Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: NOX2 enhances FADDosome-induced gastric epithelial mitochondrial fission and dysfunction in PHG. (A) Two-dimensional hierarchical clustering results for the enzyme NADPH oxidase (NOX) family were presented for the comparison between PHG patients and healthy volunteers (n = 3 per group). The fold changes and P values of the indicated mRNAs in PHG tissues relative to those in normal (Uninvolved) tissues from the microarray experiment were also presented. (B) NOX1, NOX2 and NOX4 IHC staining (brown) showed that NOX2, but not NOX1 or NOX4, was upregulated in the gastric mucosal samples of PHG patients, and the NOX1, NOX2 and NOX4 areas were also analyzed. n = 6 per group. * P < 0.05. (C) Representative images of IHC staining (brown) for NOX1, NOX2 and NOX4 in mouse models revealed that Nec-1 (an inhibitor of RIPK1) repressed NOX2 expression in mice with PVL. n = 6 per group. (D) The NOX1, NOX2 and NOX4 areas detected by IHC staining in (C) were presented. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (E) The colocalization of TOMM20 (red) and NOX2 (green) determined by confocal imaging in GES-1 cells confirmed that TNF-α increased the mitochondrial localization of NOX2 (white arrows indicate colocalization). Nuclei (blue) were counterstained with DAPI. (F) The levels of and interaction between NOX2 and TOMM20 were determined by western blotting, and IP showed that Nec-1 decreased the epithelial expression of total and mitochondrial NOX2 in the primary epithelial cells of mice with PVL. TEM also revealed that Nec-1 partly normalized the abnormal or damaged mitochondria in the mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (G) The ATP concentration in the mouse models verified that Nec-1 and GSK2795039 (GSK, a NOX2 inhibitor) improved the production of ATP in mice with PVL. n = 6 per group. * P < 0.05. (H) Representative 4-HNE IHC staining (brown) and quantification analysis in SO mice and mice with PVL suggested that GSK2795039 (GSK) reduced the level of 4-HNE in mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (I) Representative MitoSox staining (red) and the MitoSox intensity in mouse model revealed that GSK2795039 alleviated mitochondrial ROS (mtROS) accumulation in the primary epithelial cells of mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (J) Related protein levels in primary epithelial cells from mouse models treated with the indicated agents ( shFADD , Nec-1 or BAY) showed interactions among TAK1, NF-κBp65, FADD and NOX2 signaling. (K) GES-1 cells were transfected with either the control vector or shFADD and then treated with TNF-α or vehicle, and the NOX2 luciferase reporter activities were presented (left panel). * P < 0.05 versus the vehicle group and TNF-α-treated cells transfected with shFADD . The NOX2 luciferase reporter activities in GES-1 cells transfected with pcDNA3.1-p65 -vector or pcDNA3.1 -vector were also shown (right panel), * P < 0.05.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Comparison, Microarray, Immunohistochemistry, Expressing, Imaging, Western Blot, Concentration Assay, Staining, Transfection, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: The alteration of glycolysis associated with dysfunction of the mitochondrial electron transport chain contributes to oxidative stress in the PHG. (A) Western blotting showed that the protein levels of LDHA and HK2 were increased in mice with PVL but were decreased by GSK2795039 (GSK, a NOX2 inhibitor) in mice with PVL. The ratios of the normalized LDHA/β-actin and HK2/β-actin densitometric units and the relative level of lactic acid were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL not treated with GSK2795039. (B) Oxygen consumption rate (OCR) analysis of primary epithelial cells isolated from mouse models suggested that increased nonmitochondrial oxygen consumption and reduced maximum respiration were present in mice with PVL but were reversed by GSK2795039. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (C) Examination of the glycolytic rate (for the extracellular acidification rate [ECAR]) showed that GSK2795039 treatment reduced basal and compensatory glycolysis in mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (D) Two-dimensional hierarchical clustering results of the genes of electron transport chain (ETC)-related elements between PHG patients (as PHG) and healthy volunteers (as Uninvolved) (n = 3 per group). (E) Western blotting analysis (left panel) of protein levels of representative ETC complex subunits in the primary epithelial cells of PHG patients and healthy volunteers (as Uninvolved) revealed that the levels of the mitochondrial complex I, II, III, IV and V subunits were decreased in PHG patients. The activity of the antioxidant enzyme SOD and the ratio of reduced (GSH) to oxidized (GSSG) states (GSH/GSSG) detected by assay kits (right panel) were found to be decreased in PHG. n = 6 per group. * P < 0.05. (F) Western blotting analysis of representative ETC complex subunits from primary epithelial cells isolated from mouse models suggested that Nec-1 (an inhibitor of RIPK1) mitigated the defects in ETC subunit expression and decreased ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (G) SOD activity and the GSH/GSSG ratio, as detected by assay kits, were inhibited in mice with PVL but were restored by Nec-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (H) Western blotting analysis and quantification of protein levels for representative ETC complex subunits from primary epithelial cells isolated from mouse models showed that Mdivi-1 reversed the decrease in mitochondrial complex I, II, III, IV and V subunit levels and reduced ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also presented. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment. (I) Decreases in SOD activity and the GSH/GSSG ratio detected by assay kits were found in mice with PVL but were reversed by Mdivi-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Western Blot, Isolation, Activity Assay, Expressing

