txnip inhibitor  (MedChemExpress)

Journal: Cell Death & Disease

Article Title: BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

doi: 10.1038/s41419-023-05811-2

Figure Lengend Snippet: A Volcano plot representing the genes significantly differentially expressed in K562 and K562G ( P < 0.05). B Relative mRNA levels of TXNIP in the PB and BM were compared among samples from normal healthy donors and patients as described in the figure. Each individual dot represents 1 sample. CP, chronic phase; AP, accelerated phase; BP, blast phase. C , D TXNIP protein ( C ) and mRNA ( D ) levels after BCR-ABL transduction (MIG210) in normal CD34 + cells from 2 independent healthy donors. Empty vector MIGR1 was used as a control. E , F TXNIP protein ( E ) and mRNA ( F ) levels in indicated CML cells 24 h after 0.5 μM imatinib treatment. G , H TXNIP protein ( G ) and mRNA ( H ) levels in K562 and K562G cells 24 h after 0.5 μM imatinib treatment. I TXNIP protein levels in K562 and KCL22 cells after BCR-ABL knockdown by shABL lentiviral vector. *** P < 0.001. ** P < 0.01.

Article Snippet: To assess the mitochondrial membrane potential and mitochondrial mass, K562G and K562R cells with or without TXNIP knockdown were stained with the JC-1 reagent (2 μM, #HY-15534, MedChemExpress) and MitoTracker Green (0.1 μM, #HY-135056, MedChemExpress) at 37 °C for 30 min, respectively.

Techniques: Transduction, Plasmid Preparation, Control, Knockdown

Journal: Cell Death & Disease

Article Title: BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

doi: 10.1038/s41419-023-05811-2

Figure Lengend Snippet: A GSEA analysis of the RNAseq data predicts the gene enrichment in c-Myc targets in K562G and K562 cells. B The amount of c-Myc bound to the TXNIP promoter was determined by ChIP in K562 and KCL22 cells 24 h after imatinib treatment. C , D The activity of TXNIP promoter (−1185 ~ +334) was determined in K562 and KCL22 cells 24 h after imatinib ( C ) or JQ1 ( C ) treatment. E – G TXNIP protein ( E ), mRNA ( F ), and promoter activity ( G ) were determined, respectively, in K562 and KCL22 cells 48 h after the transfection of Miz-1 overexpression vector. H The indicated TXNIP promoter truncations were transfected into K562 cells with or without Miz-1 overexpression. The luciferase activity were determined. I K562 cells were transfected with the TXNIP reporter construct (−751 ~ +25) with or without a c-Myc expression plasmid. Increasing amounts (0.02, 0.1, and 0.2 μg) of Miz-1 construct were co-transfected as indicated. J K562 cells were transfected with the TXNIP reporter construct (−751 ~ +25) together with wild-type Miz-1 and its mutant construct, as indicated. K K562 cells were transfected with indicated constructs with TXNIP reporter truncation (−751 ~ +25). 24 h after transfection, cell were treated with 0.5 μM imatinib for additional 24 h. L K562 cells were transfected with Miz-1 constructs and treated with 0.5 μM imatinib. Immunoprecipitation was performed with Miz-1 antibody. Indicated proteins were further determined by western blot. M – P The mRNA expression associations of TXNIP with Miz-1 or c-Myc in the PB and BM were determined among samples from normal healthy donors and patients. Each individual dot represents 1 sample. Q The mRNA expression associations of TXNIP with Miz-1 were analyzed by GEPIA according to the whole blood samples from normal human in GETx dataset. R The mRNA expression associations of TXNIP with c-Myc were analyzed by GEPIA according to the Pan-Cancer samples from normal human in TCGA dataset. B , C , K , L IM, imatinib. *** P < 0.001. ** P < 0.01.

Article Snippet: To assess the mitochondrial membrane potential and mitochondrial mass, K562G and K562R cells with or without TXNIP knockdown were stained with the JC-1 reagent (2 μM, #HY-15534, MedChemExpress) and MitoTracker Green (0.1 μM, #HY-135056, MedChemExpress) at 37 °C for 30 min, respectively.

