Journal: Cell metabolism

Article Title: Telomere dysfunction induces sirtuin repression that drives telomere-dependent disease

doi: 10.1016/j.cmet.2019.03.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After washing in PBS, slides were incubated with ImmPRESS HRP Anti-Mouse IgG Polymer (Vector Laboratories) at room temperature for 1 hour.

Techniques: Construct, Plasmid Preparation, Recombinant, Protease Inhibitor, Bradford Assay, Fractionation, Reporter Assay, Mutagenesis, Activity Assay, Staining, In Situ, Knock-Out, Transgenic Assay, Software, RNA Sequencing Assay

Journal: The EMBO Journal

Article Title: Acidity suppresses CD8 + T-cell function by perturbing IL-2, mTORC1, and c-Myc signaling

doi: 10.1038/s44318-024-00235-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The 293T and C1498 cell lines (ATCC) were grown in DMEM medium containing 4.5 g/L glucose, sodium pyruvate, and glutamax (Gibco), supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 10 mM HEPES, 50 U/mL penicillin, and 50 μg/mL streptomycin (Gibco).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Sequencing, Staining, Lysis, Software, Cell Isolation, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: STAR Protocols

Article Title: XACT-seq: A photocrosslinking-based technique for detection of the RNA polymerase active-center position relative to DNA in Escherichia coli

doi: 10.1016/j.xpro.2021.100858

Figure Lengend Snippet:

Article Snippet: Add 50 μL of 1 × Taq DNA polymerase buffer (NEB) to wash the beads and to prepare samples for the next step.

Techniques: Recombinant, Staining, Purification, Sequencing, Plasmid Preparation, Software, Electroporation

Journal: STAR Protocols

Article Title: XACT-seq: A photocrosslinking-based technique for detection of the RNA polymerase active-center position relative to DNA in Escherichia coli

doi: 10.1016/j.xpro.2021.100858

Figure Lengend Snippet:

Article Snippet: Add 50 μL of 1 × Taq DNA polymerase buffer (NEB) to wash the beads and to prepare samples for the next step.

Techniques: Concentration Assay

Journal: eLife

Article Title: Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF

doi: 10.7554/eLife.67549

Figure Lengend Snippet:

Article Snippet: After blocking with PBS containing 5% normal goat/donkey serum and 0.1% Triton X-100 (blocking buffer) at RT for 1 hr, the sections were incubated at 4°C overnight with the following antibodies in the blocking buffer: mouse anti-RORC (1:800; catalog no. PP-H3925-00, Perseus Proteomics; although this antibody recognizes RORα, β and γ, RORγ is not expressed in the postnatal brain, and RORα expression generally overlaps with RORβ but is weak in the cortex [in Allen Brain Atlas]), rabbit anti-GFP (1:800; #A6455, Invitrogen), rabbit anti-Iba1 (1:500; catalog no. 019-19741, Wako), rabbit anti-5HTT (1:10,000; catalog no. 24330, ImmunoStar), goat anti-Brn2 (1:50; catalog no. sc-6029, Santa Cruz), rat anti-Ctip2 (1:200; catalog no. ab18465, Abcam), rabbit anti-Tbr1 (1:500; catalog no. ab31940, Abcam), rabbit anti-RFP (1:1000; catalog no. PM005, MBL), mouse anti-FLAG (1:1000, catalog no. F1804, Sigma-Aldrich), rabbit anti-ssDNA (1:300; catalog no. 18731, MBL), rabbit anti-RORβ (1:5000; catalog no. pAb-RORβHS-100, Diagenode), mouse anti-NeuN (1:400; catalog no. MAB377, Chemicon), rabbit anti-Cux1 (1:100, catalog no. sc-13024, Santa Cruz), rabbit anti-NRN1 (1:50; catalog no. sc-25261, Santa Cruz), and goat anti-VGF (1:50; catalog no. sc-10381, Santa Cruz).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Expressing, Sequencing, Imaging, Software, Staining

Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet:

Article Snippet: Incubate neurons with primary antibodies (PI3P, Z-P003, Echelon, 1:50, PI4P, Z-P004, Echelon, 1:200, or PI(4,5)P 2 , Z-A045, Echelon, 1:150 and Rab8, R5530, Sigma-Aldrich, 1:150 or TGN46, ab50595, Abcam, 1:200) in buffer A containing 5% NGS for 1 h. 41.

