Journal: Cell Reports Medicine

Article Title: The SGLT2 inhibitor dapagliflozin ameliorates renal fibrosis in hyperuricemic nephropathy

doi: 10.1016/j.xcrm.2024.101690

Figure Lengend Snippet: Effects of dapagliflozin on renal fibrosis of patients with HN (A and B) Representative images of H&E, Masson staining and quantitation of tubular injury score, and Masson staining-positive area in kidney sections from each patient group. (C) Representative images of αSMA (green), FN1 (red), and LTL (white) immunofluorescent staining and quantitation of αSMA and FN1 fluorescence intensity in kidney sections from each patient group. (D) Representative TEM images of mitochondria and quantitation of aspect ratio, circularity, and roundness in kidney sections from each patient group. Red outlines indicate mitochondria. (E) Representative images of TOM20 (red) and LTL (white) immunofluorescent staining and quantitation of TOM20 fluorescence intensity in kidney sections from each patient group. (F) Schematic diagram of the RNA-seq using kidneys from HN-animals, UA-treated cells. (G and I) Heatmap showing the upregulation of fibrotic pathway and inflammation in kidneys from HN mice (GEO: GSE190205) or UA-treated cells (GEO: GSE198133) versus the control group. (H, J, and K) Representative quantitation of αSMA and FN1 mRNA levels normalized to β-actin in isolated renal tubules, TECs, or HK-2 cells per group. (L) Chemical structure formula of dapagliflozin in 2D and 3D conformer. (M and N) Representative images of H&E, Masson staining and quantitation of tubular injury score, and Masson staining-positive area in kidney sections from each patient group. (O) Renal survival percent (an increase in serum Cr levels to twice the baseline value) (Kaplan-Meier curve) in each patient group over time. Log-rank test p < 0.001. A, adenine; P, potassium oxonate; Dapa, dapagliflozin. control patients ( N = 6), patients with HN ( N = 12), and patients with HN + Dapa ( N = 12); n = 3 cultures per group; ns: not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; five visual fields for each section analyzed.

Article Snippet: SGLT2-KO HK2 cell , Cyagen , N/A.

Techniques: Staining, Quantitation Assay, Fluorescence, RNA Sequencing, Control, Isolation

Journal: Cell Reports Medicine

Article Title: The SGLT2 inhibitor dapagliflozin ameliorates renal fibrosis in hyperuricemic nephropathy

doi: 10.1016/j.xcrm.2024.101690

Figure Lengend Snippet: Effects of dapagliflozin or ERRα overexpression in UA-treated TECs and HK-2 cells (A and B) Representative western blots of ERRα, αSMA, and FN1 protein and quantification of protein levels normalized to β-actin in each TEC group. (C) Representative images of ERRα (green) and FN1 (red) immunofluorescent staining and quantitation of fluorescence intensity in each TEC group. (D and E) Representative TEM images of mitochondria and quantitation of inner mitochondrial membrane (IMM)/outer mitochondrial membrane (OMM) ratio, total cristae length/mitochondrial area, and cristae junction/mitochondrial area in each TEC group. Red outlines indicate the zoom area. (F) Representative traces show OCR in each TEC group. (G) Representative quantitation of ATP levels in each TEC group. (H) Representative images of MitoTracker (red) immunofluorescent staining and quantitation of mitochondrial morphology in each TEC group. White outlines indicate the zoom area. (I) Representative images of ROS (green) immunofluorescent staining and quantitation of fluorescence intensity in each TEC group. (J) Diagram of the spatial binding pattern of dapagliflozin and ERRα protein. (K) Dapagliflozin’s bond with ERRα analyzed by surface plasmon resonance (SPR). (L) Dapagliflozin’s bond with ERRα analyzed by microscale thermophoresis (MST). (M and O) Representative images of αSMA (green) and FN1 (red) immunofluorescent staining and quantitation of fluorescence intensity in each TEC and HK-2 cell group. (N, P, and Q) Representative western blots of ERRα, αSMA, and FN1 protein and quantification of protein levels normalized to β-actin in each TEC and HK-2 cell group. (R and S) Representative TEM images of mitochondria and quantitation of IMM/OMM ratio, total cristae length/mitochondrial area, and cristae junction/mitochondrial area in each HK-2 cell group. Red outlines indicate the zoom area and mitochondria. Dapa, dapagliflozin; n = 3 cultures per group; MitoTracker, mitochondrial marker; Hoechst, nucleus marker; ns: not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; five visual fields for each section analyzed.

