Journal: Aging Cell

Article Title: Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction

doi: 10.1111/acel.13004

Figure Lengend Snippet: DKK1 and mitoQ preserve mitochondrial functions and inhibit cellular senescence in an established aging model. (a) Experimental design. Red arrows indicate the starting point of d ‐gal injection. Green arrows indicate the injections of pFlag‐DKK1 plasmid. Green bar indicates treatment with mitoQ. (b–e) Representative (b) Western blots and graphical representations of (c) DKK1, (d) active β‐catenin, and (e) TOMM20 are presented. * p < .05 versus control mice; † p < .05 versus d ‐gal‐treated mice ( n = 5–6). (f) Representative MitoTracker and mitoSOX staining micrographs show the mitochondrial mass loss and ROS production induced by d ‐gal were blocked by DKK1 or mitoQ. Arrows indicate positive staining. Scale bar, 50 µm. (g) Representative staining micrographs show renal expression of SA‐β‐gal activity, γH2AX, fibronectin, PDGFRβ, and Sirius red staining. Frozen kidney sections were stained for SA‐β‐gal activity, fibronectin, and PDGFRβ. Paraffin sections were immunostained with an antibody against γH2AX or performed with Sirius red staining. Arrows indicate positive staining. Scale bar, 50 μm. (h–k) Representative (h) Western blots and graphical representations of (i) γH2AX, (j) fibronectin, and (k) α‐SMA protein expression levels in kidneys are presented. * p < .05 versus control mice; † p < .05 versus d ‐gal‐treated mice ( n = 5–6). (l) Quantitative determination of renal fibrotic lesions in different groups. * p < .05 versus control mice; † p < .05 versus d ‐gal‐treated mice ( n = 5–6)

Article Snippet: Antibodies used were as follows: Klotho (AF1819; R&D Systems), Wnt1 (ab15251; Abcam), AT1 (AB15552; Merck Millipore), ACE (ab75762; Abcam), AGT (SAB2100072; Sigma‐Aldrich), p16 INK4A (ab189034; Abcam), γ‐H2AX (ab2893; Abcam), PGC‐1α (ab54481; Abcam), and DKK1 (BM4554; Boster).

Techniques: Injection, Plasmid Preparation, Western Blot, Control, Mouse Assay, Staining, Expressing, Activity Assay

