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: 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

Journal: Biomedicines

Article Title: Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis

doi: 10.3390/biomedicines9050545

Figure Lengend Snippet: TBK1 and HK2 directly interact with MPT64. ( A ) Identification of TBK1 and HK2 by mass spectrometry analysis in THP-1 cell lysates treated with rMPT64 or rVector. ( B ) THP-1 cells were stimulated with rMPT64 (5 μg mL −1 ) for the indicated times, followed by IP with αHis-agarose bead and IB with αTBK1, αP-TBK1 (S172), αHK2, αHis, αActin. ( C , D ) Titration of fluorescently labelled MPT64 with TBK1 and HK2 (left), with K d (193 and 134 nM), determined by curve fit analysis (right). ( E , G ) Binding mapping. Schematic diagrams of the structures of MPT64 (upper). At 48 h after transfection with GST or GST-MPT63 and truncated mutant constructs together with Flag-TBK1 or V5-p47phox. 293T cells were used for GST pull down, followed by IB with αFlag or αV5. WCLs were used for IB with αFlag or αV5, αGST, and αActin. ( F , H ) 293T cells expressing Myc-MPT63 and Flag-TBK1 or V5-p47phox and treated with several Tat-MPT64-N or MPT64-C peptides (10 µM) for 6 h, followed by IP with αMyc and IB with αFlag. WCLs were used for IB with αMyc, αFlag, and αActin. The data are representative of four independent experiments with similar results ( A – H ).

Article Snippet: A HK2 KO THP-1 cell line was established by CRISPR/Cas9-mediated genome editing by HK2 Human Gene Knockout Kit (CRISPR) (KN209482) from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Mass Spectrometry, Titration, Binding Assay, Transfection, Mutagenesis, Construct, Expressing

Journal: Biomedicines

Article Title: Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis

doi: 10.3390/biomedicines9050545

Figure Lengend Snippet: HK2 peptide’s role in signal peptide for targeting the MTB-infected macrophages. ( A ) Schematic design of HK2 peptide (upper). Empty or HK2 KO THP-1 and BMDM cells were treated with Cy5.5 labelled-HK2 peptide for 1 h in various concentrations. THP-1 and BMDMs were used for IB or counting the number of HK-HK2 peptide + cells by FACS. ( B ) Empty or HK2 KO THP-1 cells were infected with MTB for 4 h and treated Cy5.5 labelled-HK2 peptide in various concentrations for 1, 18 or 72 h. After 1 h, the THP-1 cells were used for IB and the number of HK2-Hk2 peptide + cells were counted by FACS (top). The HK2 peptide treated-supernatants of THP-1 for 18 h were used for ELISA to measure the level of TNF-α and IL-6 (middle). The colony forming units (CFU) of intracellular MTB in THP-1 cells were measured after 3 d (bottom). ( C ) Mice was infected by MTB through intranasal infection (1 × 10 3 /per mice) and intranasally treated Cy5.5-labelled HK2 peptide (1 mg kg −1 ) after 3 wks. Lung harvests were used for analysis of the number of HK2 peptide + cells by FACS. The data are representative of four independent experiments with similar results ( A – C ).

Article Snippet: A HK2 KO THP-1 cell line was established by CRISPR/Cas9-mediated genome editing by HK2 Human Gene Knockout Kit (CRISPR) (KN209482) from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis

doi: 10.3390/biomedicines9050545

Figure Lengend Snippet: rMPT regulates MTB infection through enhancing the inflammation with declining the expression of IFN-β and increasing the level of ROS in macrophages. ( A ) Schematic in design of rMPT. ( B ) Bacterially purified 6xHis-rMPT and rVehicle were analyzed by coomassie blue staining (left) or immunoblotting (IB) with αHis (right). ( C ) BMDMs were incubated with rVehicle and rMPT for the indicated times and concentrations, then cell viability was measured with MTT assay. ( D ) BMDMs were treated with rVehicle or rMPT and immunolabelled with αHis (Alexa 586), αHK2, α p47phox, αTBK1 (Alexa 488), and DAPI. Scale bar, 10 μm. ( E ) BMDMs were treated with rVehicle or rMPT for 1 h. BMDMs were used for IP by αHis, followed by IB with αHK2, αp47phox, αP-p47phox (S345 and S359), αTBK1, αP-TBK1 (S172). WCLs were used for IB with αHK2, αp47phox, αTBK1, αHis, and αActin. ( F ) BMDMs were infected by MTB for 4 h and treated rMPT in various concentrations for 1 h. BMDMs were used for IP by αTBK1 and αIRF3, followed by IB with αIRF3 and αSTING1. WCLs were used for IB with αTBK1, αIRF3, αSTING1, αHis, and αActin. ( G ) BMDMs were used for IP by αp47phox, followed by IB with αp22phox and αp67phox. WCLs were used for IB with αp47phox, αp22phox, αp67phox, αHis, and αActin. ( H ) WT, TBK − / − , or p47phox − / − BMDMs were infected by MTB for 4 h and treated rMPT in various concentrations for 18 h. The supernatant of BMDMs were used for ELISA to measure the level of IFN-β, TNF-α, and IL-6. ( I ) The load of intracellular bacteria was measured after 3 d from treating the rVehicle or rMPT in WT, TBK − / − or p47phox − / − (Upper) and HK2 fl/fl LysM-Cre ‑ , or HK2 fl/fl LysM-Cre + BMDMs. The data are representative of four independent experiments with similar results ( C – I ). Significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) compared with rVehicle-treated BMDMs.

