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: 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: 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

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM expression is increased in HCC and fetal liver tissues and is associated with prognosis. A , Normalized (Norm) HNRNPM expression levels during mouse liver development from GSE57824 data. B , HNRNPM expression levels during mouse liver development from GSE13149 data. C , Western blot analysis of HNRNPM protein levels in human fetal liver and adult liver tissues. D , Real-time qPCR analysis of HNRNPM mRNA levels in human fetal liver and adult liver tissues. Data are mean ± standard deviation of n = 3 independent samples. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001 by the Student t test. E , Norm HNRNPM expression in HCC and normal liver tissues. ∗∗ P < .01 by the Student t test. F , Real-time qPCR analysis of HNRNPM mRNA levels in 60 paired HCC and normal liver tissues. G , Representative images of HNRNPM by IHC in HCC and normal tissues. H , Kaplan-Meier analysis of HNRNPM in HCC cohort. I , Kaplan-Meier analysis of HNRNPM in TCGA cohort.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM was associated with clinopathological characteristics and poor prognosis in patients with HCC. A , Oncomine analysis showed the prognostic splicing factors from TCGA datasets. B , The selected prognostic splicing factors validated by real-time PCR in portal vein tumor thrombosis (PVTT) HCC, non-PVTT HCC, and normal liver tissues. C , The HNRNPM protein expression in metastasis and metastasis-free HCC tissues. D , The HNRNPM protein expression in tumor grade I/II and III/IV. E , The HNRNPM protein expression in no-microvascular invasion and microvascular invasion HCC tissues. F , The relative HNRNPM expression in tumor stage I/II/III/IV. G , The correlation analysis between Ki-67 and HNRNPM in TCGA database. H , Kaplan-Meier analyses of the correlations between HNRNPM level and overall survival in HCC tumor stage I/II and III/IV from our HCC cohort. I , Kaplan-Meier analyses of the correlations between HNRNPM level and OS in HCC tumor grade I/II and III/IV from our HCC cohort.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Association of HNRNPM Expression With Clinical Characteristics in 240 Patients With HCC

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Association of HNRNPM Expression With Clinical Characteristics in 371 Patients With HCC

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Virus

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of Overall Survival for HNRNPM (n = 240)

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of Overall and Disease-free Survival for HNRNPM (n = 370) From TCGA Database

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Cell stem cell transcriptional factors SOX2 and OCT4 bind with promoter and upregulate the expression of HNRNPM. A , The basic expression of HNRNPM in different HCC cell lines. B-C , Western blot analysis of HNRNPM expression when overexpressing ( B ) or depletion of ( C ) OCT4 and SOX2. D , The predicted binding site for OCT4 and SOX2 with HNRNPM promoter. E , OCT4 directly binds with HNRNPM promoter by ChIP assays and luciferase assays. F , SOX2 directly binds with HNRNPM promoter by ChIP assays and luciferase assays. G-H , Correlation analysis between OCT4 ( G ), SOX2 ( H ), and HNRNPM from TCGA database.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Western Blot, Binding Assay, Luciferase