Journal: Nature Communications

Article Title: Hypoxia induced responses are reflected in the stromal proteome of breast cancer

doi: 10.1038/s41467-023-39287-7

Figure Lengend Snippet: Multivariate survival analysis (proportional hazards regression model) of breast cancer patients (METABRIC-Discovery cohort)

Article Snippet: Of relevance for secretome studies, we found that 40 of the PAM50 signature genes/proteins have been reported in serum or plasma (plasma proteome database; PPD: http://www.plasmaproteomedatabase.org/ ) , and/or the Human Protein Atlas—blood protein (Human Protein Atlas proteinatlas.org) .

Techniques:

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: The expression of SMARCAD1 is significantly higher in pancreatic cancer and positively correlated with poor prognosis. A. The mRNA expression level of SMARCAD1 in pancreatic cancer is much higher than that of normal tissues from GSE16515, GSE11838 and GSE15471. B-C. Expression levels of SMARCAD1 by immunohistochemistry performed with tissue microarray of PC (n=69) and adjacent normal tissues (n=68). Representative images showed positive expression of SMARCAD1 in PC and negative expression in paired normal tissues, respectively. Scale bars=50μm. D. Kaplan-Meier analysis shows the correlation between SMARCAD1 expression and overall survival in patients. Patients with high SMARCAD1 expression had poorer overall survival than those with low expression. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 enhances proliferation of PANC-1 cells. A-B. The efficiency of SMARCAD1 knockdown (A) or overexpression (B) in PANC-1 cells was detected by western blotting. β-actin was used as an internal control. C-D. CCK8 assay was performed to determine the proliferation of PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) at the indicated time points after plated. Cell viability was measured at 450nm. E-F. The effect of SMARCAD1 knockdown (E) or overexpression (F) on Colony-forming of PANC-1 cells was shown in the top panels. Number of foci was counted as shown in the bottom panels. All data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Knockdown, Over Expression, Western Blot, Control, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 promotes migration and invasion of PANC-1 cells. A-B. Effect of SMARCAD1 knockdown (A) or SMARCAD1 overexpression (B) on cell migration was detected by wound healing at indicated time points after scratching. The wound healing was measured by ImageJ software. C-D. Motility ability of PANC-1 cells with SMARCAD1 depletion (C) or overexpression (D) was assessed by transwell assay at 24h. Representative images of migration were photographed at 24h (Top panel). The number of migrated cells was counted from 5 randomly selected fields under microscope (Bottom panel). E-F. Invasion ability of PANC-1 cells with SMARCAD1 depletion (E) or overexpression (F) was assessed by transwell assay at 48h. Representative images of invasion were photographed at 48h (Top panel). The number of invaded cells was counted from 5 randomly selected fields under microscope (Bottom panel). Scale bars=150um. Data were presented as mean ±SEM. *p<.05, **p<.01.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Migration, Knockdown, Over Expression, Software, Transwell Assay, Microscopy

