Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A) RT-qPCR showing up-regulation of metabolic genes in mDM (above) or BMDM (below) treated with LPS or MSUc for 5h (n≥4/condition). (B) WB of lysates of BMDM treated with LPS or MSUc in control PBS, LPS or MSUc at 8h or 24h showing up-regulation of SLC2A1 and PFKBP3 in BMDM treated with LPS or MSUc and down-regulation of LDHB in BMDM treated with MSUc.

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A) Bar plot showing up-regulation of genes involved in glucose metabolism in macrophages treated with MSUc or LPS. (B) Protein analysis by IF showing up-regulation of SLC2A1 in macrophages treated with MSUc or LPS at 5h. (C-D) Analysis of the concentration of glycolytic metabolites by 1D 1 H-NMR in the pellet (C) or supernatant (D) or BMDM treated with LPS or MSUc for 4h or 8h showing activation of glycolysis in macrophages treated with MSUc to a higher degree than LPS (n≥4/condition). (E) 1D 1 H-NMR showing increased levels of acetate, citrate and lactate and reduced levels of succinate in the supernatant of mDM treated with MSUc for 4h or 8h (n=3 donors/condition). (F) Bar graphs showing up-regulation of several amino acid transporters (top) or genes involved in glycine/serine/threonine metabolism (bottom) in macrophages treated with MSUc or LPS for 5h assessed by RNA-Seq. (G,H) 1D 1 H-NMR showing increased levels of glycine, threonine and tryptophan in the pellet (G) and alanine, glutamate, phenylalanine and tryptophan in supernatant (H) of BMDM treated with MSUc or LPS for 4h or 8h (n≥4/condition). (I) 1D 1 H-NMR showing increased levels of glutamate and glycine, and reduced levels of aspartate, glutamine, isoleucine and leucine in the supernatant of mDM treated with MSUc for 4h or 8h (n=3 donors/condition). (#=P<0.10;*=P<0.05; **=P<0.01).

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Concentration Assay, Activation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A,B) Quantification of pJNK expression in BMDM treated with LPS or MSUc at various time points showing increased total pJNK expression in BMDM treated with LPS for 30 min (A) but prolonged pJNK expression in BMDM treated with MSUc (A,B)(n=2 replicates/condition). (C) Pie chart showing the degree of amelioration of gene expression in BMDM treated with LPS+JNKi vs. LPS or MSUc+JNKi vs. MSUc for 5h. (D) HOMER analysis of the promoter [-2000;+500 bp, TSS] of genes up-regulated by LPS or MSUc and further down-regulated by treatment with JNKi (>50% left, 33%-50% center, <33% right). Data shows enrichment in IRFs motifs in genes ameliorated by JNKi in LPS and motifs for AP-1, MYC, NRF2 and circadian clock proteins in genes ameliorated by JNKi in MSUc. (E) GSEA analysis using REACTOME of genes between > 50% reduccion of expression in MSUc+JNKi vs. MSUc or LPS+JNKi vs. LPS. Data shows enrichment in inflammatory gene sets in LPS and MSUc and metabolic gene sets in MSUc. (F,G) Protein analysis by IF of JUN, pJUN Ser63 , FOSL1, SLC2A1 and PFKBP3 in BMDM treated with MSUc or MSUc+JNKi for 5h. Quantification of G corresponds to experiment shown in F and shows downregulation of protein expression in MSUc+JNKi vs. MSUc (n=3/condition). (#=P<0.10;*=P<0.05; **=P<0.01). Broken lines in E represents the cutoff for significance –log 10 (0.05)=1.30.

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Expressing, Gene Expression

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A,B) Protein analysis by IF of JUN, pJUN Ser63 , FOSL1, SLC2A1, FOSB in mDM treated with MSUc or MSUc+JNKi for 5h. Quantification of B corresponds to experiment shown in A, and shows downregulation of protein expression in MSU+JNKi vs. MSUc (n=3/condition). (C) Protein analysis by ELISA of cytokines in the supernatant of BMDM treated with LPS or MSUc w/wo JNKi showing complete reduction in MSUc+JNKi vs. MSUc and partial reduction in LPS+JNKi vs. LPS (n=3/group). (D) Analysis of metabolites by 1D 1 H-NMR in the culture media of BMDM treated with MSUc or MSUc+JNKi showing varying degree of recovery (n=3/condition). (E,F) JUN ChIP in BMDM (E) or mDM (F) treated with LPS, MSUc, LPS+JNKi or MSUc+JNKi and qPCR over regulatory regions of genes up-regulated by LPS or MSUc. Data shows higher levels of JUN binding in macrophages treated with MSUc that is reduced upon treatment with JNKi (n=4/condition). (#=P<0.10;*=P<0.05; **=P<0.01).