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The tumorigenesis effects of HNRNPM overexpression in MHCC97L and HepG2 cells. A-B , The mRNA and protein levels of HNRNPM in MHCC97L ( A ) and HepG2 cells ( B ) stably overexpressing HNRNPM. C-D , The cell proliferation by CCK-8 assays stably MHCC97L ( C ) and HepG2 cells ( D ) stably overexpressing HNRNPM. ∗∗∗∗ P < .0001 as compared with control. E-F , The cell apotosis by flow cytometry stably MHCC97L ( E ) and HepG2 cells ( F ) stably overexpressing HNRNPM. Data were from 3 independent experiments. ∗∗ P < .01 by the Student t test. G-H , The in vivo effects in BALB/c nude mice in MHCC97L ( G ; n = 6) and HepG2 cells ( H ; n = 6) stably overexpressing HNRNPM. ∗∗ P < .01 by the Student t test. I , The CSC frequency was determined from a limiting dilution assay performed with HCC cells depleting HNRNPM from the third transplant recipient mice (n = 6). The ELDA web tool was used to calculate the frequency of CSCs.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Over Expression, Stable Transfection, CCK-8 Assay, Control, Flow Cytometry, In Vivo, Limiting Dilution Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM drives HCC tumorigenesis and maintains CSC properties. A-B , Sphere formation and limiting dilution assays when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. The number of spheroids formed as a fraction of the number of cells seeded per well is given. Data are from 3 independent experiments. C-D , Cell cycle detected by flow cytometry when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. E-F , Colony formation assay when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. G-H , Cell migration assay when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05 by the Student t test. I , Cell invasion assays when overexpressed HNRNPM in MHCC97L and HepG2 cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05 by the Student t test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Flow Cytometry, Colony Assay, Cell Migration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The oncofetal properties of HNRNPM in hepatocyte differentiation model. A , The model scheme in hepatocyte differentiation model. B , The expression of OCT4, E2F1, SOX2, and HNRNPM in different stages from hepatocyte differentiation model. C , The correlation analysis between HNRNPM and E2F1 from TCGA databases. D , The potential binding site for E2F1 to HNRNPM promoter. E , E2F1 directly bind with HNRNPM promoter by ChIP assays. Data were from 3 independent experiments. ∗∗ P < .01 by the Student t test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM is required for tumorigenesis of HCC cells. A , The mRNA and protein levels of HNRNPM in MHCC97H cells stably depleting HNRNPM. B , The protein levels of HNRNPM by immunofluorence stably depleting HNRNPM. C , Sphere formation and limiting dilution assays when depleting HNRNPM in MHCC97H cells. The number of spheroids formed as a fraction of the number of cells seeded per well is given. Data are from 3 independent experiments. ∗∗ P < .01 by the Student t test. D , The cell proliferation by CCK-8 assays stably depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. E , The cell apotosis by flow cytometry stably depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. F , Cell cycle detected by flow cytometry when depleting HNRNPM in MHCC97H cells. G , Colony formation assay when depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. H , Cell migration assay when depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗∗∗ P < .001 by the Student t test. I , Cell invasion assays when depleting HNRNPM in MHCC97H cells. J-K , The in vivo effects in BALB/c nude mice (n = 6 per group) when overexpressed and depleted HNRNPM. Results are presented as mean ± standard error of the mean, n = 6. ∗ P < .05; ∗∗ P < .01 by the Student t test. L-M , The number of liver metastasis in BALB/c nude mice when overexpressed and depleted HNRNPM. Results are presented as mean ± standard error of the mean, n = 6. ∗ P < .05; ∗∗ P < .01 by the Student t test. N , The CSC frequency was determined from a limiting dilution assay performed with HCC cells from the third transplant recipient mice. The ELDA web tool was used to calculate the frequency of CSCs.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Stable Transfection, CCK-8 Assay, Flow Cytometry, Colony Assay, Cell Migration Assay, In Vivo, Limiting Dilution Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The genome-wide landscape and global alternative splicing of HNRNPM. A , Kyoto Encyclopedia of Genes and Genomes analysis of HNRNPM-targeted splicing events. B , Quantification of the different AS events regulated by HNRNPM. A3SS , alternative 3′ splicing site; A5SS , alternative 5′ splicing site; MXE , mutually exclusive exon; RI , retained intron; SE , skipped exon. ∗∗∗ P < .001 by the Student t test. C-D , The quantification of significant AS events regulated by HNRNPM ( P < .05). E , HNRNPM-RIP-seq peaks were enriched in 5′UTR, promoter and 3′ UTR. All RIP-seq peaks were categorized according to the distribution on different genomic elements andcompared with the genomic background. F , De novo motif analysis identifying GU-repeat motif as the only enriched motif within the top HNRNPM RIP-seqpeaks. G , Schematic diagram of MBD2 molecular model. H , The RIP experiment showed HNRNPM directly binded with MBD2. I , The shift of MBD2a and MBD2c between HNRNPM overexpressed stably transduced and control MHCC97H cells. J , The shift of MBD2a and MBD2c between HNRNPM shRNA stably transduced and control MHCC97H cells. K , The RMMs of HNRNPM bind to MBD2 by RIP experiments. L , The potential binding of HNRNPM to MBD2 pre-mRNA by CLIP assay.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Genome Wide, Alternative Splicing, Stable Transfection, Control, shRNA, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Significant Alternative Splicing Events by Comparing Depletion of HNRNPM With Wild-type HCC Cells

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Alternative Splicing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Results of HNRNPM-RIP Analysis

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Intersection Results of HNRNPM-RIP Analysis and Transcriptomic Sequencing