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1 induces EMT in PANC-1 cells. A-B. The morphology changes of PANC-1 cells: cells lose contact with each other with SMARCAD1 depletion (A) or gain more contact with SMARCAD1 overexpression (B), Scale bars=250μm. C-D. Changes in mRNA level of EMT relative markers were tested by Quantitative real-time PCR in SMARCAD1 knockdown (C) or overexpression (D) cells. The results were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. (D). E-F. The protein levels of EMT relative markers in SMARCAD1 knockdown (E) or overexpression (F) cells were assessed by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Knockdown, Control, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SMARCAD1 Promotes Pancreatic Cancer Cell Growth and Metastasis through Wnt/β-catenin-Mediated EMT

doi: 10.7150/ijbs.29562

Figure Lengend Snippet: SMARCAD1-induced EMT was regulated by Wnt/beta-catenin signaling pathway. A-B. The mRNA level of β-catenin was detected by Quantitative real-time PCR in PANC-1 cells with SMARCAD1 knockdown (A) or overexpression (B) respectively. The data were presented as mean ±SEM. All values were normalized to the level (=1) in NC or control cells. *p<.05, **p<.01. C-D. β-catenin, cyclin-D1, c-Myc and survivin protein levels were assayed by western blotting in PANC-1 cells with SMARCAD1 knockdown (C) or overexpression (D) respectively. E. PANC-1 cells with SMARCAD1 depletion were treated with CHIR99021 (6μM/ml) for 24h. The protein levels of EMT markers and Wnt/β-catenin target genes (β-catenin, cyclin-D1, c-Myc and survivin) were detected by western blotting. β-actin was used as an internal control.

Article Snippet: The specimens were incubated with anti-SMARCAD1 antibody (1:50), and staining results were observed with a Nikon ECLIPSETs2R microscope.

Techniques: Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Control, Western Blot

Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

Article Title: Reparameterization of PAM50 expression identifies novel breast tumor dimensions and leads to discovery of a genomewide significant breast cancer locus at 12q15

doi: 10.1158/1055-9965.EPI-17-0887

Figure Lengend Snippet: Example Utah high-risk breast cancer pedigree 1817. a. Confirmed and sampled breast cancer cases are indicated in black (55 sampled out of 138 total confirmed UCR cases). Star, triangle and hexagon symbols indicate pedigree branches. b. shows only those cases from (a) with tumor expression data available and indicates PAM50 intrinsic subtype by color. Cases whose tumors are extreme for PC3 are indicated by ‘3’; extreme for PC5 are indicated by ‘5’. c. shows only the PC3-extreme cases from (b). A ‘+’ indicates those cases that share the genomewide significant region at 12q15.

Article Snippet: Gene expression data was generated using the PAM50 RT-qPCR research assay( 12 ) in the Bernard Lab at the Huntsman Cancer Institute( 15 ).

Techniques: Expressing

Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

Article Title: Reparameterization of PAM50 expression identifies novel breast tumor dimensions and leads to discovery of a genomewide significant breast cancer locus at 12q15

doi: 10.1158/1055-9965.EPI-17-0887

Figure Lengend Snippet: Summary of the 11 high-risk Utah pedigrees Tumor count by intrinsic subtype per pedigree.

Article Snippet: Gene expression data was generated using the PAM50 RT-qPCR research assay( 12 ) in the Bernard Lab at the Huntsman Cancer Institute( 15 ).

Techniques:

Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology

Article Title: Reparameterization of PAM50 expression identifies novel breast tumor dimensions and leads to discovery of a genomewide significant breast cancer locus at 12q15

doi: 10.1158/1055-9965.EPI-17-0887

Figure Lengend Snippet: A slice from a three-dimensional scatterplot of PC1, PC2, and PC4 shows that they recapitulate the PAM50 intrinsic subtypes. Reinforcing the constrasts illustrated in Figure 2, PC1 here clearly distinguishes Basal-like from Luminal A tumors. PC2 and PC4 aid in distinguishing HER2-enriched from Luminal B tumors. Combined, these three components define clusters corresponding to the intrinsic subtypes.

Article Snippet: Gene expression data was generated using the PAM50 RT-qPCR research assay( 12 ) in the Bernard Lab at the Huntsman Cancer Institute( 15 ).

Techniques:

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Content of coronavirus antigen microarray.