Article Snippet: SGLT2-KO HK2 cell , Cyagen , N/A.

Techniques: Over Expression, Western Blot, Staining, Quantitation Assay, Fluorescence, Membrane, Binding Assay, SPR Assay, Microscale Thermophoresis, Marker

Journal: Cell Reports Medicine

Article Title: The SGLT2 inhibitor dapagliflozin ameliorates renal fibrosis in hyperuricemic nephropathy

doi: 10.1016/j.xcrm.2024.101690

Figure Lengend Snippet: Effects of dapagliflozin in UA-treated SGLT2- or ERRα-KO HK-2 cells (A and H) Diagram of the SGLT2 or ERRα gene KO by CRISPR-Cas9 technology and sequencing identification in HK-2 cells. (B and I) Representative western blots of SGLT2, ERRα, αSMA, and FN1 protein and quantification of ERRα, αSMA, and FN1 protein levels normalized to β-actin in each HK-2 cell group. (C and J) Representative images of ERRα (green) and FN1 (red) immunofluorescent staining and quantitation of fluorescence intensity in each HK-2 cell group. White outlines indicate the zoom area. (D and K) Representative images of ROS (green) immunofluorescent staining and quantitation of fluorescence intensity in each HK-2 cell group. White outlines indicate the zoom area. (E and L) Representative images of MitoTracker (red) immunofluorescent staining and quantitation of mitochondrial morphology in each HK-2 cell group. White outlines indicate the zoom area. (F and M) Representative traces show OCR in each HK-2 cell group. (G and N) Representative quantitation of ATP level in each HK-2 cell group. Dapa, dapagliflozin; n = 3 cultures per group; MitoTracker, mitochondrial marker; Hoechst, nucleus marker; ns: not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; five visual fields for each section analyzed.

Article Snippet: SGLT2-KO HK2 cell , Cyagen , N/A.

Techniques: CRISPR, Sequencing, Western Blot, Staining, Quantitation Assay, Fluorescence, Marker

Journal: Cell Reports Medicine

Article Title: The SGLT2 inhibitor dapagliflozin ameliorates renal fibrosis in hyperuricemic nephropathy

doi: 10.1016/j.xcrm.2024.101690

Figure Lengend Snippet: ERRα-OAT1 axis in HK-2 cells (A) Chord plots showing KEGG-enriched items of DEGs from HK-2 cells of the control group versus the si-ERRα group. (B) ChIP-seq analysis: the binding site of OAT1 with ERRα in HK-2 cells. (C) ChIP-qPCR analysis: representative agarose gel image showing OAT1 gene fragments enrichment on ERRα protein. (D) Motif analysis: the complementary sequence of the ERRα-targeted motif. (E) Diagram of the spatial binding pattern of ERRα protein and OAT1 gene. (F and G) Representative quantitation of OAT1 mRNA and protein levels normalized to β-actin, representative western blots of ERRα and OAT1 protein, and quantification of protein levels normalized to β-actin in each TEC and HK-2 cell group. (H) Schematic diagram showing the location of the ERRα-targeted motif (in the OAT1 gene’s promoter region) when inserted upstream of the luciferase gene. Dual-luciferase reporter assay: relative luciferase activity in each group of HK-2 cells. (I and J) Representative western blots of ERRα and OAT1 protein and quantification of protein levels normalized to β-actin in each TEC and HK-2 cell group. n = 3 cultures per group; ns: not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: SGLT2-KO HK2 cell , Cyagen , N/A.