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: DNA methylation controls MBD2-mediated FZD3 transcription. A , The shematic diagram of HNRNPM domains. B , The specific binding site for MBD2 with HNRNPM by CLIP assay. C , The luciferase assay for FZD3 transcription activity when overexpressing MBD2a or MBD2a and MBD2c. Data were from three independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. D-H , qPCR analysis of MBD2a, MBD2c, FZD3, β-catenin, and Snail1 mRNA transcripts in MHCC97H cells stably expressing NC, shRNAs targeting HDAC1, HDAC2, RBBP7, or MTA2. Immunoblot analysis showed the knockdown efficiency of shRNAs targeting HDAC1, HDAC2, RBBP7, or MTA2 in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. I-J , β-catenin promotes the expression of OCT4 ( I ) and SOX2 ( J ) by binding its promoter. Data were from 3 independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: DNA Methylation Assay, Binding Assay, Luciferase, Activity Assay, Comparison, Stable Transfection, Expressing, Western Blot, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: MBD2a induces, whereas MBD2c represses, HCC tumorigenesis and CSC properties. A , Sphere formation and limiting dilution assays when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. B , The cell proliferation by CCK-8 assays when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. C , Cell migration and migration assay when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. D , Colony formation assay when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. E , The cell apotosis by flow cytometry when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. F , The protein expression of HNRNPM, MBD2a, MBD2c when downregulating SOX2, OCT4, and together with overexpressing HNRNPM by Western blot experiments. G , The in vivo effects in BALB/c nude mice when overexpressing MBD2a (n = 5) or with HNRNPM depletion (n = 5), MBD2c (n = 5). ns, Non-significant. ∗ P < .05, ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: CCK-8 Assay, Migration, Colony Assay, Flow Cytometry, Expressing, Western Blot, In Vivo, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The coregulated genes by MBD2a and MBD2c in HCC cells. A-B , Venn diagram of the RNA-seq data showing the genes commonly regulated by MBD2a and MBD2c. C-D , Gene Ontology (GO) enrichment analysis. The top 5 GO terms in the indicated categories with the lowest P values are shown. E , The expression of Snail1, OCT4, SOX2 mRNA, and proteins was measured by qPCR and Western blot in MHCC97H cells expressing shRNAs targeting MBD2a, and in MHCC97H cells stably expressing MBD2c. Data were from 3 independent experiments. ∗ P < .05 as compared with controls. F , The expression of β-catenin by nuclear/cytoplasmic protein fractionation and TOP/FOP-flash reporter assays when silencing MBD2a or overexpressing MBD2c. Data were from 3 independent experiments. ∗ P < .05; ∗∗ P < .01 by the Student t test. G , The expression of Snail1, OCT4, SOX2 mRNA, and proteins was measured by qPCR and Western blot in MHCC97H cells expressing HNRNPM and shRNA targeting MBD2a. Data were from 3 independent experiments. ∗∗∗ P < .001 as compared with controls; ns, Not significant; P > .05. H , The expression of β-catenin by nuclear/cytoplasmic protein fractionation and TOP/FOP-flash reporter assays when overexpressing HNRNPM and silencing MBD2a. Data were from 3 independent experiments. ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: RNA Sequencing, Expressing, Western Blot, Stable Transfection, Fractionation, shRNA, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: A , The relative expression of MBD2a, MBD2c in fetal liver, adult liver, HCC, and adjacent noncancerous liver tissues. ∗∗∗ P < .001 by the Student t test. B-C , The Kaplan-Meier analyses of the correlations between MBD2a ( B ), MBD2c ( C ) level and overall survival of n = 100 patients with HCC. The median MBD2a or MBD2c level was used as the cutoff. D , The multivariate analysis for MBD2a and MBD2c in patients with HCC. E , The correlation analysis between HNRNPM and MBD2a in patients with HCC (n = 30) by IHC experiments. F , FZD3 and HNRNPM expression in protein levels in HCC tissues with strong or weak HNRNPM staining intensity. The median HNRNPM staining intensity was used as the cutoff (n = 60 HCC tissues). ∗∗∗ P < .001. G-H , The correlation between the expression of HNRNPM and FZD3 ( G ), β-catenin ( H ) from TCGA datasets.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The effects of HNRNPM-specific ASO for HCC in vivo and in vitro. A , The expression of HNRNPM was significantly correlated with MBD2a. B , The IC50 of ASO-2 for MHCC97H cells. C , The protein expression of HNRNPM, MBD2a, FZD3, OCT4, SOX2, and β-catenin related assays when treated with ASO-2 in HCC cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. D , The CSC markers expression by ASO treatment. E , The CCK-8 experiment when treated with HNRNPM-specific ASO in MHCC97H cells. F , Sphere formation and limiting dilution assays when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. G , Limiting dilution assays when treated with HNRNPM-specific ASO in MHCC97H cells. H , Colony formation assay when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. I-J , Invasion assay ( I ) and cell migration ( J ) and when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. K , The HCC cell apoptosis changes by ASO treatment. Data were from 3 experiments. ∗∗ P < .01 by the Student t test. L , The schematic diagram of ASO-2 treating nude mice when inoculating the tumor cells. M , The effects of HNRNPM-specific ASO when treated ASO I.P by 25 mg/kg (n = 5). ∗∗∗ P < .001 by the Student t test. N ,. The HNRNPM expression in tumors when treating HNRNPM-ASO by IHC experiments.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: In Vivo, In Vitro, Expressing, CCK-8 Assay, Colony Assay, Invasion Assay, Migration