Journal: Aging Cell

Article Title: Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction

doi: 10.1111/acel.13004

Figure Lengend Snippet: Wnt/β‐catenin mediates age‐related renal fibrosis in vitro. (a) Real‐time PCR results show d ‐gal induced upregulation of multiple Wnt genes in cultured proximal tubular cell line (HKC‐8). HKC‐8 cells were treated with d ‐gal (10 mg/ml) for 60 hr. Total RNA was extracted and analyzed for various Wnt mRNA expression levels. * p < .05 versus control group. Ctrl, control. (b) Representative immunofluorescence micrographs show d ‐gal induced nuclear translocation of β‐catenin. HKC‐8 cells were treated with d ‐gal (10 mg/ml) for 60 hr and then stained for β‐catenin (red) and DAPI (blue). Arrows indicate nuclear staining of β‐catenin. Scale bar, 20 μm. (c) Representative electron microscopy and BrdU incorporation assay micrographs show mitochondria and cell proliferation in renal tubular cells. HKC‐8 cells were pretreated with ICG‐001 (5 μm) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. BrdU (10 μM) was added for 12 hr before collection. For TEM analyses, arrows indicate healthy mitochondria, and arrowheads indicate abnormal‐shaped mitochondria. Scale bar, 2 μm; for BrdU incorporation assay, arrows indicate BrdU incorporation positive cells. Scale bar, 75 μm. TEM, transmission electron microscopy. (d–f) Representative (d) Western blots and graphical representations of (e) phospho‐PGC‐1α and (f) fibronectin are presented. HKC‐8 cells were pretreated with ICG‐001 (5 μm) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. * p < .05 versus control group; † p < .05 versus d ‐gal group ( n = 3). (g–k) Representative (g) Western blots and graphical representations of (h) phospho‐PGC‐1α, (i) TFAM, (j) p16 INK4A , and (k) fibronectin are presented. HKC‐8 cells were pretreated with losartan (10 μM) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. * p < .05 versus control group; † p < .05 versus d ‐gal group ( n = 3). (l) Representative fluorescence micrographs show MitoTracker, BrdU, and fibronectin staining. After pretreatment with losartan (10 μM) or mitoQ (100 nM) for 1 hr, HKC‐8 cells were treated with d ‐gal (10 mg/ml) for 60 hr. BrdU (10 μM) was added for 12 hr before collection. Cells were stained with MitoTracker deep red probe (300 nM) or antibodies against BrdU and fibronectin. Arrowhead indicates the loss of mitochondrial mass and the increase in fragmentation of cristae. Arrows indicate positive staining for BrdU‐positive cells and fibronectin. Scale bar, 10 μm for images of MitoTracker staining. (m–o) Representative (m) Western blots and graphical representations of (n) p16 INK4A and γH2AX, and (o) fibronectin and α‐SMA are presented. HKC‐8 cells were pretreated with mitoQ (100 nM) for 1 hr and then treated with d ‐gal (10 mg/ml) for 60 hr. * p < .05 versus control group; † p < .05 versus d ‐gal group ( n = 3). (p) Schematic presentation depicts the potential mechanism by which Wnt/β‐catenin induces age‐related renal fibrosis. Wnt/β‐catenin signaling triggers the activation of RAS, thereby leading to the injury of mitochondrial biogenesis. This causes mitochondrial dysfunction with a loss of mass and increase in fragmentation and ROS production, which in turn induces tubular cell senescence and age‐related renal fibrosis. The mitochondrial dysfunction and activation of Wnt/β‐catenin signaling reciprocally induce each other. Multiple approaches, such as the inhibition of Wnt/β‐catenin by DKK1, ICG‐001, or Klotho, the blockade of RAS by losartan, the protection of mitochondrial biogenesis by resveratrol, and the mitochondria‐targeted antioxidant mitoQ could slow age‐related renal fibrosis

Article Snippet: Antibodies used were as follows: Klotho (AF1819; R&D Systems), Wnt1 (ab15251; Abcam), AT1 (AB15552; Merck Millipore), ACE (ab75762; Abcam), AGT (SAB2100072; Sigma‐Aldrich), p16 INK4A (ab189034; Abcam), γ‐H2AX (ab2893; Abcam), PGC‐1α (ab54481; Abcam), and DKK1 (BM4554; Boster).

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Control, Immunofluorescence, Translocation Assay, Staining, Electron Microscopy, BrdU Incorporation Assay, Transmission Assay, Western Blot, Fluorescence, Activation Assay, Inhibition

Journal: Natural Products and Bioprospecting

Article Title: Scutellarin ameliorates diabetic nephropathy via TGF-β1 signaling pathway

doi: 10.1007/s13659-024-00446-y

Figure Lengend Snippet: Scutellarin Restored Podocyte Injury of the DN Mice. a Representative images of immunohistochemistry for NPHS1 and NPHS2 of the mice treated with vehicle, scutellarin or empagliflozin (× 200; scale bar = 50 µm). b Representative images of Western-blotting for NPHS1, NPHS2. c Quantitative plot of the expression of NPHS1 of the mice. d Quantitatification of NPHS1 expression of the mice. e Representative images of Western-blotting for β-catenin, Axin2, snail and DKK1 of the mice. f – i Quantifications of the protein levels for β-catenin, Axin2, snail and DKK1 from E. All data are presented as the mean ± S.D.; n = 4–6 for each group, “n” stands for the number of animals; p vs. the model group (STZ)