Techniques: Control, ChIP-sequencing, Binding Assay, ChIP-qPCR, Agarose Gel Electrophoresis, Sequencing, Quantitation Assay, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: Cell Reports Medicine

Article Title: The SGLT2 inhibitor dapagliflozin ameliorates renal fibrosis in hyperuricemic nephropathy

doi: 10.1016/j.xcrm.2024.101690

Figure Lengend Snippet:

Article Snippet: SGLT2-KO HK2 cell , Cyagen , N/A.

Techniques: Virus, Control, Recombinant, SYBR Green Assay, Lysis, CCK-8 Assay, ATP Assay, BIA-KA, cDNA Synthesis, Reporter Gene Assay, Plasmid Preparation, Software, Clinical Proteomics

Journal: Stem cell research & therapy

Article Title: TMT-based quantitative proteomic analysis of spheroid cells of endometrial cancer possessing cancer stem cell properties.

doi: 10.1186/s13287-023-03348-x

Figure Lengend Snippet: Fig. 8 Functional assays of selected targets, PFKFB3, GPRC5A and HK2. A–C The mRNA levels of PFKFB3, GPRC5A and HK2 were detected by qRT-PCR. D The expression levels of PFKFB3, GPRC5A and HK2 were detected by Western blot (Full-length blots are presented in supplementary material). E qRT-PCR results for Ishikawa and HEC1A transfected with HK2-siRNA-1, HK2-siRNA-2, HK2-siRNA-3 and NC. F Western blot analysis revealed significant downregulation in HK2-siRNA cells in comparison with the controls (Full-length blots are presented in supplementary material 1 and 2). G CCK-8 assays for Ishikawa and HEC1A cells transfected with HK2-siRNA or NC. H The number of spheroids for Ishikawa and HEC1A transfected with HK2-siRNA or NC in spheroid formation assay. I KEGG biochemical classification for HK2-siRNA endometrial cancer cells (*P < 0.05, **P < 0.01, *** P < 0.001)

Article Snippet: Primary antibodies of western blotting including HK2 (1:1,000, rabbit anti-human, 2A11C3, #66,974–1-Ig; Proteintech Group, INC, USA), GPRC5A (1:1,000, rabbit anti-human, #10,309–1-AP; Proteintech Group, INC, USA), PFKFB3 (1:1,000, rabbit anti-human, #13,763– 1-AP; Proteintech Group, INC, USA), and GAPDH (1:5,000, mouse anti-human, conjugated with HRP, 1E6D9, #60,004–1-Ig; Proteintech Group, INC, USA).

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection, Comparison, CCK-8 Assay, Tube Formation Assay

Journal: Cardiovascular Research

Article Title: ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection

doi: 10.1093/cvr/cvae026

Figure Lengend Snippet: List of plasmid sources for ISGylation targets

Article Snippet: Hk2 , Hexokinase 2 (HK2) , NM_013820.3 , pCMV6 , C-terminal FLAG , OriGene, Rockville, MD, USA.

Techniques: Plasmid Preparation

Journal: Cardiovascular Research

Article Title: ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection

doi: 10.1093/cvr/cvae026

Figure Lengend Snippet: Primer list for K to R mutagenesis of HK2 K419 and PFK1 K373 and K727

Article Snippet: Hk2 , Hexokinase 2 (HK2) , NM_013820.3 , pCMV6 , C-terminal FLAG , OriGene, Rockville, MD, USA.

Techniques: Mutagenesis

Journal: Cardiovascular Research

Article Title: ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection

doi: 10.1093/cvr/cvae026

Figure Lengend Snippet: Glycolytic control enzymes are targets of ISGylation. ( A ) Schematic representation of glycolysis depicting identified ISGylated enzymes and modification sites within CVB3-infected mouse hearts. (B + C) Validation of ISG15–modification of hexokinase-2 (HK2) and phosphofructokinase (PFK). HeLa cells were transfected with a four-plasmid combination (HA-ISG15, Ube1L, Ube2L6, Herc5) and FLAG-tagged HK2 ( B ) or PFK ( C ). FLAG-immunoprecipitation was performed prior to Western blot analysis. Arrows point toward enriched target and modification sites, as indicated. (D + E) R mutants of HK2 ISGylation site K419 ( D ) and PFK ISGylation sites K372/K727 ( E ) were generated. Transfection and immunoprecipitation were performed as described in (B + C). ISGylation patterns of HK2 K419R ( D ) and PFK K372R/K727R (E) were compared by Western blotting. Targets and modification bands are indicated by arrows and brackets.