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A,B) Protein analysis by IF showing expression of SLC2A1 (A) and pJNK (B) in macrophages in the subcutaneous cavity of the air pouch when injected with MSUc (n=2/group). (C) Histological analysis by HE showing the recruitment of inflammatory cells (arrows) induced by MSUc in the air pouch is reduced upon treatment with JNKi (n≥4/group). (D,E) Pathological assessment of inflammatory cell infiltrates of the air pouch (D) and neutrophil count of the air pouch lavage (E) showing reduction in MSUc+JNKi vs. MSUc (n≥4/group). (F) Histological analysis by HE showing the recruitment of inflammatory cells (arrows) induced by MSUc in the air pouch is reduced upon treatment with BAY-876 (n≥5/group). (G,H) Pathological assessment of inflammatory cell infiltrates of the air pouch (G) and neutrophil count of the air pouch lavage (H) showing reduction in MSUc+BAY-876 vs. MSUc (n≥10/group). (I,J) Histological analysis by H&E showing reduction in the recruitment of inflammatory cells (arrow) in the synovial cavity of mice injected with MSUc+JNKi vs. MSUc (n≥10/group). (K,L) Histological analysis by H&E showing reduction in the recruitment of inflammatory cells (arrow) in the synovium of mice injected with MSUc+BAY-876 vs. MSUc (n≥7/group). (M) Diagram showing that MSUc induces a unique transcriptional program with up-activation of inflammatory –but not interferon stimulated genes- and metabolic genes, which is regulated through up-regulation of AP-1 via JNK but not IRFs. (#=P<0.10;*=P<0.05; **=P<0.01).

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Expressing, Injection, Activation Assay

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: The impact of DBI-2 and BAY-876 on cell signaling pathways in LS174T ( A ) and HCT116 ( B ) CRC cells. Combination of DBI-2 (3 μmol/L) and GLUT1 inhibitor BAY-876 (1 μmol/L) resulted in enhanced depletion of Axin2 and c-Myc, increase in p-AMPK and p-ACC, and depletion of p-P70S6K and p-S6. Data are presented as the means ± SEM. * p < 0.05, and ** p < 0.01, n = 3. One-way ANOVA followed by Tukey’s multiple comparisons test were applied as the statistical method.

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques:

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: DBI-2 and BAY-876 synergically inhibited CRC cells in vitro. ( A , B ) DBI-2 inhibited colorectal cancer cell LS174T ( A ) and HCT116 ( B ) proliferation. The IC 50 of DBI-2 to inhibit the proliferation of LS174T cells and HCT116 cells was 1.14 μmol/L and 0.53 μmol/L, respectively. ( C , D ) Combinatorial effects of DBI-2 and BAY-876 on CRC cell proliferation. The cells were exposed to DBI-2 or BAY-876 at specified concentrations for a duration of 5 days, and the number of viable cells was determined. Data are presented as the means ± SEM. ** p < 0.01, n = 3. One-way ANOVA followed by Tukey’s multiple comparisons test were applied as the statistical method.

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques: In Vitro

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: Synergistic effects of DBI-2 and BAY-876 on LS174T and HCT116 CRC cells. ( A , B ) DBI-2 and BAY-876 synergistically inhibited the proliferation of colorectal cancer cells. The cells were exposed to DBI-2 and BAY-876 at specified concentrations over a period of 5 days, and the number of viable cells was determined. Synergy scores were measured utilizing SynergyFinder, with Bliss serving as the benchmark model. The synergy scores for drug combinations of DBI-2 and BAY-876 were 25 for LS174T cells ( A ) and 15 for HCT116 cells ( B ).