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Expression of HNRNPM correlated with immune checkpoint in human HCC. A-E , Expression correlation between HNRNPM and immune checkpoint gene RNA amounts in the TCGA HCC database, n = 370, HNRNPM (HNRNPM), PD-L1 (CD274), B7-H3 (CD276), B7-H4 (VTCN1), LAG-3 (LAG3), and TIM-3 (HAVCR2). B , Pearson correlation analysis of HNRNPM and CD276 immune checkpoint expressions in human HCC tissue microarray based on the IHC results, n = 240.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Microarray

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM inhibition curbs immune escape and enhances PD-1 blockade by promoting CD8+ T cells activation phenotype. A , Schematic diagram of Hep1-6-OVA cells co-cultured with OTI cells. B , The flow cytometry analysis of IFN-γ+ or granzyme B+ CD8+ T cells between control and shHNRNPM groups. Data were from 3 independent experiments. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. C , Schematic diagram of ASO and anti-PD-1 therapy in C57/BJ6 mice. D , Tumor inhibition by IgG (n = 6), HNRNPM-ASO (n = 6), anti-PD-1 (n = 6), or combination therapy (n = 6) in C57/BJ6 mice. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. E , Survival analysis of IgG (n = 6), HNRNPM-ASO (n = 6), anti-PD-1 (n = 6), or combination therapy (n = 6) in C57/BJ6 mice. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. F , The profiles of immune cells in tumors by HNRNPM-ASO, anti-PD-1 or combination therapy. G , CD8+ T cells infiltration in HNRNPM-ASO, anti-PD-1 or combination therapy groups. ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. H , The changes of Treg, IFNG+, GMZB+ CD8+ T cells in control, HNRNPM-ASO, anti-PD-1 or combination therapy groups in tumor-bearing C57/BJ6 mice. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. I , The immune cells infiltration landscape of spleen in control, HNRNPM-ASO, anti-PD-1, or combination therapy groups in tumor-bearing C57/BJ6 mice. J , The mice weight between controls and HNRNPM-ASO group. ns , Non-significant. K , The relative expression of β-catenin in HNRNPM-ASO, anti-PD-1, or combination therapy groups. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. L-M , The distribution of CTNNB1 mutation in PD-1 responders or non-responders. N , The study model diagram.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Inhibition, Activation Assay, Cell Culture, Flow Cytometry, Control, Comparison, Expressing, Mutagenesis

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: List of Antibodies Used in This Research

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: List of Primers Sequences and shRNA Sequences Used in this Research

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: shRNA, Sequencing, Control, Negative Control