Article Snippet: Hk2 , Hexokinase 2 (HK2) , NM_013820.3 , pCMV6 , C-terminal FLAG , OriGene, Rockville, MD, USA.

Techniques: Control, Modification, Infection, Biomarker Discovery, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Generated

Journal: Cardiovascular Research

Article Title: ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection

doi: 10.1093/cvr/cvae026

Figure Lengend Snippet: Impact of ISG15/ISGylation on HK2 and PFK1 activity. ( A–C ) ISG15-deficient HeLa cells were transfected with a four-plasmid combination (HA–ISG15 or GFP, together with Ube1L, Ube2L6, and Herc5) and FLAG-tagged HK2 ( B ) or PFK1 ( C ) or their respective K to R site mutants. HK2 and PFK1 were enriched by FLAG-immunoprecipitation prior to enzyme activity measurement of HK2 ( n = 4) and PFK1 ( n = 3). Measurements [mU/µg] were normalized to baseline activity. Statistical comparisons were achieved by one-tailed and two-tailed t -tests. ( D–E ) Lysine 419 is located close to the substrate (Glc) binding site in HK2 (surface representation), as revealed by an already determined enzyme structure (PDB ID 5hg1 . Consequently, ISG15 (cartoon representation, PDB 1z2m bound to K419 with its C-terminal domain would cover this substrate binding site. The provided visualization is not a complex model between HK2 and ISG15, rather a putative orientation is implied. Computational docking of ISG15 to K419 is not feasible using known template structures, since HK2 is always in a substrate bound conformation, while ISG15 would likely bind to an unbound (apo-) HK2 conformation, which can be drastically different. Such structural template is not available; therefore, this scheme is an approximation. However, specific ISG15-bound protease structures (PDB: 5w8u . ( F ) shows that ISG15 can be bound into a cleft-like structure arrangement of the target protein, as would be the case supposed here for the HK2-ISG15 assembling at K419. ( G ) A similar observation can be made at a structural PFK1 model (surface representation, two subunits (orange, beige)) with bound ligands (e.g. Frc6P) where several lysine residues are located close to the ligand-binding sites (red). ISG15 fused with the C-terminal domain to one of these lysine residues would hamper substrate binding, whereby two ISG15 molecules should be bound into cleft-like structural arrangements in a spatial fit-in manner. The visualization again does not show a computational fully fused and modelled ISG15-PFK1 complex, but is a by-hand oriented approximation due to a missing enzyme template structure in a non-substrated state. Therefore, the structural PFK1 conformation accessible for ISG15 is unknown (as for HK2) and cannot be simulated without further information. ( H ) The lysine residues K372 and K727 identified in this study as ISGylation sites were experimentally excluded to have a functional impact on PFK1 activity and are indeed more distantly located to the substrate binding regions. Note: The visualized PFK1 protein model is derived from a phosphofructokinase structure of Staphylococcus aureus (PDB 5xz8 . The sequence of the protein was substituted by mouse PFK1 amino acid sequence for homology (sequence similarity ∼83%, BLOSUM 62 matrix). Model representations were created using the PyMol Molecular Graphics System Version 1.3 (Schrödinger, LLC, New York, NY).

Article Snippet: Hk2 , Hexokinase 2 (HK2) , NM_013820.3 , pCMV6 , C-terminal FLAG , OriGene, Rockville, MD, USA.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation, One-tailed Test, Two Tailed Test, Binding Assay, Ligand Binding Assay, Functional Assay, Derivative Assay, Sequencing