Techniques: Activity Assay, Transfection, Over Expression, Plasmid Preparation, Luciferase, Construct, Expressing, Mutagenesis, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

doi: 10.1038/s41419-023-05811-2

Figure Lengend Snippet: A Schematic strategy of evaluation of the in vivo effect of TXNIP knockout on CML transformation. B Kaplan–Meier-style survival curves for recipients of BCR-ABL-Cre-GFP or BCR-ABL-GFP transduced BM cells from TXNIP fl/fl ( n = 10) mice. C Apoptosis of BCR-ABL or MIGR1 (CTRL) transduced normal CD34 + cells with or without Cre overexpression in the presence of 2 μM imatinib. The double-transduced CD34 + cells were sorted and then analyzed for annexin V + cells after 48 h of culture. D , E Analysis of total cell numbers ( D ) and colony-forming cell (CFC) formation ( E ) of BCR-ABL-Cre-GFP or BCR-ABL-GFP transduced normal CD34 + cells with 2 μM imatinib treatment. F The indicated protein levels were determined by western blot in K562 and KCL22 cells after TXNIP knockdown. G The cell viabilities of K562 and KCL22 cells were determined after TXNIP knockdown in indicated days. H The cell viability was determined in K562 and KCL22 cells following indicated doses of imatinib treatment for 72 h, and normalized to the control group. I Edu incorporation assay was performed for cell viability determination in K562 and KCL22 cells after TXNIP knockdown with or without 2 μM imatinib treatment. J K562 and KCL22 cells were plated in methylcellulose-containing medium after TXNIP knockdown, with or without imatinib treatment. The CFC were scored after 1 week. K Apoptosis was analyzed 48 h after imatinib treatment. L Representative bioluminescence imaging photographs of tumor burden in nude mice ( n = 6) 24 days after the subcutaneous injection of indicated cells. sh#2: shTXNIP#2. M The Tumor size was measured and tumor volume was calculated by the formula (width 2 × length × 0.5). IM: imatinib. *** P < 0.001. ** P < 0.01.

Article Snippet: To assess the mitochondrial membrane potential and mitochondrial mass, K562G and K562R cells with or without TXNIP knockdown were stained with the JC-1 reagent (2 μM, #HY-15534, MedChemExpress) and MitoTracker Green (0.1 μM, #HY-135056, MedChemExpress) at 37 °C for 30 min, respectively.

Techniques: In Vivo, Knock-Out, Transformation Assay, Over Expression, Western Blot, Knockdown, Control, Imaging, Injection

Journal: Cell Death & Disease

Article Title: BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

doi: 10.1038/s41419-023-05811-2

Figure Lengend Snippet: A Schematic strategy of evaluation of the in vivo effect of TXNIP overexpression on CML transformation. B Kaplan–Meier-style survival curves for recipients of BCR-ABL-TXNIP-GFP or BCR-ABL-GFP transduced BM cells from C57BL/6 ( n = 12) mice. C Apoptosis of BCR-ABL or MIGR1 (CTRL) transduced normal CD34 + cells with or without TXNIP overexpression. The double-transduced CD34 + cells were sorted and then analyzed for annexin V + cells after 48 h of culture. D , E Analysis of total cell numbers ( D ) and CFC formation ( E ) of BCR-ABL-TXNIP-GFP or BCR-ABL-GFP transduced normal CD34 + cells. F The cell viability was determined in K562, K562G, and K562R cells following indicated doses of imatinib treatment for 72 h, and normalized to the control group. G TXNIP and c-Myc expressions in K562, K562G, and K562R cells. H TXNIP and c-Myc expressions in TXNIP overexpressed K562G and K562R cells. I – L In TXNIP overexpressed K562G and K562R cells, the cell viabilities were determined by CCK8 ( I ) and Edu staining ( J ) assay. The CFC formation ( K ) and apoptosis ( L ) were analyzed, respectively. M Survival curves of mice receiving xenografted CML cells. K562G or K562R cells with or without TXNIP overexpression were inoculated into nude mice. Mice were euthanized when the tumor volume reached 1000 mm 3 . N The human primary CML CD34 + cells were infected with TXNIP lentivirus, and then injected into sublethally irradiated (300 cGy) NSI mice. After 12 weeks, human multilineage engraftment was analyzed by flow cytometry. O , P The percentage (O) and the absolute number ( P ) of human CD45 + cells engrafted in the BM after transplantation of human CML CD34 + cells (1 × 10 6 cells/mouse) for 12 weeks. Q The proportion of human CD45 + cells engrafted in the spleen at 12 weeks. R The CFC formation after TXNIP overexpression. *** P < 0.001. ** P < 0.01.

Article Snippet: To assess the mitochondrial membrane potential and mitochondrial mass, K562G and K562R cells with or without TXNIP knockdown were stained with the JC-1 reagent (2 μM, #HY-15534, MedChemExpress) and MitoTracker Green (0.1 μM, #HY-135056, MedChemExpress) at 37 °C for 30 min, respectively.