Techniques: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting

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: International Journal of Biological Sciences

Article Title: The Cyclin-Dependent Kinase 8 Inhibitor E966-0530-45418 Attenuates Pulmonary Fibrosis In Vitro and In Vivo

doi: 10.7150/ijbs.105826

Figure Lengend Snippet: E966-0530-45418 significantly attenuates the TGFβ1-induced increase of EMT proteins, fibrotic markers, and cell migration. ( A - J ) A549 cells were exposed to the indicated concentrations of E966-0530-45418 (μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of TGFβ1 (10 ng/mL) for 24 h. The protein levels of E-cadherin, N-cadherin, snail, α-SMA, and COL1A1 were determined by western blot in A549 cells (n = 4 independent samples per group, except for COL1A1, where n = 6) (A-D). The mRNA levels of E-cadherin, snail, α-SMA, COL1A1, TGFβ1, and CTGF were analyzed by RT‒qPCR in A549 cells (n = 3 independent samples per group, except for COL1A1, where n = 5) (E‒J). ( K , L ) A549 cells were treated with E966-0530-45418 at the indicated concentrations (μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of TGFβ1 (10 ng/mL), allowed to migrate into the wound area for 24 h and photographed; the cyan solid line depicts the edge between the cell-occupying region and the wound area (40× magnification) (Scale bar: 100 μm) (K). Cell migration into the wound was quantified using ImageJ software (L). (n = 4 independent samples per group). The results are shown as the mean ± SEM. P values were determined using one-way ANOVA followed by Tukey's post hoc test (B-J, and L).

Article Snippet: Senexin A (HY-15681) and Pirfenidone (HY- B0673) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Migration, Western Blot, Software

Journal: International Journal of Biological Sciences

Article Title: The Cyclin-Dependent Kinase 8 Inhibitor E966-0530-45418 Attenuates Pulmonary Fibrosis In Vitro and In Vivo

doi: 10.7150/ijbs.105826

Figure Lengend Snippet: E966-0530-45418 markedly mitigated TGFβ1/Smad3/RNA polymerase II signal transduction, subsequently lowering the transcription of EMT and fibrosis-related genes. ( A - C , E - G ) A549 cells were treated with E966-0530-45418 (5 μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of TGFβ1 (10 ng/mL) for 3 h and then subjected to nuclear-cytosolic fractionation. The protein levels in the cytosol and nucleus were detected by western blotting and quantified (n = 5 independent samples per group) (A-C). The nuclear proteins were immunoprecipitated with an anti-CDK8 antibody and then subjected to immunoblotting to assess the interactions between CDK8 and p-Smad3 T179, p-RNA Pol II S2/5, MED12, Pin 1, and cyclin C (n = 4 independent samples per group) (E-G). ( D ) Protein-protein interaction (PPI) network analysis was established by the STRING online database. The network nodes represent proteins, and the colored edges denote evidence of interactions between different proteins in the PPI network. Strong connections and networks were clustered using the k-means cluster algorithm with default parameters and are indicated by solid lines. POLR2A: RNA polymerase II. ( H ) Schematic illustrating Smad3 binding to the Smad-binding element (sequence logo obtained from the UCSC JASPAR joint website). ( I - K ) A549 cells were incubated with E966-0530-45418 (5 μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of TGFβ1 (10 ng/mL) for 6 h and then subjected to a Smad3 ChIP assay with RT‒qPCR analysis of the promoter sequences of N-cadherin, Snail (I), COL1A1, α-SMA (J), TGFβ1 and CTGF (K). The histone H3 antibody (Ab) group was used as a positive control, and the rabbit IgG group was used as a negative control (n = 3 independent samples per group). ( L ) Schematic illustration of the human E-cadherin and TGFβ1 promoter luciferase reporter genes, the function of which might be affected by TGFβ1 treatment. ( M ) A549 cells were transfected with 7TFP CDH1 or pGL3- TGFB1 reporter plasmid (1 μg) for 6 h, followed by subsequent treatment with TGF-β1 (10 ng/mL) with E966-0530-45418 (5 µM), senexin A (5 µM), pirfenidone (1 mM), or no inhibitor for 24 h, and luciferase expression was subsequently determined (n = 5 independent samples per group). The results are shown as the mean ± SEM. P values were determined using one-way ANOVA followed by Tukey's post hoc test (B, C, F, G, I-K, and M).