Article Snippet: Scutellarin (Yunnan Phytopharmaceutical Co., LTD., China); Empagliflozin (Cat. C14295412, Macklin Biochemical, China), Streptozotocin (Cat. S8050, Solarbio, China); goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (Cat. AS014, Abclonal, China); anti-mouse IgG HRP-linked antibody (Cat. 7076S, Cell Signaling Technology, USA); Methenamine Silver Plating Stain Kit (Cat. G1790, Solarbio); Glycogen Periodic Acid Schiff (PAS) Stain Kit (Cat. G1281, Solarbio); Masson’s Trichrome Stain Kit (Cat. G1340, Solarbio); Mouse MAU enzyme-linked immunosorbent assay (ELISA) Kit (Cat. JL20493, JONIN, China); Anti-DKK1 antibody (Cat. A00632, Boster Biological Technology, China); Anti-SNAIl antibody (Cat. BP0449, Boster Biological Technology); Anti-α-SMA antibody (Cat. BM0002, Boster Biological Technology); Anti-TGFB1 antibody (Cat. BA0290, Boster Biological Technology); Anti-NPHS1 antibody (Cat. BA1669, Boster Biological Technology); Anti-NPHS2 antibody (Cat. BA1688, Boster Biological Technology); Anti-SMAD2/3 antibody (Cat. BA1395, Boster Biological Technology); Anti-Phospho-SMAD2(s250) antibody (Cat. BM4693, Boster Biological Technology); Anti-FN1 antibody (Cat. BA1772, Boster Biological Technology); AXIN2 antibody (Cat. Ab32197, Abcam, USA); Anti-Phospho-SMAD3(ser425) antibody (Cat. AF3362, Affinity Biosciences, China); Anti-COL3A1 antibody (Cat. M00788, Boster Biological Technology); Anti-extracellular signal-regulated kinase (ERK)1/2 antibody (Cat. ET1601-29, HUABIO, China); Anti-ERK1(PT202/PY204) + ERK2(PT185/PY187) antibody (Cat. ET1610-13, HUABIO); Anti-P38 antibody (Cat. ET1602-26, HUABIO); Anti-Phospho-P38(Thr180 + Tyr182) antibody (Cat. ER1903-01, HUABIO); Anti-β-Actin antibody (Cat. EM21002, HUABIO); Anti-SMAD4 antibody (Cat. A5657, Abclonal); Anti-β-catenin antibody (Cat. 610154, BD Biosciences, China); Creatinine (Cr) Assay kit (Cat. C011-2-1, Nanjing Jiancheng Bioengineering Institute, China); Glucose Assay Kit (Cat. F006-1-1, Nanjing Jiancheng Bioengineering Institute); microalbunminuria (MAU) ELISA kit (Cat. JL20493, JONIN).

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: Oncogene

Article Title: A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway

doi: 10.1038/s41388-025-03445-6

Figure Lengend Snippet: A Two epitope bins are apparent amongst the Twist anti-DKK1 leads from this epitope binning analysis. The formation of Antibody-Antigen-Antibody complexes indicates the antibodies are not binding to the same epitope of DKK1. B Anti-DKK1 lead antibodies bind to hDKK1 cysteine-rich domain CRD1 or CRD2 or both CRD1 and CDR2 (in the instance of bispecific antibodies), and cross-reactivity with mouse and cynomolgus monkey DKK1. The assays were repeated in triplicate.

Article Snippet: Anti-DKK1 antibodies were tested for in vivo tumor-suppressing activity in HuCD34-NCG mice (Jackson Laboratory) inoculated with PC3 cells.

Techniques: Binding Assay

Journal: Oncogene

Article Title: A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway

doi: 10.1038/s41388-025-03445-6

Figure Lengend Snippet: A Epitope Mapping methodology: Differences in solvent accessibility of various regions of the antigen (DKK1) using PLIMB and hydroxyl radical labeling were measured. Each antibody was added individually at a 1:1 antibody/antigen molar ratio. For epitope mapping, peptide level-analysis shows the detection of larger-order structural changes and identification of regions of interest showing “protection” and “deprotection” upon complexation. Peptides with a decreased level of modification upon binding with the antibody indicate “protection”. Peptides with an increased level of modification upon complexation represent “deprotection” or areas of conformational change, which take place due to antibody binding. The peptides of interest were further validated by visually inspecting MS2 spectra and adjusting extracted ion chromatogram (XIC) windows across peaks that showed consistency between samples and whose modifications could be identified with MS2 hits. The peptide-level analysis was utilized for peptides in the candidate epitope regions to determine which of the individually labeled amino acids show the greatest changes in solvent accessibility due to binding. B Principal component analysis (PCA) of all quantified log2 fold-changes between unbound and bound states of all eight antibodies. Colors are representative of clusters as determined by partition around medoids (PAM). C Structural representation of DKK1 epitope groups. Blue: N-terminal cysteine-rich domain, Pink: C-terminal cysteine-rich domain, Green: unstructured. Structure from AlphaFold (AF- O94907 -F1).