Techniques: In Vivo, Over Expression, Transformation Assay, Control, Staining, Infection, Injection, Irradiation, Flow Cytometry, Transplantation Assay

Journal: Cell Death & Disease

Article Title: BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

doi: 10.1038/s41419-023-05811-2

Figure Lengend Snippet: A 2 NBDG assays for glucose uptake in K562 and KCL22 cells with TXNIP knockdown 24 h after 1 μM imatinib treatment. B Lactate production in K562 and KCL22 cells with TXNIP knockdown 24 h after 1 μM imatinib treatment. C – F The high throughput Seahorse assays to monitor extracellular acidification rate (ECAR) in K562 ( C , D ) and KCL22 ( E , F ) cells with TXNIP knockdown. G – J The high throughput Seahorse assays to monitor cellular oxygen consumption rate (OCR) in K562 ( G , H ) and KCL22 ( I , J ) cells with TXNIP knockdown. K – N The high throughput Seahorse assays to monitor ECAR in K562G ( K , L ) and K562R ( M , N ) cells with TXNIP overexpression. O – R The high throughput Seahorse assays to monitor cellular OCR in K562G ( O , P ) and K562R ( Q , R ) cells with TXNIP overexpression. S , T The intracellular ATP concentrations were determined in indicated cells with TXNIP knockdown after 1 μM imatinib treatment ( S ), or TXNIP overexpression ( T ). U The transmission electron microscope assays for assessing the mitochondrial shape in K562G and K562R cells after TXNIP overexpression. V MitoTracker-based flow cytometry assays for assessing mitochondrial number. W JC1-mitochondrial membrane potential assays. BR basic respiration, AP ATP production, MR maximal respiration, SC spare capacity, NGA non-glycolytic acidification, GLY glycolysis, GLC glycolytic capacity, GLR glycolytic reserve. *** P < 0.001. ** P < 0.01.

Article Snippet: To assess the mitochondrial membrane potential and mitochondrial mass, K562G and K562R cells with or without TXNIP knockdown were stained with the JC-1 reagent (2 μM, #HY-15534, MedChemExpress) and MitoTracker Green (0.1 μM, #HY-135056, MedChemExpress) at 37 °C for 30 min, respectively.

Techniques: Knockdown, High Throughput Screening Assay, Over Expression, Transmission Assay, Microscopy, Flow Cytometry, Membrane

Journal: Cell Death & Disease

Article Title: BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

doi: 10.1038/s41419-023-05811-2

Figure Lengend Snippet: A The heatmap represents the expression of glycolytic-related genes in K562 cells, K562 cells with TXNIP knockdown (K562sh), and K562G cells according to the RNAseq data. B HK2 and LDHA mRNA levels in K562 and KCL22 cells with or without TXNIP knockdown. C , D c-Myc mRNA levels in indicated cells with TXNIP knockdown or overexpression. E Indicated protein levels were determined in K562 cells with or without TXNIP overexpression, or TXNIP-overexpressed K562 cells with Fbw7 knockdown. F K562 cells with or without TXNIP expression were treated with 20 μM MG132 for 2 h. Levels of indicated proteins were determined by western blot. G Levels of indicated proteins in indicated K562 cells following 10 μg/ml CHX treatment for indicated time. H c-Myc expression levels were further quantified by ImageJ and normalized to β-actin to determine the protein degradation rate. I Levels of indicated proteins in K562 cells with or without TXNIP overexpression. J – M We overexpressed the wild type or activating form of Akt1 (myr-Akt1) in TXNIP overexpressed K562 cells. J The indicated proteins levels were determined by western blotting. K Immunoprecipitation was performed to determine the interaction between c-Myc and Fbw7 in indicated K562 cells. After the Fbw7 protein were immunoprecipitated with an anti-Fbw7 antibody, indicated proteins were detected by western blotting. L , M c-Myc enrichment on HK2 ( L ) and LDHA ( M ) promoter were determined by ChIP. N , O c-Myc enrichment on HK2 ( N ) and LDHA ( O ) promoter were determined by ChIP in K562 cells with or without TXNIP knockdown after AKT inhibitor Capivasertib (Cap) or c-Myc inhibitor JQ1 treatment. *** P < 0.001.

Article Snippet: To assess the mitochondrial membrane potential and mitochondrial mass, K562G and K562R cells with or without TXNIP knockdown were stained with the JC-1 reagent (2 μM, #HY-15534, MedChemExpress) and MitoTracker Green (0.1 μM, #HY-135056, MedChemExpress) at 37 °C for 30 min, respectively.

Techniques: Expressing, Knockdown, Over Expression, Western Blot, Immunoprecipitation

Journal: Diseases

Article Title: Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy

doi: 10.3390/diseases9040091

Figure Lengend Snippet: HG induces TXNIP expression and triple drug combination treatment reduces TXNIP in rMC1. ( A ) On Western blots, TXNIP expression increases ~4.62 +/− 0.34 folds by HG (lane 2 #, mean +/− SE versus LG, n = 3). Lysosome-targeted drug ML-SA1 (20 μM) or TXNIP-IN1 (TXNIPi, an inhibitor of TXNIP-Trx interaction, 5 μM) has no effect on HG-induced TXNIP expression, while mitochondria-targeted anti-oxidant mito-Tempo (5 μM) and triple drug combination, TMM, reduces TXNIP level by 69% (lane 6 *) and 50% (lane 10 *), respectively, versus HG (2), n = 3). ( B ) Similarly, TXNIP level also increases in the mitochondrion under HG (lane 2 #, ~2.3-folds versus LG, n = 3), while combination drug (TMM) treatment reduces ~70% (Lane 2 versus Lane 4 *, n = 3).