Journal: Cell

Article Title: Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer

doi: 10.1016/j.cell.2018.08.065

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A complete list of cell lines can be found in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal Ki-CT45-2 antibody Dr. Hans-Jürgen Heidebrecht (Kiel, Germany) N/A Mouse monoclonal anti-HLA-ABC antibodies W6/32 Thermo Fisher Scientific #MA1-19027; RRID: AB_1076699 Rabbit monoclonal anti-γH2AX Cell Signaling #9718; RRID: AB_2118009 Rabbit polyclonal anti-Cleaved CASP-3 Cell Signaling #9661; RRID: AB_2341188 Rabbit anti-IgG-HRP Cell Signaling #7074; RRID: AB_2099233 Mouse anti-IgG-HRP Cell Signaling #7076; RRID: AB_330924 Rabbit polyclonal anti-PPP4C Bethyl #A300-835A; RRID: AB_597901 Rabbit polyclonal anti-SMEK2 Bethyl #A300-842A; RRID: AB_597905 Rabbit polyclonal anti-KAP1 Bethyl #A300-274A; RRID: AB_185559 Mouse monoclonal anti-Actin Sigma #A5441; RRID: AB_476744 Rabbit polyclonal anti-CT45A Sigma #SAB1301842 Rabbit polyclonal anti-PPP4R2 Sigma #HPA034695; RRID: AB_10671231 Rabbit polyclonal anti-PPP4R3α Sigma #HPA002568; RRID: AB_1079207 Rabbit monoclonal anti-FANCD2 Abcam #ab108928; RRID: AB_10862535 Mouse monoclonal anti-γH2AX Life Technologies #MA1-2022; RRID: AB_559491 Mouse monoclonal anti-V5 Life Technologies #MA5-15253; RRID: AB_10977225 Mouse monoclonal anti-GST Abcam #3G10/1B3; RRID: AB_524046 Biological Samples Ovarian cancer specimens, Tables S1 and S2 This paper N/A Tumor infiltrating T cells This paper N/A Chemicals, Peptides, and Recombinant Proteins Carboplatin Hospira N/A Growth factor reduced Matrigel BD Biosciences (Rockville, MD) 356231 Synthetic HLA-I peptides of CT45 Core facility, Max Planck Institute of Biochemistry, Martinsried, Germany N/A MHC class-I tetramers NIH Tetramer Core Facility N/A Recombinant PP4R3β Dr. Jakob Nilsson (University of Copenhagen, Denmark) N/A Recombinant GST-CDC20 Dr. Jakob Nilsson (University of Copenhagen, Denmark) N/A Critical Commercial Assays Ser/Thr phosphatase assay kit Millipore #17-127 Deposited Data Raw and analyzed data This paper http://proteomecentral.proteomexchange.org/cgi/GetDataset (dataset identifier PXD010372) Patient information Tables S1 and S2 This paper N/A Ovarian cancer proteome data, Table S3 This paper N/A Patient information of tissue microarray, Table S4 This paper N/A HLA-I peptidomes of cell lines, Table S5 This paper N/A RNaseq analysis of 37 tissues Uhlén et al., 2015 https://www.proteinatlas.org/about/download RNAseq expression analysis of HGSOC and across cancers TCGA IlluminaHiseq BC and HiSeqV2_PANCAN Experimental Models: Cell Lines SKOV3ip1, female origin.

Techniques: Recombinant, Phosphatase Assay, Microarray, Expressing, Plasmid Preparation, Sequencing, Software

Journal: EMBO Molecular Medicine

Article Title: PlexinD1 is a driver and a therapeutic target in advanced prostate cancer

doi: 10.1038/s44321-024-00186-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Acetyl-Histone H3 (ChIP: 1/50) , Cell Signaling Technology , 9649.

Techniques: Recombinant, Sequencing, Control, Transfection, Membrane, Reverse Transcription, Software, Microarray, Subcloning, shRNA, Cell Viability Assay, Viability Assay, Reporter Assay, In Situ, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Extraction, Bicinchoninic Acid Protein Assay, Magnetic Beads, Gel Extraction, Purification, Ligation, Mutagenesis, Chromatin Immunoprecipitation

Journal: Genome Biology

Article Title: RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells

doi: 10.1186/s13059-016-0952-x

Figure Lengend Snippet: a ES cell in vitro neuralization. DIV days of in vitro differentiation. 0DIV corresponds to the time of leukemia inhibitory factor (LIF) withdrawal. N2 and B27 are the supplements used in the minimal medium of differentiation. Example of bright-field microphotographs of cells at different DIV are shown on the bottom. ELA epiblast-like aggregates, NPC neural progenitor cells, NPC/Neu neural precursors, Neu differentiated neurons. b RT-PCR gene expression analysis. Values are relative to β-actin mRNA expression. Highest and lowest expression levels were normalized to 1 in the left / middle histograms and in the right histogram, respectively. c , d Oct4 and Nanog immunodetection in ES cells ( c ) or ELA cells ( d ). e Violin plot shows the distribution of green fluorescent protein (GFP) intensity in a TNG-A Nanog::GFP line in LIF/serum (ES cells, red ) and 24 h ( green ) or 48 h ( blue ) after LIF/serum withdrawal ( ELA ) or Activin/fibroblast growth factor (FGF)2 induction ( EpiSC ), respectively. f , g Derivation of epiblast stem cells (EpiSC) and ELA-EpiSC from ES and ELA cells, respectively. h , i EpiSC and ELA-EpiSC bright-field images. j Expression correlation of markers of pluripotency and priming between EpiSC ( y-axis ) and ELA-EpiSC ( x-axis ). Values are expressed as log 2 ΔCt of RT-PCR assay; R 2 coefficient of determination. k Hierarchical clustering analysis on Spearman correlation between different microarray samples. l Flow cytofluorimetric analysis of Sox1::GFP cells (46C line), indicating the ratio of GFP-positive cells ( y-axis ) in different cell types or times of differentiation ( x-axis ). m , n Immunodetection of neural markers at 7 days of ELA-EpiSC neuralization. o RT-PCR gene expression analysis as in b in ELA-EpiSC after 4 ( +4DIV ) or 8 ( +8DIV ) days from FGF2/Activin A withdrawal. Error bars in b , l , and o show standard error. In b and o * p = 0.05, ** p = 0.01 (REST randomization test). Scale bars are 30 microns in a , c , and d , 40 microns in h , i , m , and n