Article Snippet: Senexin A (HY-15681) and Pirfenidone (HY- B0673) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Transduction, Fractionation, Western Blot, Immunoprecipitation, Binding Assay, Sequencing, Incubation, Positive Control, Negative Control, Luciferase, Transfection, Plasmid Preparation, Expressing

Journal: International Journal of Biological Sciences

Article Title: The Cyclin-Dependent Kinase 8 Inhibitor E966-0530-45418 Attenuates Pulmonary Fibrosis In Vitro and In Vivo

doi: 10.7150/ijbs.105826

Figure Lengend Snippet: E966-0530-45418 diminished M2 macrophage polarization induced by IL6 in PMA-treated THP-1 cells. ( A ) GSEA of IL6/STAT3 signaling pathway genes in human IPF vs. normal lung samples from the GSE49072 dataset. The P value was computed using the 2-sided permutation test with the Benjamini-Hochberg adjustment for multiple comparisons. FDR: false discovery rate. ( B ) PPI network validating the interaction of IL6/STAT3/CDK8/RNA polymerase II (POLR2A) signals. Network nodes represent proteins; the colored edges denote evidence of different PPI types. Solid interactions and networks were clustered using the k-means cluster algorithm with default parameters and are indicated by solid lines. ( C ) PMA-induced THP-1 cells were treated with E966-0530-45418 (5 μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of IL6 (5 ng/mL) for 3 h and then subjected to nuclear-cytosolic fractionation. The protein levels in the cytosol and nucleus were detected by western blotting analysis and quantified (n = 5 independent samples per group). ( D - H ) PMA-induced THP-1 cells were treated with the indicated concentration of E966-0530-45418 (μM), senexin A (5 μM), pirfenidone (1 mM), or no inhibitor in the presence of IL6 (5 ng/mL) for 48 h (D and E) or 24 h (F-H). The CD206 levels were evaluated by flow cytometric analysis (n = 4 independent samples per group) (D and E). The mRNA levels of TGFβ1 were determined by RT‒qPCR (n = 3 independent samples per group) (F). The protein levels of arginase I were assessed by western blotting (n = 5 independent samples per group) (G and H). The results are shown as the mean ± SEM. P values were determined using one-way ANOVA followed by Tukey's post hoc test (C, E, F, and H).

Article Snippet: Senexin A (HY-15681) and Pirfenidone (HY- B0673) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Fractionation, Western Blot, Concentration Assay

Journal: Cell reports

Article Title: Interferon-γ induces combined pyroptotic angiopathy and APOL1 expression in human kidney disease

doi: 10.1016/j.celrep.2024.114310

Figure Lengend Snippet:

Article Snippet: BX795 (DMSO, 1 μM) , MedChemExpress , HY-10514.

Techniques: Plasmid Preparation, Recombinant, High Molecular Weight, Cell Culture, RNA sequencing, Derivative Assay, Modification, Software

Journal: iScience

Article Title: Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution

doi: 10.1016/j.isci.2022.105802

Figure Lengend Snippet:

Article Snippet: ImmPRESS® HRP Horse Anti-Mouse IgG Polymer Detection Kit , Vector laboratories , Cat# MP-7402.

Techniques: Virus, Recombinant, Saline, Staining, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Activity Assay, Polymer, Sequencing, Expressing, Software, Imaging, Cell Culture, Microscopy, Blocking Assay

Journal: The EMBO Journal

Article Title: The integrated stress response drives MET oncogene overexpression in cancers

doi: 10.1038/s44318-024-00338-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: When indicated, cells were treated with 50 ng/ml of carrier-free recombinant human HGF protein (Catalogue number 294-HG/CF, R&D Systems) for 15 min.

Techniques: Recombinant, Sequencing, Transfection, Western Blot, Luciferase, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, 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