Article Snippet: Influenza , H3N2 , A/Texas/50/2012 , HA1 , AGL07159.1 , HEK293 , Sino Biological , N-(AA1-345)-His-C , 40354-V08H1.

Techniques: Microarray

Journal: bioRxiv

Article Title: Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

doi: 10.1101/2020.03.24.006544

Figure Lengend Snippet: Non-coronavirus respiratory virus antigens on microarray.

Article Snippet: Influenza , H3N2 , A/Texas/50/2012 , HA1 , AGL07159.1 , HEK293 , Sino Biological , N-(AA1-345)-His-C , 40354-V08H1.

Techniques: Microarray, Expressing, Construct

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Joint swelling and clinical scores in wild-type (WT), Fas lpr/lpr , Fasl gld/gld , and Fas –/– mice (n = 6 per group). ( B ) Joint swelling and clinical scores in WT, Fasl Δm/Δm , Fasl Δs/Δs , and Fasl Δs/Δs mice injected with sFasL (n = 6 per group). ( C , D ) Gross and microscopic examination of arthritis (magnified 10× in the upper panel and 200× in the lower panel). Scale bars: 1 cm ( C ), 200 μm ( D , upper panel), and 100 μm ( D , lower panel). ( E ) Tandem mass spectra of unique DR5 peptides. ( F ) Transcript levels of Tnfrsf10b in synovial CD45 + immune cells and CD45 – non-immune cells from WT mice with or without AIA. ( G ) Immunohistochemistry of DR5 expression in joint tissue from a healthy control subject and a patient with rheumatoid arthritis (n = 3; magnified 400×, scale bar: 50 μm). ( H ) Flow cytometric analysis of biotinylated protein binding to EL4 cells transfected with human WT TNFRSF10B preincubated with recombinant hTRAIL, or simultaneously incubated with anti-FasL, or anti-DR5 antibodies. ( I ) Flow cytometric analysis of biotinylated FasL binding on hFLSCs with FAS and/or TNFRSF10B knockout, and TNFRSF10B and/or FAS overexpression in FAS and TNFRSF10B double knockout (DKO) cells. ( J ) hLFSCs were preincubated with TNF-α (as a negative control), FasL, or TRAIL and cross–linked with BS 3 . Lysates from these cells were immunoprecipitated with anti–DR5 or control IgG antibody and immunoblotted with anti-DR5, TNF-α, FasL, or TRAIL antibodies. ( K ) Flow cytometric analysis of DR5–Fc binding on EL4 cells transfected with human WT FASLG in the presence of recombinant hTRAIL, anti-DR5, or FasL antibodies. Data were pooled from three ( A , B , and D–G ) or four ( H, K ) independent experiments and are presented as mean ± standard error of the mean (SEM). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way analysis of variance (ANOVA). Figure 1—source data 1. Numerical data obtained during experiments represented in , , and .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection, Immunohistochemistry, Expressing, Control, Protein Binding, Transfection, Recombinant, Incubation, Binding Assay, Knock-Out, Over Expression, Double Knockout, Negative Control, Immunoprecipitation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Schematic diagram showing AP–MS analyses. ( B ) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of biotinylated Fc (control) or sFasL–Fc cross-linked protein complexes. The fractions used in in-gel digestion are separated by red lines. ( C ) Expression of DR5 in CD45 + and CD45 – cells from the joint tissues of WT mice with arthritis. ( D ) Flow cytometry analyses of the expression of Fas and DR5 in human (h) fibroblast-like synovial cells (FLSCs). ( E ) Flow cytometry analyses of FasL–Fc binding to hFLSCs in the presence of anti-DR5 and/or anti-Fas antibodies. ( F , G ) Flow cytometry analyses of human DR5 ( F ) and Fas ( G ) expression in EL4 cells transfected with human WT tumor necrosis factor receptor superfamily (TNFRSF)10B ( F ) and FAS ( G ). ( H ) Expression of TNFRSF10B and FAS in EL4 mouse T cells transfected with various human genes. ( I ) Flow cytometry analyses of biotinylated protein binding to EL4 cells transfected with human WT FAS preincubated with recombinant human (h) TNF-related apoptosis-inducing ligand (TRAIL) or treated simultaneously with anti-Fas and DR5 antibodies. ( J ) Flow cytometry analyses of FasL–Fc binding to hFLSCs after preincubation with recombinant sTRAIL or sFasL. All experiments were performed four times independently.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Protein-Protein interactions, Polyacrylamide Gel Electrophoresis, Control, Expressing, Flow Cytometry, Binding Assay, Transfection, Protein Binding, Recombinant