Article Snippet: Primary antibodies used for microscopy included Oct3/4 (1:200; Santa Cruz Biotechnology C-10; sc-5279), Nanog (1:300; Novus Biologicals; NB100-58842), acetylated N-tubulin (clone 6-11B-1; 1:500; Sigma; T7451), neuronal class III β-tubulin (1:500; Covance; MRB-435P), Nestin (1:200; Millipore; MAB353), Pax6 (1:400; Covance; PRB-278P), and GFP (1:1000; Life Technologies; A-6455).

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Immunodetection, Microarray

Journal: Neuro-Oncology

Article Title: Autocrine activation of the IFN signaling pathway may promote immune escape in glioblastoma

doi: 10.1093/neuonc/nox051

Figure Lengend Snippet: MxA is expressed in gliomas in vivo. (A) MxA mRNA expression levels in gliomas of different WHO grades were analyzed using data from the database of TCGA (left). Overall survival analysis within the TCGA database for glioblastoma patients with high versus low MxA expression was performed by Kaplan–Meier analysis. The median was used as cutoff (right). (B) MxA protein levels were assessed by immunohistochemistry on a glioma tissue microarray and quantified by H scoring (left). Phospho-STAT1 protein levels were analyzed by immunohistochemistry on a TMA and quantified by H scoring. A correlation analysis of pSTAT1 H scores with MxA H scores is shown (right). (C) Representative images of normal brain and glioblastoma specimens with low, intermediate, and high MxA levels are shown (scale bar, 100 µm or 10 µm for 20x or 40x magnification, respectively). (D) MxA/CD45 costaining was performed on a glioma TMA and the number of double-positive cells was counted.

Article Snippet: 20 , 21 For costainings, TMA sections were stained with primary antibodies to MxA (rabbit, 1:200) and CD45 (mouse, 1:50) and visualized using mouse anti-rabbit immunoglobulin (Ig)G–horseradish peroxidase secondary antibody (Santa Cruz) with the ImmPact DAB kit followed by anti-mouse IgG–alkaline phosphatase secondary antibody (Vector Laboratories) with the HighDef green IHC chromogen kit (Enzo).

Techniques: In Vivo, Expressing, Immunohistochemistry, Microarray

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Quantification of the retinal nerve fiber layer thickness (RNFLT). To quantify the damage to the retina caused by the chronically elevated IOP in vivo, the retina was examined by optical coherence tomography (OCT). This allows retinal cross-sections to be imaged. The retinal nerve fiber layer was defined and quantified using a semi-automatic segmentation algorithm. For quantification, a 12° diameter circular B-scan was acquired. The Heidelberg Eye Explorer software divides the retina into the main sectors, superior (S), temporal (T), inferior (I), and nasal (N), as well as into the intermediate sectors, temporal superior (TS), temporal inferior (TI), nasal inferior (NI), and nasal superior (NS) ( A ). The scheme shows the interventions performed during the study on the eye, episcleral vein occlusion (EVO), and intravitreal injection (IVI) (scheme modified from ). The mean RNFLT of the whole B-scan was used for follow-up quantification ( B ). Further quantification of the RNFLT at week 10 was performed for the individual sectors ( C–H ). Legend: black (IgG OS), blue (HMGB1 OS), light grey (IgG OD), dark grey (HMGB1 OD). * p < 0.05, ** p < 0.01, *** p < 0.001, not significant (ns), one-way ANOVA, Tukey’s HSD post hoc test.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: In Vivo, Tomography, Software, Injection, Modification

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Summary of OCT analysis including RNFLTs of the IgG OS and HMGB1 groups and their differences.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Ganzfeld electroretinogram (ERG) pattern and quantification of B-wave and PhNR amplitude at week 0 and week 10. ERG recordings were acquired at different time points. The mean values of the ERG recordings of the individual animals were plotted for the time points week 0 (before EVO) and week 10 after EVO ( A – D ). The flash intensity 1.37 log 10 cd·s·m −2 was used for the ERG recordings shown. The B-wave amplitude (*) and the amplitude of the photopic negative response (PhNR, **) were used for quantification. Quantification was performed separately for the B-wave amplitude of the IgG ( E ) and the HMGB1 group ( F ) as well as for the PhNR amplitude of the IgG ( G ) and the HMGB1 group ( H ). The millivolt decrease was calculated as a percentage difference (Δ = % change).

