Journal: Cell

Article Title: A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain

doi: 10.1016/j.cell.2018.05.057

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Pdf (1:50 dilution) , Developmental Studies Hybridoma Bank , Cat# PDF C7; RRID: AB_760350 , AB_2315084.

Techniques: Staining, Multiplex Assay, Isolation, Purification, Microarray, Sequencing, Software

Journal: Cell stem cell

Article Title: Phospholipid remodeling and cholesterol availability regulate intestinal stemness and tumorigenesis

doi: 10.1016/j.stem.2017.12.017

Figure Lengend Snippet:

Article Snippet: Biotinylated Goat Anti-Rat IgG Antibody (Vector laboratories, # BP-9400) at room temperature for 1 h and developed with ImmPACT DAB Peroxidase (HRP) Substrate (Vector, #SK4105).

Techniques: Plasmid Preparation, Recombinant, Amplex Red Cholesterol Assay, Microarray, Generated, Transgenic Assay, Software

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: Over Expression, Western Blot, 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, 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, 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, Over Expression, 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: Cell reports

Article Title: Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment

doi: 10.1016/j.celrep.2021.109088

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Each 50 μL PCR reaction consisted of a maximum of 2 μg gDNA, 0.5 μL of Takara Ex Taq DNA polymerase (Takara RR001C), 5 μL of 10x Ex Taq buffer, 4 μL dNTPs, 0.25 μL of 100 μM P5 stagger-mix forward primer, 5 μL of 5 μM P7 barcode reverse primer, and water to 50 μL total.

Techniques: Peptide Microarray, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, TUNEL Assay, Software, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, CRISPR

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: 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: Scientific reports

Article Title: Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome.

doi: 10.1038/s41598-017-02403-x

Figure Lengend Snippet: Figure 1. RPL23 knockdown blocks cell proliferation and induces apoptosis and cell cycle arrest. (a,b) The cell proliferation capacity of SKM-1/K562 cells was measured by a CCK-8 assay, and cellular viability was inhibited after RPL23 knockdown. (c) The representative flow graphic for apoptosis detection 72 h after efficient transfection of LV-RPL23-RNAi-GFP (KD group) and LV-RPL23-NC-GFP (NC group) in both SKM-1 and K562 cell lines is shown. The early-stage (d) and late-stage (e) apoptotic ratio increased after RPL23 knockdown. (f) The representative flow graphic for cell cycle detection 72 h after efficient transfection in both SKM-1 and K562 cell lines is shown. (g,h) The knockdown of RPL23 induced increases in the proportion of cells in the G0/G1 phase and a decreased proportion of cells in the S phase. Each assay was performed in triplicate. Unpaired Student’s t tests were used to calculate all p values shown in the figure. The data are expressed as the means ± S.E.M. WT: wild type; NC: RPL23-NC; KD: RPL23-KD. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following primary antibodies were used: a mouse mAb against c-Myc (5605, CST, 1:800; cytoplasmic staining), a rabbit pAb against Miz-1 (sc-22837, Santa Cruz, 1:50; cytoplasmic staining) and a rabbit pAb against RPL23 (16086-1-AP, ProteinTech, 1:50; cytoplasmic staining).

Techniques: Knockdown, CCK-8 Assay, Transfection

Journal: Scientific reports

Article Title: Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome.

doi: 10.1038/s41598-017-02403-x

Figure Lengend Snippet: Figure 2. Global gene expression profiles and GSEA analysis. A DNA microarray analysis was performed using three RPL23-KD DNA samples and three NC DNA samples of infected SKM-1 cells. (a) A volcano plot of all assayed probes shows the distribution of DEGs based on the expression data from RPL23-KD and NC SKM-1 samples. The X-axis is the log2 (fold-change), and the Y-axis is the p-value on a −log10 scale. Entities with significant differences are highlighted in red. (b) Unsupervised hierarchical clustering of six samples shows distinct segregation based on treatment (pink row for RPL23-KD, blue row for NC SKM-1 cells). The expression of 753 probes was significantly altered (>1.5-fold) in RPL23-KD cells compared with NC samples (p < 0.05). A total of 305 probes (upper left region) were upregulated, and 448 (lower left region) were downregulated. Each row represents a gene, and each column represents a sample. The coloured bars on the right represent the absolute mean expression (absolute fold change) of each gene in the KD/NC groups. The indicated DEGs for our target molecules are shown on the right side of the bars. (c) Identified GO gene sets categorized by biological process, cellular component and molecular function. A GSEA analysis indicates the overrepresented pathways enriched in RPL23-KD SKM-1 cells: enrichment plots and heatmaps of leading edge genes in KEGG_ PATHWAY IN CANCER (NES = 2.09) (d) and KEGG_APOTOSIS (NES = 1.65) (e). The asterisks mark DEGs involved in Miz-1/c-Myc function and PI3K/AKT signalling. GO ID/Term: Gene Ontology ID/term; NES: normalized enrichment score; FDR: false discovery rate.