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques:

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: Synergistic effects of DBI-2 and BAY-876 on inducing apoptosis in LS174T cells and HCT116 cells through flow cytometry analysis using Annexin-V-FITC and propidium iodide (PI) dual staining. ( A , E ) Cells were treated with DMSO. The apoptosis rate was 16% and 17%, respectively. ( B , F ) Cells were treated with DBI-2 at 3 μmol/L. The percentage of apoptotic cells was 26% and 22%, respectively. ( C , G ) Cells were treated with BAY-876 at 1 μmol/L. The apoptosis rate was 34% and 23%, respectively. ( D , H ) Cells were treated with the combination of DBI-2 (3 μmol/L) with BAY-876 (1 μmol/L). The apoptosis rate was 70% and 75%, respectively. ( I , J ) Percentage of normal, early apoptotic, and late apoptotic cells in LS174T cells and HCT116 cells. Data are presented as the means ± SEM. * p < 0.05, and ** p < 0.01 versus control group, ## p < 0.01 versus DBI-2 or BAY-876 group, n = 3. One-way ANOVA followed by Tukey’s multiple comparisons test were applied as the statistical method.

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques: Flow Cytometry, Staining

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: Synergistic effects of DBI-2 and BAY-876 on inducing autophagy and necrosis in LS174T cells and HCT116 cells. ( A ) Detection of autophagic vacuoles by MDC staining in LS174T cells treated with DBI-2 and BAY-876 alone and in combination for 8 h (a: DMSO; b: DBI-2; c: BAY-876; and d: DBI-2 and BAY-876). ( B ) Expression of LC3-Ⅰ and LC3-Ⅱ in LS174T cells and HCT116 cells after 8 h treatment. ( C ) The LDH activity in the culture medium of LS174T cells and HCT116 cells. Data are presented as the means ± SEM. * p < 0.05, and ** p < 0.01, n = 3. One-way ANOVA followed by Tukey’s multiple comparisons test were applied as the statistical method.

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques: Staining, Expressing, Activity Assay

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: The combination of DBI-2/KD and the GLUT1 inhibitor BAY-876 exhibited a synergistic inhibitory effect on colon cancer cell xenografts in vivo. ( A – C ) The combination of DBI-2 (40 mg/kg/day, IP) with BAY-876 (3 mg/kg/day, PO) or ketogenic diet feeding significantly inhibited tumor growth in RAG1 −/− γc − mice. Notably, this treatment regimen did not induce any noticeable adverse effects based on body weight. ( D ) Pictures of tumors after treatment for 12 days. ( E ) H&E staining of tumor sections (arrows). ( F ) IHC staining of Ki67, p-AMPK, and AMPK. Data are presented as the means ± SEM. * p < 0.05, and ** p < 0.01, n = 12. One-way ANOVA followed by Tukey’s multiple comparisons test were applied as the statistical method.

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques: In Vivo, Staining, Immunohistochemistry

Journal: Cancers

Article Title: Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells

doi: 10.3390/cancers16071399

Figure Lengend Snippet: Combination of DBI-2 with BAY-876 reduced blood glucose, plasma triglycerides (TG), T-CHO, and LDL-C. ( A ) Blood glucose tested from RAG1 −/− γc − mice tails. ( B – E ) The plasma TG, T-CHO, LDL-C, and HDL-C tested in tumor-bearing animals. Blood obtained via retro orbital eye bleeds prior to sacrifice. Blood samples spun and plasma separated. Data are presented as the means ± SEM. * p < 0.05, and ** p < 0.01, n = 6. One-way ANOVA followed by Tukey’s multiple comparisons test were applied as the statistical method.

Article Snippet: BAY-876 (i.e., N4-[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide) was acquired from Medkoo Bioscience (Durham, NC, USA) (Cat No. 530485).

Techniques:

Journal: Journal of Oral Pathology & Medicine

Article Title: Transcriptional regulation of glucose transporters in human oral squamous cell carcinoma cells

doi: 10.1111/jop.13342

Figure Lengend Snippet: Fold change difference between 50% and 100% confluence of GLUT mRNA expression in OKF6 and oral squamous cell carcinoma cells when treated with BAY876 or WZB117. Each data point was obtained from 3 biological replicates and error bars represent the SD. The control was baseline expression with no inhibitor present and the SD is an averaged SD from all groups.

Article Snippet: Cells were treated with 500 nM BAY876 (GLUT1 inhibitor; A17216; ADOOQ Bioscience) and 25 μM WZB117 (GLUT1, GLUT3 and GLUT4 inhibitor; 19900; Cayman Chemical Company) 24 h before they were expected to reach the chosen confluency.

Techniques: Expressing, Control