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Expression pattern of selected retinal proteins analyzed by an antibody microarray. For the analysis, 10 µg of protein lysate was used. This was labeled, and a size exclusion chromatography column removed excess unbound dye and buffers unfavorable for the array incubation. The altered proteins with respective expressions in the elevated-IOP eyes were shown. ** p < 0.01, * p < 0.05, other p values as indicated, Student’s t -test ( A ). The protein expression of the high-mobility group box protein 1 (HMGB1) was examined in the IgG animals (OS, OD) and the antibody-treated HMGB1 animals (OS, OD) ( B – G ).

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Microarray, Labeling, Size-exclusion Chromatography, Incubation

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Schematic illustration of a proposed mechanism of anti-HMGB1 Ab application. ( A ) Schematic section of the healthy retina. HMGB1 is mainly located in the nucleus. ( B ) Schematic section of glaucomatous retina. HMGB1 translocates from the nucleus to the cytoplasm. Additionally, HMGB1 is secreted by cells and binds its receptors, such as Toll-like receptors (TLRs) or receptor for advanced glycation endproducts (RAGE). As a result, the expression of cytokines such as CXCL8 is induced and leads to an inflammatory response. ( C ) Glaucomatous retina with anti-HMGB1 Abs. Expression and distribution of HMGB1 is comparable to ( B ). By application of anti-HMGB1 Ab, HMGB1 is mainly captured by the Ab, which suppresses binding to TLRs or RAGE. Thereby, signaling through these receptors is reduced and thus the expression of inflammatory cytokines such as CXCL8 is also reduced, consequently reducing the inflammatory response. Abbreviations: RNFL—retinal nerve fiber layer, GCL—ganglion cell layer, IPL—inner plexiform layer, INL—inner nuclear layer, OPL—outer plexiform layer, CXCL8—C-X-C motif chemokine ligand 8/ interleukin 8 (IL-8). ( D ) Legend for subfigures ( A – C ). This figure was created with BioRender.com.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma

doi: 10.3390/ijms23084107

Figure Lengend Snippet: Antibodies used for antibody-based microarray.

Article Snippet: The treatment group (HMGB1 OS, n = 6) received 50 μg of a mouse-derived anti-HMGB1 antibody (1F3, NBP2-25148, Novus Biologicals, Littleton, CO, USA).

Techniques: Microarray

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: Statistically significant changed categories (control vs. PJ34 in wild-type or Trp53 −/− )

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: Control, Binding Assay

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: Change of mRNA expression profiles in the p53 signaling pathway. a The mRNA expression profiles were generated by microarray analysis. The genes, the expression level of which increased more than 2-fold after PARP inhibition, are indicated as a black box in the p53 signaling pathway published by the KEGG database. b The mRNA expression profiles shown in ( a ) were confirmed by RT-PCR. Several genes downstream to Trp53 (p53) were upregulated after PARP inhibition, whereas Trp53 itself was constant regardless of the presence of the PARP inhibitors. c Changes in the levels of p53 and phosphorylated p53 by the PARP inhibitors (PJ34, DHIQ, or 3AB) were observed in NSPCs. p21 protein as well as p21 mRNA, as shown in ( b ), were upregulated by all of the PARP inhibitors. Cleaved fragment of caspase-3 at Asp 175 (p17), which is a marker of apoptosis, was also increased by PARP inhibition. d Quantitative mRNA analyses after the addition of PJ34, DR2313 (PARP-1-specific inhibitor), or UPF1069 (PARP-2-specific inhibitor) were performed for the genes in the p53 signaling pathway. DR2313 as well as PJ34 upregulated the genes in the p53 signaling pathway, while UPF1069 did not change the expression of these genes, except Bbc3 (PUMA). Data shown in ( d ) are expressed as the ratio of the mean value of the control ( vehicle alone ). Data represent the mean value ± SEM (n = 3). * p < 0.05 by comparison against control using one-way ANOVA followed by Tukey’s post hoc test

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: Expressing, Generated, Microarray, Inhibition, Reverse Transcription Polymerase Chain Reaction, Marker, Control, Comparison