Article Snippet: A HK2 KO THP-1 cell line was established by CRISPR/Cas9-mediated genome editing by HK2 Human Gene Knockout Kit (CRISPR) (KN209482) from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Infection, Expressing, Purification, Staining, Western Blot, Incubation, MTT Assay, Enzyme-linked Immunosorbent Assay, Bacteria

Journal: Biomedicines

Article Title: Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis

doi: 10.3390/biomedicines9050545

Figure Lengend Snippet: rMPT is a potential therapeutic agent against MTB in mice. ( A ) Schematic of TB model treated with rMPT or rVehicle. Mice ( n = 10 per group) were intranasally infected by MTB H37Rv (1 × 10 4 CFU/mice). After 3 wks, mice were treated with rMPT or rVehicle for 7 d. Immunological analysis conducted in 5 wks. ( B ) Bacterial loads, the number of granuloma, and the level of inflammation were analyzed in each group of mice lungs (upper). Histopathology scores were obtained from H&E stained lung sections (bottom). Scale bar, 500 μm. ( C ) Bacterial loads were counted in WT, TBK −/ − , and p47phox −/ − HK2 fl/fl LysM-Cre − , and HK2 fl/fl LysM-Cre + mice lung. ( D ) Lung harvests in each group of mice were used for IP with His-agarose bead, followed by IB with αHK2, αp47phox, αP-p47phox (S345 and S359), αTBK1, and αP-TBK1 (S172). WCLs were used for IB with αHK2, αp47phox, αTBK1, αHis, and αActin. ( E ) Fluorescence images of the lung, liver, and spleen of the mice intranasally administrated with Cy5.5 labelled-rMPT (left), and quantitative fluorescence intensities of the organs measured by an IVIS spectrum-chromatography (CT) system. The data are representative of four independent experiments with similar results ( B – E ). Significant differences (* p < 0.05, ** p < 0.01; *** p < 0.001) compared with rVector-treated mice.

Article Snippet: A HK2 KO THP-1 cell line was established by CRISPR/Cas9-mediated genome editing by HK2 Human Gene Knockout Kit (CRISPR) (KN209482) from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Infection, Histopathology, Staining, Fluorescence, Chromatography

Journal: Biomedicines

Article Title: Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis

doi: 10.3390/biomedicines9050545

Figure Lengend Snippet: Schematic model for the roles in rMPT against MTB infection. ( A ) Domain screening of interacting site between MPT63 or MPT64 with TBK1, p47phox and HK2. ( B ) Construction of rMPT combined TBK1, p47phox, and HK2 interacting domains in MPT63 and MPT64. ( C ) Regulatory pathway of rMPT in macrophages.

Article Snippet: A HK2 KO THP-1 cell line was established by CRISPR/Cas9-mediated genome editing by HK2 Human Gene Knockout Kit (CRISPR) (KN209482) from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Infection

Journal: bioRxiv

Article Title: Intermittent hypoxia enhances the expression of HIF1A by increasing the quantity and catalytic activity of KDM4A-C and demethylating H3K9me3 at the HIF1A locus

