tgms inhibitor tgi  (MedChemExpress)

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: Effects of different TG2 inhibitors on the cross-linking activity of recombinant TG2, cell viability, and extracellular TG2 activity. (A) Fluorometric quantitation of the cross-linking of biotin-conjugated gliadin peptide P56-88 by recombinant TG2 ( n = 3, three technical replicates per experiment). (B) Fluorometric quantitation of cell viability after 24 h of incubation with the corresponding substances using the resazurin-based assay ( n = 3, two technical replicates per experiment). (C) Fluorometric quantitation of extracellular cross-linking of the TG2 substrate 5BP by Caco-2 cells ( n = 3, with three technical replicates per experiment). All data are shown as mean ± SD. Statistical significance was tested by using univariate ANOVA (A) or Student’s t -test with Welch correction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 5BP = 5-(biotinamido)-pentylamine, ZnCl 2 = zinc chloride, AA = ascorbic acid.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Activity Assay, Recombinant, Quantitation Assay, Incubation, Resazurin Assay

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: Effect of IFN-γ on the expression of ERp57, TRX and TG2 in Caco-2 cells. Analysis of relative expression of (A) ERp57, (B) TRX, and (C) TG2 by Western blotting after stimulation with different concentrations of IFN-γ over 48 h. No significant change in the expression of ERp57 and TRX. Dose-dependent increase in TG2 expression. All data are shown as mean ± SD. Statistical significance was tested by using Student’s t -test. n = 4. * P < 0.05.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Expressing, Western Blot

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: SiRNA knockdown of ERp57, TRX, and TG2, and the effect on cellular/extracellular TG2 activity. Caco-2 cells were incubated with specific siRNAs or mock siRNA for 48 h. The efficacy of the siRNA knockdown was determined by using Western blotting. Expression of ERp57, TRX, and TG2 was normalized against the housekeeping protein GAPDH. (A–C) SiRNA treatment significantly reduced expression of (A) ERp57, (B) TRX, and (C) TG2. Incubation with mock siRNA did not affect protein expression ( n = 4). (D) Treatment with ERp57-siRNA und TRX-siRNA did not show any off-target effects on TG2 expression ( n = 3). (E) Cellular expression of ERp57, TRX, and TG2 determined by fluorometry after siRNA knockdown. Protein levels of all three target proteins were reduced by siRNA-mediated knockdown. (F) Cellular TG2 activity was determined by fluorometry after siRNA treatment. SiRNA knockdown of TRX and TG2 reduced cellular TG2 activity ( n = 5). (G) Extracellular expression of ERp57, TRX, and TG2 was investigated in unpermeabilized Caco-2 cells by fluorometry. Protein levels of all three target proteins were significantly reduced by siRNA-mediated knockdown. (H) Extracellular TG2 activity was investigated in unpermeabilized Caco-2 cells by fluorometry. SiRNA knockdown of TRX and TG2 significantly reduced extracellular TG2 activity ( n = 5). All data are shown as mean ± SD. Statistical significance was tested by using Student’s t -test with Welch correction where appropriate. * P < 0.05, ** P < 0.01, *** P < 0.001. GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Knockdown, Activity Assay, Incubation, Western Blot, Expressing

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: TG2 is expressed in the lamina propria and the epithelium of CD biopsies. (A) Fluorescence staining revealed TG2 to be nearly absent in the epithelium of control biopsies and low levels were expressed in the lamina propria (arrows). In CD biopsies, TG2 expression was increased in the epithelium (arrowheads) and the lamina propria (arrows). (B) Detailed images of the duodenal mucosa reveal high levels of TG2 in the lamina propria (arrows) and the epithelium (arrowheads) of CD patients. (C) TG2 expression was significantly increased in the epithelium and the lamina propria of CD patients. All data are shown as median. Statistical significance was tested by using Student’s t -test. n = 5. * P < 0.05; scale: 20 µm.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Fluorescence, Staining, Control, Expressing

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 1. Effect of TGase2 inhibition on reducing pIgA1 binding to HMC. (A) Western blot analysis of TGase2 expression in HMC after various concen- tration of pIgA1 stimulation for 48 hours. Quantification is shown in the bar graph as mean ± SD (n = 3, 1-way ANOVA). (B) HMC (1.6 × 105 cells in 200 μL 0.5% FBS-1640 medium) was preincubated with or without 10 μM LDN-21279 (LDN) at 37°C for 1 hour and then treated with pIgA1 from patients with IgAN (10 μg/mL) for another 1 hour. The stained cells were analyzed on FACScan. Representative plots and bar graph show the percentages of IgA1 staining cells. Data represent the mean ± SD (n = 3). Significant difference was determined by 1-way ANOVA. (C) Western blot analysis of fibronectin (Fn), collagen type I (Col-I), and TGase2 expression in HMC, which were pretreated with LDN-27219 (LDN, 10 μM) for 1 hour, followed by pIgA1 (10 μg/mL) treatment for 48 hours. Data are shown as mean ± SD (n = 3, 1-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Inhibition, Binding Assay, Western Blot, Expressing, Staining