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: Effects of PARP-1 or PARP-2 knockdown on the p53 signaling pathway in NSPCs. Quantitative mRNA analyses after silencing Parp1 or Parp2 by shRNA-expressing lentivirus were performed for Parp1 and Parp2 ( a ) and the genes in the p53 signaling pathway ( b ). Genes in the p53 signaling pathway were upregulated by the suppression of Parp1 expression, but not by Parp2 suppression. Data shown in ( a , b ) are expressed as the ratio of the mean value of the sh Control group. Data represent the mean value ± SEM (n = 3). † p < 0.01, ‡ p < 0.005, and § p < 0.001 by comparison against control using Student’s t test

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: Knockdown, shRNA, Expressing, Control, Comparison

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: Higher amounts of p53 protein and phosphorylated p53 in NSPCs after PARP inhibition. a Cleaved fragments of caspase-3 (p17) and caspase-8 (p43/p41/p18) were clearly detectable by western blotting with specific antibodies. The cleaved fragment of pro-caspase-9 (p37) was also detectable, although the band density of this fragment was low. b Phase-contrast images were shown on the top. Higher amounts of p53 protein and phosphorylated p53 in NSPCs after PARP inhibition were also revealed by immunocytochemistry. Representative images from 4 separate experiments are shown. c Western blot analysis demonstrated that PARP-1 was abundant and activated in NSPCs in contrast to MEFs. Amounts of p53 protein and phosphorylated p53 at Ser 18 were remarkably increased by PJ34. Scale bars in ( b ), 20μm

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: Inhibition, Western Blot, Immunocytochemistry

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: Effects of PJ34 on neurosphere formation and cell viability of Trp53 +/− and Trp53 −/− NSPCs. a Western blot analysis demonstrated that the amounts of p53 protein and phosphorylated p53 at Ser18 were increased by PJ34 in Trp53 +/− NSPCs as well as wild-type NSPCs. Automodification of PARP-1 was detectable irrespective of Trp53 genotype. b Neurospheres were detectable in wild-type NSPCs after a 2-day incubation in the absence of PJ34, but were scarcely detectable with 10 or 20 μM PJ34. In contrast, neurospheres were still detectable with 10 μM PJ34 in Trp53 +/− NSPCs, and with 10 or 20 μM PJ34 in Trp53 −/− NSPCs. c The increase of MTS-reduction activity of wild-type NSPCs was suppressed by 10 or 20 μM PJ34. No suppressive effect of PJ34 was observed in Trp53 −/− NSPCs. Data represent the mean value ± SEM (n = 3). * p < 0.05 and † p < 0.01 by one-way ANOVA followed by Tukey’s post hoc test. Scale bars in ( b ), 50μm

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: Western Blot, Incubation, Activity Assay

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: Poly(ADP-ribosyl)ation of ATM and ATR, but not p53. a Poly(ADP-ribosyl)ated proteins were isolated from cell lysates by highly specific macrodomain poly(ADP-ribose) affinity resin. The resin-bound proteins were eluted and analyzed by western blotting with an anti-p53 antibody. Immunoprecipitation was carried out using an anti-p53 antibody. The resulting immunocomplexes were subjected to western blot analysis with anti-p53 and anti-poly(ADP-ribose) antibodies. Poly(ADP-ribosyl)ated p53 was undetectable irrespective of PARP inhibition. b The same membrane on which the proteins eluted from poly(ADP-ribose) affinity resin were transferred was analyzed by western blotting with anti-ATM and anti-ATR antibodies, resulting in the detection of ATM and ATR. c Immunoprecipitation was performed using an anti-ATM or anti-ATR antibody. Immunocomplexes were subjected to western blot analysis with an anti-ATM or anti-ATR antibody as well as an anti-poly(ADP-ribose) antibody. Poly(ADP-ribosyl)ated ATM and ATR were detectable in the absence of PJ34

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: Isolation, Western Blot, Immunoprecipitation, Inhibition, Membrane

Journal: BMC Neuroscience

Article Title: Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells

doi: 10.1186/s12868-016-0333-0

Figure Lengend Snippet: A model of cell fate decision by poly(ADP-ribosyl)ation in NSPCs. In normal conditions, both ATM and ATR are poly(ADP-ribosyl)ated and inactivated, leading to dephosphorylation of p53 at Ser18. The resulting inactivation of p53 promotes the self-renewal of NSPCs and neurosphere formation. Under PARP inhibition, ATM and/or ATR without poly(ADP-ribosyl)ation are activated, leading to the phosphorylation and activation of p53. The activated p53 signaling pathway delays cell cycle progression and induces apoptosis by different mechanisms

Article Snippet: The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling).

Techniques: De-Phosphorylation Assay, Inhibition, Phospho-proteomics, Activation Assay