doi: 10.1101/2021.07.25.453726

Figure Lengend Snippet: (A) Nuclear HIF-1α in MCF7, MDA- MB-231, and PC3 cells following exposure to normoxia, chronic hypoxia and intermittent hypoxia over 18 h. (B-D) mRNA expression of HIF-1 target genes involved in glycolysis ( SLC2A1, HK2, LDHA ), extracellular matrix remodeling ( PLOD2, P4HA1, P4HA2 ) and the HIF pathway ( EGLN1, EGLN3 ) in MCF7 (B), MDA-MB-231 (C), and PC3 (D) cells after 18 h exposure to oxygen conditions. All values are normalized to normoxic expression levels which is set to 0 on the Log2 scale. Results are the mean ± S.E.M of 3 independent experiments run in duplicate. (E) HIF1A mRNA levels in HCT116 cells and MCF7 cells (F) after 18 h exposure to normoxia, chronic hypoxia and intermittent hypoxia. Results are the mean ± S.E.M of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Asterisks above a data point (without line) indicate significance as compared to normoxia. Asterisks above a line compare data points connected by the line. (G) Nuclear HIF-1α protein levels in HCT116 cells exposed to normoxia, chronic hypoxia and intermittent hypoxia treated with DMSO or 10 μg/ml cycloheximide (CHX, protein synthesis inhibitor). (H) Nuclear HIF-1α in HCT116 cells exposed to normoxia, chronic hypoxia and intermittent hypoxia with DMSO or 1 µM MG262 (proteasome inhibitor). (I) Nuclear HIF- 1α in MCF7 cells exposed to 18 h normoxia, chronic hypoxia, and intermittent hypoxia with or without an additional 4 h exposure to chronic hypoxia. Nuclear histone H3 is used as a loading control for (A), (G), (H) and (I).

Article Snippet: Antibodies used were as follows: HIF-1α (NB100-479, 1:500, Novus), Hexokinase II (TA325030, 1:500, Origene), Glut1 (ab115730, 1:1000, Abcam), LDHA (3582T, 1:1000, Cell Signaling Technologies), JMJD2A (5328, 1:1000, Cell Signaling Technologies), JMJD2B (8639, 1:1000, Cell Signaling Technologies), JMJD2C (ab226480, 1:500, Abcam), Tri- Methyl-Histone H3 (Lys9) (13969, 1:1000, Cell Signaling Technologies), and total Histone H3 (ab1791, 1:2000, Abcam).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Intermittent hypoxia enhances the expression of HIF1A by increasing the quantity and catalytic activity of KDM4A-C and demethylating H3K9me3 at the HIF1A locus

doi: 10.1101/2021.07.25.453726

Figure Lengend Snippet: (A) CA9 mRNA and (B) HK2 mRNA expression following exposure to normoxia, chronic hypoxia and intermittent hypoxia treated with DMSO or cycloheximide (CHX, protein synthesis inhibitor).

Article Snippet: Antibodies used were as follows: HIF-1α (NB100-479, 1:500, Novus), Hexokinase II (TA325030, 1:500, Origene), Glut1 (ab115730, 1:1000, Abcam), LDHA (3582T, 1:1000, Cell Signaling Technologies), JMJD2A (5328, 1:1000, Cell Signaling Technologies), JMJD2B (8639, 1:1000, Cell Signaling Technologies), JMJD2C (ab226480, 1:500, Abcam), Tri- Methyl-Histone H3 (Lys9) (13969, 1:1000, Cell Signaling Technologies), and total Histone H3 (ab1791, 1:2000, Abcam).

Techniques: Expressing

Journal: bioRxiv

Article Title: Intermittent hypoxia enhances the expression of HIF1A by increasing the quantity and catalytic activity of KDM4A-C and demethylating H3K9me3 at the HIF1A locus

doi: 10.1101/2021.07.25.453726

Figure Lengend Snippet: MCF7 cells were transfected with combined siKDM4A, siKDM4B, and siKDM4C (referred to as siKDM4 in figure) followed by exposure to normoxia, chronic hypoxia (2%) and intermittent hypoxia for 18 h. mRNA expression of (A) KDM4A, (B) KDM4B, (C) KDM4C, (D) HIF1A, (E) HK2, and (F) PLOD2. All values are normalized to normoxic expression levels which is set to 0 on the Log2 scale. Results are the mean ± S.E.M n = 4 independent experiments. (G) Nuclear extracts of KDM4A, KDM4B, KDM4C, HIF-1α, H3K9me3, and histone H3 (loading control), and cytoplasmic extracts of HK2 and β-actin (loading control).

Article Snippet: Antibodies used were as follows: HIF-1α (NB100-479, 1:500, Novus), Hexokinase II (TA325030, 1:500, Origene), Glut1 (ab115730, 1:1000, Abcam), LDHA (3582T, 1:1000, Cell Signaling Technologies), JMJD2A (5328, 1:1000, Cell Signaling Technologies), JMJD2B (8639, 1:1000, Cell Signaling Technologies), JMJD2C (ab226480, 1:500, Abcam), Tri- Methyl-Histone H3 (Lys9) (13969, 1:1000, Cell Signaling Technologies), and total Histone H3 (ab1791, 1:2000, Abcam).