Article Snippet: Drugs used in this study were purchased from commercial vendors, such as TXNIP-IN1 (Cat# HY-115688, MedChemEXpress, Monmouth Junction, NJ, USA) and Mito-Tempo (Cat# SML0737), and ML-SA1 (Cat# SML0627) and Tranilast (Cat# T0138) from Sigma, St. Louis, MO, USA.

Techniques: Expressing, Western Blot

Journal: Diseases

Article Title: Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy

doi: 10.3390/diseases9040091

Figure Lengend Snippet: Combination triple drug treatment reduces mitophagy in rMC1 under HG in live-cell imaging. rMC1 cells were transduced with 2×mt8a-CG (mCherry-EGFP) for 3 days under HG or LG. When drug treatments were performed, they were present for 24 h before the confocal live-cell imaging. We observed that HG increases red mitolysosome formation (white arrows, HG second panel) when compared to LG. Active mitochondria show yellow (red and green), while damaged mitochondria after flux to lysosomes yield red due to EGFP (green) quenching. Interestingly, combination triple drug treatment with AST (amlexanox, SS-31 and tranilast) or with TMM (TXNIP-IN1+Mito-Tempo+ML-SA1) reduces mitophagic flux in rMC1 under HG (last two panels, respectively). A representative of n = 3 is shown here, captured at 630× magnification in a Zeiss confocal microscope.

Article Snippet: Drugs used in this study were purchased from commercial vendors, such as TXNIP-IN1 (Cat# HY-115688, MedChemEXpress, Monmouth Junction, NJ, USA) and Mito-Tempo (Cat# SML0737), and ML-SA1 (Cat# SML0627) and Tranilast (Cat# T0138) from Sigma, St. Louis, MO, USA.

Techniques: Live Cell Imaging, Transduction, Microscopy

Journal: Diseases

Article Title: Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy

doi: 10.3390/diseases9040091

Figure Lengend Snippet: Combination triple drug treatment reduces mitophagy in rMC1 under HG in fixed-cell imaging. rMC1 cells were transduced with 2×mt8a-CG (mCherry-EGFP) for 3 days under HG or LG similar to live-cell imaging. When drug treatments were performed, they were added 24 h before fixing the cells. Nuclei were counter-stained with DAPI (blue). HG increases red mitolysosome formation (while arrows, HG second panel) in rMC1 cells than in LG. Active mitochondria show yellow (red and green), while damaged mitochondria emit red in lysosomes due to EGFP (green) quenching. AST (amlexanox, SS-31 and tranilast) or TMM (TXNIP-IN1+Mito-Tempo+ML-SA1) reduces mitophagic flux in rMC1 under HG (last two panels, respectively). A representative of n = 3 is shown here, captured at 630× magnification in a Zeiss confocal microscope.

Article Snippet: Drugs used in this study were purchased from commercial vendors, such as TXNIP-IN1 (Cat# HY-115688, MedChemEXpress, Monmouth Junction, NJ, USA) and Mito-Tempo (Cat# SML0737), and ML-SA1 (Cat# SML0627) and Tranilast (Cat# T0138) from Sigma, St. Louis, MO, USA.

Techniques: Imaging, Transduction, Live Cell Imaging, Staining, Microscopy

Journal: Diseases

Article Title: Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy

doi: 10.3390/diseases9040091

Figure Lengend Snippet: Summary of HG-induced TXNIP expression, redox stress, and the mitochondrial–lysosomal axis dysregulation in Müller cells. A potential therapeutic approach for targeting multiple steps at the cellular stress and organelle damage in the pathogenesis of DR. TXNIP-IN1, an inhibitor of the TXNIP-Trx interaction, Mito-Tempo, a mitochondrial matrix-specific superoxide scavenger, and ML-SA1, an agonist of the mucolipin transient receptor potential calcium channel 1 (MCOLN1/TRPML1), description in Discussion.

Article Snippet: Drugs used in this study were purchased from commercial vendors, such as TXNIP-IN1 (Cat# HY-115688, MedChemEXpress, Monmouth Junction, NJ, USA) and Mito-Tempo (Cat# SML0737), and ML-SA1 (Cat# SML0627) and Tranilast (Cat# T0138) from Sigma, St. Louis, MO, USA.

Techniques: Expressing