Article Snippet: The following primary antibodies were used: a mouse mAb against c-Myc (5605, CST, 1:800; cytoplasmic staining), a rabbit pAb against Miz-1 (sc-22837, Santa Cruz, 1:50; cytoplasmic staining) and a rabbit pAb against RPL23 (16086-1-AP, ProteinTech, 1:50; cytoplasmic staining).

Techniques: Gene Expression, Microarray, Infection, Expressing

Journal: Scientific reports

Article Title: Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome.

doi: 10.1038/s41598-017-02403-x

Figure Lengend Snippet: Figure 3. Validation of screened mRNAs identified by microarray analysis through qRT-PCR and western blotting of SKM-1 cells. (a) The mRNA expression levels of RPL23-KD-associated genes were analysed by qRT- PCR. The Miz-1 (2.50 ± 0.14, p = 0.009), p15Ink4b (2.44 ± 0.23, p = 0.003), and p21Cip1 (6.46 ± 0.37, p = 0.005) expression levels were increased in the KD group compared with the NC group, whereas the RPL23 (0.02 ± 0.01, p < 0.001), c-Myc (0.58 ± 0.08, p = 0.031) and PI3KCG (0.15 ± 0.05, p = 0.004) expression levels were decreased with RPL23 knockdown. (b,c) The western blotting results revealed altered protein levels, specifically increased expression of Miz-1 (p < 0.001) and its downstream target molecules, p21Cip1 (p < 0.001) and p15Ink4b (p = 0.09), and decreased expression of c-Myc (p < 0.001) and PIK3CG (p < 0.001). The phosphorylation of AKT was also weakened (p = 0.008). (d) Histogram denoting the relative grey scale of the target stripes normalized to the GAPDH expression level using Image-Pro Plus v6.0 software (Media Cybernetics, USA). Target stripes were displayed as chopped. Original scans were provided in Supplementary Information (Supplementary Fig. S5). Each assay was performed in triplicate. Unpaired Student’s t tests were used to calculate all p values shown throughout the figure. The data are expressed as the means ± S.E.M. WT: wild type; NC: RPL23-NC; KD: RPL23-KD. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following primary antibodies were used: a mouse mAb against c-Myc (5605, CST, 1:800; cytoplasmic staining), a rabbit pAb against Miz-1 (sc-22837, Santa Cruz, 1:50; cytoplasmic staining) and a rabbit pAb against RPL23 (16086-1-AP, ProteinTech, 1:50; cytoplasmic staining).

Techniques: Biomarker Discovery, Microarray, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Phospho-proteomics, Software

Journal: Scientific reports

Article Title: Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome.

doi: 10.1038/s41598-017-02403-x

Figure Lengend Snippet: Figure 4. Expression of c-Myc overcomes RPL23-KD-induced viability suppression and apoptotic tendency in SKM-1 cells. (a) Growth curve of SKM-1 cells infected with LV-RPL23-RNAi expression vectors alone or together with CMV–c-Myc expression plasmid vectors after superinfection at the indicated time point. Suppressive proliferation of RPL23-KD cells was overcome by CMV-driven expression of c-Myc (p < 0.01). (b,c) Forty-eight hours after superinfection, cellular apoptosis was measured by Annexin V single-staining, and induced expression of c-Myc alleviated the high apoptotic potential that is mediated by depletion of L23. Unpaired Student’s t tests were used to calculate all p values shown throughout the figure. The data are expressed as the means ± S.E.M. WT: non-transfected group (used as blank control); NC: superinfected with two empty vectors (used as negative control); RPL23-KD: superinfected with LV-RPL23- RNAi and CMV-c-Myc-NC; RPL23-KD/c-Myc-OE: superinfected with LV-RPL23- RNAi and CMV-c-Myc and c-Myc-OE: superinfected with CMV-c-Myc and LV-RPL23-NC. /: non-treated; −: decreased expression; +: induced expression; NC: transfected with empty vectors. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following primary antibodies were used: a mouse mAb against c-Myc (5605, CST, 1:800; cytoplasmic staining), a rabbit pAb against Miz-1 (sc-22837, Santa Cruz, 1:50; cytoplasmic staining) and a rabbit pAb against RPL23 (16086-1-AP, ProteinTech, 1:50; cytoplasmic staining).

Techniques: Expressing, Infection, Plasmid Preparation, Staining, Transfection, Control, Negative Control

Journal: Scientific reports

Article Title: Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome.

doi: 10.1038/s41598-017-02403-x

Figure Lengend Snippet: Figure 5. RPL23, c-Myc and Miz1 mRNA and protein expression levels in the BM haematopoietic cells of MDS patients. (a) RPL23, (b) c-Myc and (c) Miz-1 mRNA expression levels in lower-risk (IPSS scores ≤1.0) and higher-risk (IPSS scores >1.0) MDS patients compared with healthy donors. Total RNA was extracted from 2 ml of BM mononuclear cells (BMNCs) from MDS patients with different risk levels using the TRIzol® reagent. The protocols used for cDNA synthesis and qRT-PCR are described in Methods. (d) Correlation between RPL23 and c-Myc mRNA levels in BMNC cells from MDS patients. