human gdf 15 elisa kit  (Elabscience Biotechnology)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: The relationship between GDF15 and tubulointerstitial fibrosis mediated by TGF β in HK2 cells. (A–D) Screening the differentially expressed genes related to TIF. (E, F) Western blot analysis and quantification of fibronectin, collagen I, and GDF15 in HK2 cells stimulated with 10 ng/mL of TGF‐β1 for 12, 24, and 48 h. (G) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in different groups as indicated. The relative expression levels of the indicated proteins were normalised to that of β‐actin ( n = 3).* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Western Blot, Quantitative RT-PCR, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 expression in the fibrotic kidney of patients with CKD and UUO mice. (A) Immunohistochemical staining for Collagen I, GDF15 and representative images of Masson's trichrome staining in the kidneys of patients with CKD. (B) Correlation between Collagen I, serum creatinine, eGFR and the expression of renal GDF15 in patients with CKD. (C, E) Western blot analysis and quantification of the GDF15 and GFRAL levels in UUO mice. The relative expression levels of the indicated proteins were normalised to that of β‐actin. (D) Immunohistochemical staining for Collagen I, GDF15 and representative images of Masson's trichrome staining, Picrosirius red staining in the kidneys of UUO mice. (F) Quantitative analysis of immunohistochemistry for GDF15 in the cortex and medulla of UUO mice ( n = 6). (G) Dynamic changes in serum GDF15 concentrations at different time points following UUO intervention in mice and GDF15 levels in the supernatant of TGF‐β1‐stimulated HK2 cells ( n = 3). (H) Co‐staining of GDF15 with AGTR1: A distinct yellow signal in the merged image indicates that GDF15 is primarily localised in AGTR1‐positive tubular epithelial cells. (I) Co‐staining of GDF15 with the proximal tubule marker LTL: Absence of a yellow overlap in the merged image suggests weak GDF15 expression in proximal tubules. (J) Co‐staining of GDF15 with the collecting duct marker AQP2: No overlap signal is observed in the merged image, indicating minimal GDF15 expression in collecting ducts. (K) Co‐staining of GDF15 with WT1, a podocyte marker: The merged image shows no co‐localisation, suggesting that GDF15 is not expressed in glomerular podocytes. (L) Co‐staining of GFRAL with NCC: Strong overlap of red and green signals in the merged image indicates predominant localisation of GFRAL in the distal tubules. (M) Co‐staining of GFRAL with AQP2: No overlapping signal is observed in the merged image, demonstrating that GFRAL is not expressed in collecting ducts. Scale bars for all merged images = 50 μm ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Immunohistochemistry, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: Downregulation of GDF15 enhances TGF β‐stimulated TIF in vitro and UUO‐induced TIF in vivo. (A) Representative images of Masson's trichrome staining, Picrosirius red staining and immunohistochemical staining for Collagen I, Fibronectin, and macrophage infiltration in the kidney homogenates from sham and UUO mice injected with shNC or sh556. (B) Quantitative analysis of immunohistochemistry for Collagen I, Fibronectin and macrophage in different groups as indicated. (C, E) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vitro ( n = 3). The relative expression levels of the indicated proteins were normalised to that of β‐actin. (G) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vitro ( n = 3). (D, F) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vivo. The relative expression levels of the indicated proteins were normalised to that of β‐actin ( n = 6). (H) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vivo ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: In Vitro, In Vivo, Staining, Immunohistochemical staining, Injection, Immunohistochemistry, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: Overexpression of GDF15 ameliorated TGF‐β‐stimulated TIF in vitro and UUO‐induced TIF in vivo. (A) Representative images of Masson's trichrome staining, Picrosirius red staining and immunohistochemical staining for Collagen I, Fibronectin, and macrophage infiltration in the kidney homogenates from sham and UUO mice injected with empty vector or pGV362‐GDF15. (B) Quantitative analysis of immunohistochemistry for Collagen I, Fibronectin and GDF15 in different groups as indicated. (C, E) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vitro ( n = 3). The relative expression levels of the indicated proteins were normalised to that of β‐actin. (G) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vitro ( n = 3). (D, F) Western blot analysis and quantification of the Collagen I, Fibronectin and GDF15 in vivo. The relative expression levels of the indicated proteins were normalised to that of β‐actin ( n = 6). (H) qRT‐PCR analysis of fibronectin, collagen I, and GDF15 levels in vivo ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Over Expression, In Vitro, In Vivo, Staining, Immunohistochemical staining, Injection, Plasmid Preparation, Immunohistochemistry, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 ameliorates renal fibrosis through enhancing autophagy. (A) Bioinformatics predicts the mechanism of GDF15: PCA; Differentially clustered volcano plot; Top 15 of GO enrichment. (B) Western blot analysis of the LC3B, Beclin1 and p62 in TGF‐β‐stimulated TIF with transfection of shNC or sh865 ( n = 3). (C, F) Western blot analysis and quantification of the LC3B, Beclin1 in UUO‐induced TIF with injecting shNC or sh556 ( n = 6). (D) Western blot analysis of the LC3B, Beclin1 and p62 in TGF‐β‐stimulated TIF with transfection of empty vector or pGV362‐GDF15 ( n = 3). (E, G) Western blot analysis and quantification of the LC3B, Beclin1 in UUO‐induced TIF with injecting empty vector or pGV362‐GDF15 ( n = 6). (H) Western blot analysis the Collagen I and Fibronectin in TGF‐β‐stimulated TIF with transfection of empty vector, pGV362‐GDF15 and chloroquine treatment ( n = 3). (I) qRT‐PCR analysis of fibronectin and collagen I levels in different groups as indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Western Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 ameliorates renal fibrosis through improving lysosomal neogenesis. (A) GSEA enrichment. (B) Downregulation of kidney GDF15 in the UUO model, electron microscopy detected the number of lysosomes (i, ii), the scale bars in the upper‐row images represent 2 μm, and those in the lower‐row images represent 500 nm and immunohistochemical staining analysis of the LAMP (iii). (C) qRT‐PCR analysis of Lamp1 and Lamp2 in different groups as indicated. (D) Overexpression of kidney GDF15 in the UUO model, electron microscopy detected the number of lysosomes (i, ii), the scale bars in the upper‐row images represent 2 μm, and those in the lower‐row images represent 500 nm and immunohistochemical staining analysis of the LAMP (iii). (E) qRT‐PCR analysis of Lamp1 and Lamp2 in different groups as indicated ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Electron Microscopy, Immunohistochemical staining, Staining, Quantitative RT-PCR, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 promoted autophagy and lysosome biogenesis by inhibiting the PI3K/Akt/mTOR pathway. (A) Downregulation of kidney GDF15 in the UUO model, Western blot analysis and quantification of TFEB. The relative expression levels of the indicated proteins were normalised to that of β‐actin. (B) qRT‐PCR analysis of TFEB and PASP in different groups as indicated. (C) Overexpression of kidney GDF15 in the UUO model, Western blot analysis and quantification of TFEB. The relative expression levels of the indicated proteins were normalised to that of β‐actin. (D) qRT‐PCR analysis of TFEB and PASP in different groups as indicated. (E) Western blot analysis of p‐mTOR, mTOR, p‐Akt, Akt, p‐PI3K, and PI3K levels in TGF β‐stimulated TIF with transfection of empty vector or pGV362‐GDF15. (F, G) Effects of GDF15 silencing and PI3K inhibition on the PI3K signalling pathway and the expression of fibrosis‐ and autophagy‐related proteins. HK‐2 cells were transfected with shGDF15 (sh865) or control vector (shNC) for 48 h, followed by treatment with the PI3K inhibitor LY294002 (final concentration 20 μM) for 24 h. Western blot and quantification was performed to detect the expression levels of p‐PI3K, PI3K, fibronectin, collagen I, TFEB, and the internal control β‐actin. All data were normalised to β‐actin ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Inhibition, Control, Concentration Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Novel Roles of GDF15 in Alleviating Renal Fibrosis: Promoting Autophagy and Lysosome Biogenesis via Inhibition of the PI3K /Akt/ mTOR Pathway

doi: 10.1111/jcmm.70951

Figure Lengend Snippet: GDF15 mediates autophagy activation and anti‐fibrotic effects through GFRAL. (A) Western blot analysis and quantification of GFRAL protein expression in HK2 cells following siRNA‐mediated knockdown. siNC served as the negative control, and siGFRAL#1, #2, and #3 represent different interference sequences. (B) Western blot analysis and quantification of fibronectin, collagen I, and GDF15 protein expression in various treatment groups (Vector, GDF15, GDF15 + siNC, GDF15 + siGFRAL) under TGF‐β stimulation. β‐actin was used as the loading control. (C) Western blot analysis and quantification of LC3B, P62, and Beclin1 protein levels in each group. (D) Western blot analysis and quantification of TFEB protein expression in each group. (E) Transmission electron microscopy was used to observe the structural changes of lysosomes in HK2 cells from each group. The scale bars in the upper‐row images represent 1 μm, and those in the lower‐row images represent 500 nm. (F) qRT‐PCR analysis of Lamp1 and Lamp2 in different groups as indicated. (G) Western blot analysis and quantification of PI3K/AKT/mTOR signalling pathway proteins, including p‐mTOR, mTOR, p‐AKT, AKT, p‐PI3K, and PI3K. β‐actin was used as the loading control ( n = 3).* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: For HK2 cell culture supernatants, a human GDF15 ELISA kit (E‐EL‐H0080, Elabscience) was used according to the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Negative Control, Plasmid Preparation, Control, Transmission Assay, Electron Microscopy, Quantitative RT-PCR