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) hFLSCs and mouse synovial fibroblasts were stimulated with human or mouse sFasL in the presence or absence of anti-mouse Fas or anti-DR5 antibodies, as well as anti-human Fas or anti-DR5 antibodies for 24 hr. CX3CL1 levels in culture supernatants were measured using ELISA. ( B , C ) Jurkat cells ( B ) or mouse splenocytes ( C ) were incubated for 24 hr with recombinant FasL and TRAIL in the presence or absence of human or mouse anti-DR5 or anti-Fas antibodies. Jurkat and gated splenic TCRβ + CD4 + T cell death was measured using flow cytometry. ( D ) Jurkat cell death was measured using flow cytometry after FasL or FasL–Fc treatment in the presence or absence of anti-Fas or anti-DR5 antibodies for 24 hr. Data are presented as mean ± SEM. All experiments were performed three times independently. *p<0.05; **p<0.01. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Flow Cytometry

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Flow cytometry analyses of FasL–Fc binding to hFLSCs that were knocked down with FAS , TNFRSF10B , TNFRSF1A , TNFRSF10A , or TNFRSF12 . ( B ) Expression of FAS , TNFRSF10B , TNFRSF10A , TNFRSF1A , and TNFRSF12 in siRNA-transfected hFLSCs. ( C ) Expression of FAS and TNFRSF10B in FAS - and/or TNFRSF10B -knockout (KO) cells and KO cells transfected with TNFRSF10B and/or FAS overexpression vector. ( D , E ) Expression of Fas and DR5 ( D ) and biotinylated TRAIL binding ( E ) in FAS - and/or TNFRSF10B -KO hFLSCs and DKO (DR5 and Fas gene double knockout) cells transfected with TNFRSF10B and/or FAS in expression vectors. Biotinylated TRAIL binding was quantified by streptavidin (sAv)–fluorescein isothiocyanate staining intensity using flow cytometry. ( F, G ) Surface plasmon resonance assays for DR5–FasL ( F ) and DR5–TRAIL ( G ) interactions. ( H ) hFLSCs were incubated with PBS (control), 6× His-tagged FasL, or 6× His-tagged TRAIL and cross-linked using BS 3 . Cell lysates were immunoprecipitated with anti-His or control IgG antibodies and then immunoblotted with anti-Fas, DR5, TRAIL, and FasL antibodies. ( I ) FASLG , TNFSF10 , and TNF expression in EL4 mouse T cells transfected with human genes. ( J ) Flow cytometry analyses of DR5–Fc and Fas–Fc binding to EL4 cells transfected with human WT FASLG in the presence of anti-FasL antibodies. ( K, L ) Flow cytometry analyses of human FasL, TNF-α, and TRAIL expression ( K ), as well as DR5–Fc and Fas–Fc binding to EL4 cells transfected with human WT FASLG , TNFA , or TRAIL ( L ) and gated on transfected cells expressing the target proteins. Data were pooled from three independent experiments and are presented as mean ± SEM (n = 4 in B ). **p<0.01; ****p<0.0005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Flow Cytometry, Binding Assay, Expressing, Transfection, Knock-Out, Over Expression, Plasmid Preparation, Double Knockout, Staining, SPR Assay, Incubation, Control, Immunoprecipitation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A , B ) FasL–Fc binding to hFLSCs or EL4 cells transfected with human FAS , or TNFRSF10B after preincubation with human sTRAIL or sFasL. ( C ) Model of FasL and DR5 derived from the crystal structure of the FasL/DcR3 complex (Protein Data Bank: 4 MSV) and TRAIL/DR5 complex (Protein Data Bank: 1D4V). ( D , E ) Flow cytometric analysis of FasL–Fc or DR5–Fc binding to EL4 cells transfected with human WT or mutated TNFRSF10B or FASLG . ( F , G ) Comparison of the effects of sFasL and sTRAIL on ( F ) apoptosis and ( G ) necroptosis in hFLSCs. ( H ) Joint swelling and clinical scores in Fasl gld/gld mice injected with Z–VAD–FMK and/or sFasL (n = 6 per group). Data were pooled from four ( A , B , and D–G ) or three ( H ) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 2—source data 1. Numerical data obtained during experiments represented in , .