Article Snippet: Anti-DKK1 antibodies were tested for in vivo tumor-suppressing activity in HuCD34-NCG mice (Jackson Laboratory) inoculated with PC3 cells.

Techniques: Solvent, Labeling, Modification, Binding Assay

Journal: Oncogene

Article Title: A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway

doi: 10.1038/s41388-025-03445-6

Figure Lengend Snippet: A Wnt TCF/LEF reporter assay screening. Wnt TCF/LEF signaling is blocked by DKK1 binding to LRP5/6. Anti-DKK1 antibodies that bind to hDKK1 CRD2 block the binding of DKK1 to the co-receptors, and lead to the reactivation of Wnt canonical signaling. B MC3T3.E1 cell differentiation detection by mineralization assay. Soluble hDKK1 suppresses pre-osteoblast cell differentiation via the Wnt canonical pathway. Anti-DKK1 antibodies that bind to hDKK1 CRD2 block the binding of DKK1 to the LRP5/6 co-receptors and restore cell differentiation. C Wnt non-canonical phospho-JNK detection. Colo205 cells were treated with Wnt, DKK1, and anti-DKK1 lead antibodies. Intracellular JNK phosphorylation level was detected with ELISA. D Primary immune cell activation. DKK1 leads to immune suppression including T cell inactivation, MDSC accumulation, and NK cell clearance. GM-CSF is the biomarker for NK cell activation. Human PBMC were treated with an immune stimulator, mWNT3a, hDKK1, and DKK1 lead antibodies. Cytokine release of GM-CSF was measured by ELISA. Antibodies binding to CRD1 of DKK1 showed stronger NK cell activation. E PC3 tumor cell cytotoxicity by activated immune cells. T cells and NK cells in human PBMC were activated and co-cultured with PC3 tumor cells for 6 days. Activated immune cells kill PC3 cells, while hDKK1 treatment inhibits cytotoxicity. Blocking the interaction of hDKK1 to the receptor with Twist DKK1 lead antibodies restores the cytotoxicity potency. Antibodies binding to CRD1 of DKK1 showed stronger cytotoxicity. F Anti-DKK1 antibody targeting DKK1 CRD1 also induced cytotoxicity in breast, gastric and colon cancer cells. Using the KILR cytotoxicity assay, a high luminescence signal was detected in cytotoxic cells. All the samples were duplicated, and the assays were repeated three times.

Article Snippet: Anti-DKK1 antibodies were tested for in vivo tumor-suppressing activity in HuCD34-NCG mice (Jackson Laboratory) inoculated with PC3 cells.

Techniques: Reporter Assay, Binding Assay, Blocking Assay, Cell Differentiation, Mineralization Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Activation Assay, Biomarker Discovery, Cell Culture, Cytotoxicity Assay

Journal: Oncogene

Article Title: A multi-faceted discovery strategy identifies functional antibodies binding to cysteine-rich domain 1 of hDKK1 for cancer immunotherapy via Wnt non-canonical pathway

doi: 10.1038/s41388-025-03445-6

Figure Lengend Snippet: A SC52-001 and SC52-005 bind to DKK1 CRD1 and elicit immune cell activation, while SC52-2 binds to DKK1 CRD2 and activates Wnt signaling. Bispecific antibodies consisting of a combination of SC52-001, 002, or 005 were generated as shown. B The potencies of Wnt signal activation of DKK1 CRD1 binders are increased with bispecific antibody treatment that combine DKK1 CRD2 binders as compared to that of monospecific antibody treatment. C NK cell activation of DKK1 CRD2 binders increased with bispecific antibody treatment that combine DKK1 CRD1 binders as compared to that of monospecific antibody treatment. D , E Tumor cell cytotoxicity effect is upregulated in DKK1 CRD2 binders with bispecific antibody treatment that combine DKK1 CRD1 binders as compared to that of monospecific antibody treatment. All the samples were duplicated, and the assays were repeated twice.

Article Snippet: Anti-DKK1 antibodies were tested for in vivo tumor-suppressing activity in HuCD34-NCG mice (Jackson Laboratory) inoculated with PC3 cells.

Techniques: Activation Assay, Generated