), α-Tubulin (GTX628802, GeneTex).

Techniques: Expressing, Inhibition