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Binding Assay, Transfection, Derivative Assay, Comparison, Injection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) hLFSCs were preincubated with FasL and TRAIL using an excess of TRAIL in lane 3 (TRAIL [4 μg/mL]+FasL) and an excess of FasL in lane 6 (FasL [4 μg/mL]+TRAIL) before cross-linking with BS 3 . Lysates from these cells were immunoprecipitated with anti-DR5 (lanes 2, 3, 5, and 6) or control IgG (lanes 1 and 4) antibodies and immunoblotted with anti-DR5, FasL, or TRAIL antibodies. ( B ) Crystal structures of the TRAIL/DR5 (Protein Data Bank: 1D4V) and FasL/DcR3 (Protein Data Bank: 4 MSV) complexes. ( C ) Alignment of the human DcR3 and DR5 as well as the human FasL and TRAIL sequences. The point mutations in the mutant huDR5–cysteine-rich domains (CRD)two and CRD3, the mutant FasL–CRD2 interacting domain, and the mutant FasL–CRD3 interacting domain are indicated by asterisks (*). ( D ) Flow cytometry analyses of human DR5 in EL4 cells transfected with human WT or mutant TNFRSF10B . ( E ) Flow cytometry analyses of human FasL in EL4 cells transfected with human WT or mutant FASLG . Experiments ( A , D , and E ) were performed three times independently.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Immunoprecipitation, Control, Mutagenesis, Flow Cytometry, Transfection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A, B ) Microarray assay using joint tissues from WT, Fas lpr/lpr , and Fasl gld/gld mice with arthritis. ( C ) Cx3cl1 transcript levels estimated in joint tissues from WT, Fas lpr/lpr , Fas –/– , Fasl gld/gld , Fasl Δs/Δs , and Tnfrsf10b KO mice with arthritis. ( D ) Cx3cl1 expression in CD45 + immune and CD45 – non–immune cells from the joints of WT mice with arthritis after sFasL treatment. ( E, F ) CX3CL1 transcript levels estimated in hFLSCs in the presence of anti-Fas and/or anti-DR5 antibodies ( E ) and FAS (Fas), TNFSF10B (DR5), or FAS , and TNFRSF10B DKO, or TNFRSF10B and FAS overexpression in DKO hFLSCs ( F ). ( G ) Cx3cl1 expression in synovial fibroblasts from WT, Fas lpr/lpr , Fas –/– , or Tnfrsf10b KO mice in the presence or absence of sFasL. ( H, I ) CX3CL1 transcript levels estimated after sFasL stimulation in hFLSCs in the presence of MEK (U0126), ERK (PD980259), p38 kinase (SB203580), and NF-κB (MG132 and BMS345541) inhibitors ( H ) or transfection with control, RELA , CHUK (IKKa), or IKBKB (IKKb) siRNA ( I ). ( J ) Synovial fibroblasts obtained from WT mice with arthritis were incubated with sFasL or sTRAIL and CX3CL1 levels were measured using ELISA. ( K ) hFLSCs were stimulated with sFasL after preincubation with various concentrations of sTRAIL for 30 min and CX3CL1 levels were measured in the culture supernatant. Data were pooled from three ( C–G and K ) or four ( H–J ) independent experiments and are presented as mean ± SEM (n = 4 for C–K ). *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 3—source data 1. Numerical data obtained during experiments represented in , and .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Microarray, Expressing, Over Expression, Transfection, Control, Incubation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) CX3CL1 was measured in culture supernatants of CD45 + immune and CD45 – non-immune cells from the joints of WT mice with arthritis after sFasL stimulation. ( B–H ) Cx3cl1 transcripts and CX3CL1 protein in culture supernatants were estimated in human ( B–F ) and mouse ( G, H ) FLSCs after stimulation with sFasL or FasL–Fc in the presence of anti-Fas or anti-DR5 antibodies. ( I ) CX3CL1 transcript of hFLSCs transfected with control, TNFRSF1A , FAS , TNFRSF12 , TNFRSF10A , and TNFRSF10B siRNA was measured after stimulation with sFasL. ( J ) FAS and/or TNFRSF10B KO hFLSCs, and DKO hFLSCs were transfected with TNFRSF10B. FAS in the expression vector was stimulated with sFasL. CX3CL1 levels were measured in the culture supernatants. ( K ) Levels of CX3CL1 in culture supernatants of synovial fibroblasts obtained from WT, Fas lpr/lpr , Fas –/– , or Tnfrsf10b KO mice with arthritis after incubation with sFasL. ( L , M ) Levels of CX3CL1 in culture supernatants of hFLSCs after sFasL stimulation for 2 hr in the presence of MEK (U0126), ERK (PD980259), p38 kinase (SB203580), and NF-κB (MG132, and BMS345541) inhibitors ( L ) or transfection with control, RELA , CHUK (IKKa), or IKBKB (IKKb) siRNA ( M ). ( N ) Blotting assay for components of the NF-κB signaling pathway in hFLSCs stimulated with sFasL for the durations indicated, all preincubated with anti-Fas antibodies. ( O , P ) CX3CL1 transcript ( O ) and CX3CL1 protein ( P ) levels in culture supernatants from hFLSCs after stimulation with sFasL in the presence of 50 μM Z–VAD–FMK. Data were pooled from four ( A–F , L , and M ) or three ( G–K , O , and P ) independent experiments and are presented as mean ± SEM (n = 5; A–M , O , and P ). NS, not significant; *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Incubation