Techniques: Transfection, Expressing, Control

Journal: bioRxiv

Article Title: Intermittent hypoxia enhances the expression of HIF1A by increasing the quantity and catalytic activity of KDM4A-C and demethylating H3K9me3 at the HIF1A locus

doi: 10.1101/2021.07.25.453726

Figure Lengend Snippet: HCT116 cells were stably transfected with a 5HRE/EGFP vector and grown as spheroids. (A) Bright field and fluorescence images of HCT116 spheroids expressing GFP after 0, 3, 7, or 10 days of growth. (B) Bright field and fluorescence images of HCT116 spheroids expressing GFP transfected with scrambled control siRNA or HIF1A siRNA. (C) Protein expression of HIF-1 target genes, HK2, Glut1, and LDHA in HCT116 cells grown as a monolayer or as spheroids grown over 3, 7, or 10 days. Total histone H3 is used as a loading control. (D) Schematic illustration of spheroid exposure to oxygen conditions using oxygen-permeable membranes; schematic illustration demonstrating how confocal microscopy is used to visualize the spheroid through multiple transverse planes. (E) Confocal fluorescence images of HCT116 cells grown as spheroids over 3 days, transferred onto oxygen-permeable membranes for 24 h, and then exposed to normoxia or intermittent hypoxia over a further 18 h. Red = nuclear red live stain; Green = GFP expression. Scale bar = 300 µm for (A), (B) and (E).

Article Snippet: Antibodies used were as follows: HIF-1α (NB100-479, 1:500, Novus), Hexokinase II (TA325030, 1:500, Origene), Glut1 (ab115730, 1:1000, Abcam), LDHA (3582T, 1:1000, Cell Signaling Technologies), JMJD2A (5328, 1:1000, Cell Signaling Technologies), JMJD2B (8639, 1:1000, Cell Signaling Technologies), JMJD2C (ab226480, 1:500, Abcam), Tri- Methyl-Histone H3 (Lys9) (13969, 1:1000, Cell Signaling Technologies), and total Histone H3 (ab1791, 1:2000, Abcam).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Expressing, Control, Confocal Microscopy, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: 3-Bromopyruvic acid regulates glucose metabolism by targeting the c-Myc/TXNIP axis and induces mitochondria-mediated apoptosis in TNBC cells

doi: 10.3892/etm.2022.11447

Figure Lengend Snippet: 3-BrPA regulates expression of c-Myc, TXNIP and HK2. (A) Expression levels of c-Myc, TXNIP, HK2 and GLUT1 were detected through western blot analysis. Grayscale values of the bands were determined using software. (B) 3-BrPA downregulated c-Myc and HK2 protein expression, whereas it upregulated TXNIP protein expression in TNBC cells ( * P<0.05 vs. control group, the same cell line with 0 µM 3-BrPA). (C) However, in non-TNBC cells, the expression levels of c-Myc, TXNIP, HK2 and GLUT1 were not significantly affected. The expression levels of GLUT1 were not significantly affected in both TNBC and non-TNBC cells. TXNIP, thioredoxin-interacting protein; HK2, hexokinase 2; GLUT1, glucose transporter 1; 3-BrPA, 3-bromopyruvic acid; TNBC, triple-negative breast cancer.

Article Snippet: Then the membrane was cultured in QuickBlock Blocking Buffer for Western Blot (Beyotime Institute of Biotechnology) at room temperature for 1 h. The membrane was incubated with antibodies against GLUT1 (1:1,000; cat. no. TA312796; OriGene Technologies, Inc.), c-Myc (1:1,000; cat. no. TA150121; OriGene Technologies, Inc.), TXNIP (1:1,000; cat. no. TA349090; OriGene Technologies, Inc.), HK2 (1:1,000; cat. no. TA500856; OriGene Technologies, Inc.), Bcl-2 (1:2,000; cat. no. TA806591; OriGene Technologies, Inc.), Bax (1:2,000; cat. no. TA810334; OriGene Technologies, Inc.), cytochrome c (Cyt-C; 1:5,000; cat. no. ab13575; Abcam), Caspase-3 (1:1,000; cat. no. TA301776; OriGene Technologies, Inc.) and β-actin (1:1,000; cat. no. TA811000; OriGene Technologies, Inc.) at room temperature overnight.

Techniques: Expressing, Western Blot, Software, Control