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 2. Identification of TGase2 expression in the membrane of human mesangial cells. (A) Representative immunofluorescence staining of TGase2 (red) and IgA1 (green) in the kidney of normal controls (Con, n = 3) and patients with IgAN (IgAN, n = 5). Total original magnification, ×630. (B) Fluorescence intensity was analyzed by ImageJ software. Bars represent the mean fluorescence intensity of TGase2 signals in glomeruli from at least 5 visual fields (×40) of each sample. Data are shown as mean ± SD. Statistical analysis between groups was performed by unpaired 2-tailed Student t test. (C) Western blot analysis of TGase2 expression in HMC with or without pIgA1 (10 μg/mL) treatment for 48 hours. Cytosolic (C) and membrane (M) fractions were vali- dated for enrichment of cytosol marker (β-tubulin) or membrane marker (Na+/K+ ATPase). (D) Quantification of C is shown in the bar graph; data are shown as mean ± SD (n = 3, 1-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Expressing, Membrane, Immunofluorescence, Staining, Fluorescence, Software, Western Blot, Marker

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 3. TGase2 interactome in the cytosol of human mesangial cells treated with pIgA1. (A) HMC were treated with or without pIgA1 (10 μg/mL) for 48 hours. Cytosolic (C) and membrane (M) fractions were separated and validated for enrichment of β-tubulin and Na+/K+ ATPase by Western blot. TGase2-asso- ciated proteins in the cytosol were immunoprecipitated using anti-TGase2 antibody (n = 2). The isotype control antibody cross-link to protein A/G magnetic beads was used as a control (n = 2). (B) The TGase2-associated proteins after IP were analyzed via MS. The number of proteins was identified as specifically associated with TGase2 after exclusion of nuclear, mitochondrial, and ribosomal proteins. (C and D) Functional distribution of TGase2-associated proteins from cytosolic fractions without (C) or with pIgA1 deposition (D). Proteins were clustered based on their functions in Homo sapiens, which were determined by pro- tein identification search in the UniProtKB database (https://www.uniprot.org/). Column charts display the distribution of different functions of TGase2-as- sociated proteins with numbers and the percentage of proteins falling in the various functional categories. (E) The protein-interaction network was built from TGase2-associated proteins from cytosolic fraction without pIgA1 treatment (208 proteins) or with pIgA1 treatment (253 proteins). The protein-interaction network was mapped against the Homo sapiens reference database using the STRING tool. Candidates were selected using both known and predicted protein interactions with a threshold confidence level of 0.5. Networks were imported into Cytoscape software, and the direct partners of TGase2 were shown. The size and color shade of circles are proportional to the number of protein interactions, while the thickness and color shade of the lines are proportional to the confidence of the interactions. Here, only 1 interactome of cytosolic TGase2 is shown because it remained the same in HMC without or with pIgA1 treatment.

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Membrane, Western Blot, Immunoprecipitation, Control, Magnetic Beads, Functional Assay, Software

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 4. TGase2 is present in cellular vesicles of plasma membrane origin in HMC. (A) Flowchart of the centrifugation and filtration steps for the isola- tion of cellular vesicles. S1 and S2 indicate supernatants. S2 is total lysate (TL). C1 indicates purified cellular vesicles. F1 and F2 indicate filtrate. (B) HMC were planted in a 6-well plate overnight and then grown in serum-free medium with supplementation of 10 μg/mL pIgA1 or PBS for 24 hours. Cellular vesi- cles were purified as shown in A. The microparticle size distribution in fractions C1 from HMC treated with PBS (NC) or pIgA1 was obtained using nanoparti- cle tracking analysis (n = 3). (C) Expression of FLOT2 and TGase2 in purified cellular vesicles (C1), filtrate fractions (F1 and F2), and cell lysate (TL) from HMC treated with or without pIgA1 were measured by Western blot in equal amounts of proteins from different fractions.