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A ) Joint swelling and clinical scores in WT mice injected with anti-DR5 or anti-Fas antibodies to measure AIA (n = 5 per group). ( B, C ) Joint swelling and clinical scores in WT and Tnfrsf10b KO mice injected with sFasL or phosphate-buffered saline (PBS) to measure AIA (n = 5 per group). ( D ) Cx3cl1 transcript levels in the joints were estimated in WT, Tnfrsf10b KO, Fasl Δs/Δs , and Fasl Δm/Δm mice injected with sFasL or PBS to measure AIA (n = 5). ( E, F ) Joint swelling and clinical scores ( E ), and transcript levels of various cytokines and chemokines in joint tissues of Tnfrsf10b KO mice injected with CX3CL1 or PBS to measure AIA ( F ) (n = 6 per group). ( G ) Joint swelling and clinical scores of WT and Cx3cr1 KO mice in the presence or absence of sFasL to measure AIA (n = 6 per group). Data were pooled from three independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA. Figure 4—source data 1. Numerical data obtained during experiments represented in , .

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection, Saline

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet: ( A, B ) Joint swelling and clinical scores together with transcript levels of various cytokines and chemokines in joint tissues from Fasl gld/gld mice injected with sFasL, as well as anti-Fas, or anti-DR5 antibodies (n = 6 per group). ( C, D ) Joint swelling and clinical scores in Fasl Δs/Δs ( C ) and Fasl gld/gld ( D ) mice injected with CX3CL1 or PBS. ( E, F ) Transcript levels of various cytokines and chemokines in joint tissues from Fasl Δs/Δs ( E ) or Fasl gld/gld ( F ) mice injected with CX3CL1 or PBS to measure AIA (n = 6 per group). Data were pooled from four ( A, B ) or three ( C–F ) independent experiments and are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.005. Data were analyzed using one-way ANOVA.

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Injection

Journal: eLife

Article Title: Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

doi: 10.7554/eLife.48840

Figure Lengend Snippet:

Article Snippet: The cell lysates (500 μg) were diluted with six volumes of non-denaturing cell lysis buffer and incubated with anti-His or anti-DR5 antibodies (1:50; Cell Signaling Technology, Inc, Beverly, MA), as well as isotype control rabbit mAb IgG (DA1E; Cell Signaling Technology, Inc) for 1 hr at 4°C.

Techniques: Immunohistochemistry, Cytometry, Purification, Recombinant, In Vitro, Western Blot, In Vivo, Injection, Neutralization, Control, Cell Isolation, Enzyme-linked Immunosorbent Assay, Gene Expression, Negative Control, Staining, Sequencing, Software