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Clinical Proteomics, Membrane, Centrifugation, Filtration, Purification, Expressing, Western Blot

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 5. Polymeric IgA1 deposition on HMC and the membrane transfer of cytosol TGase2 are reduced by vesicular trafficking inhibition. (A) Exponentially growing HMC were cultured in serum-free medium with Exo1 (30 μM) or dissolve for 24 hours. Cellular vesicles were purified by centrif- ugation (5,000g for 5–8 minutes at room temperature) and filtration as shown in Figure 4. The microparticle size distribution of cellular vesicles was obtained using nanoparticle tracking analysis (n = 3). (B) HMC (1.6 × 105 cells in 200 μL 0.5% FBS-1640 medium) was preincubated with 30 μM Exo1 or dissolve (Ctl) at 37°C overnight and then treated with pIgA1 (20 μg/mL) for 1 hour. Cells were washed with 0.5% BSA-PBS and then stained with goat anti–human IgA antibody. Cells were washed with 0.5% BSA-PBS and then stained with goat anti–human IgA antibody, followed by secondary stain- ing with Alexa Fluor 647–conjugated donkey anti-goat antibody. Cells stained without anti-IgA antibody were used as negative control. The stained cells were analyzed on FACScan. (C) The results were expressed as mean ± SD of the percentage of stained cells from 3 individual experiments. Data were analyzed by 1-way ANOVA. (D) Exponentially growing HMC planted in a 6-well plate were treated with Exo1 (30 μM), dissolve (Ctl), or blank (NC) in serum-free medium. After 24 hours, Western blot was used to detect the expression of TGase2 in the cytosolic (C) and membrane (M) fraction. (E) Quantification of D is shown in the bar graph; data are shown as mean ± SD (n = 3, 1-way ANOVA). *P < 0.05, ***P < 0.001.

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Membrane, Inhibition, Cell Culture, Purification, Filtration, Staining, Negative Control, Western Blot, Expressing

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 6. TGase2 transfers to membrane dependently on RhoA-mediated vesicle-trafficking in HMC. (A) Western blot analysis of RhoA in HMC with or without pIgA1 (10 μg/mL) treatment for 48 hours. Quantification is shown in the bar graph; data are shown as mean ± SD (n = 3, 2-tailed Student t test). (B) Cellular vesicles were purified from HMC with pIgA1 (10 μg/mL) or not for 48 hours by centrifugation and filtration as shown in Figure 4. RhoA was immunoprecipitated from vesicular fractions (C1) with rabbit anti-RhoA antibody. The presence of TGase2 and RhoA in immu- noprecipitants were detected by Western blot (n = 2). (C) HMC were transiently transfected with RhoA overexpression plasmid (RhoAOE) or empty vector (Control) for 48 hours. Cellular vesicles were purified from as shown in Figure 4. The microparticle size distribution of cellular vesicles was obtained using nanoparticle tracking analysis (n = 3). (D) Lysates of vesicular fraction (C1) isolated from HMC with overexpression of RhoA obtained as described in C, total lysate (TL) and filtrate fractions (F1 and F2) were tested for RhoA, TGase2 and FLOT2 by Western blot in equal amounts of proteins from different fractions (n = 3). (E) Western blot analysis of RhoA and TGase2 in the cytosolic (C) and membrane (M) fractions of HMC

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Membrane, Western Blot, Purification, Centrifugation, Filtration, Immunoprecipitation, Transfection, Over Expression, Plasmid Preparation, Control, Isolation

Journal: JCI insight

Article Title: RhoA vesicle trafficking-mediated transglutaminase 2 membrane translocation promotes IgA1 mesangial deposition in IgA nephropathy.

doi: 10.1172/jci.insight.160374

Figure Lengend Snippet: Figure 7. The proposed pathway of TGase2 from cytosol to membrane through RhoA-mediated vesicle trafficking promotes pIgA1 deposition on human mesangial cells. (A) TGase2 is weakly expressed in normal kidney mesangial cells, and it translocates from the cytosol to the membrane using RhoA+ vesicles. The inhibitor of vesicle trafficking pathway, Exo1, significantly reduces the transport of TGase2 to the membrane. (B) Polymeric IgA1 (pIgA1) from the blood in patients with IgA nephropathy enter into the kidney and can be deposited in the mesangium through binding to TGase2 and the IgA1 receptor TFRC; this activates more expression of TGase2, RhoA, and RhoA+ vesicles. Increased TGase2 are transported to the membrane through RhoA+ vesicles, leading more and more pIgA1 sticks to the mesangial cell membrane. (C) The interaction of pIgA1 with mesangial cells induces cell proliferation and pro- duction of ECM, such as fibronectin and collagens, which will further increase the disease progression.

Article Snippet: HMC suspension was preincubated with or without TGase2 inhibitor LDN-21279 (MedChemExpress, HY-16693) or exocytosis inhibitor Exo1 (MedChemExpress, HY-112670); it was then incubated with pIgA1 isolated from patients with IgAN.

Techniques: Membrane, Binding Assay, Expressing, Biomarker Discovery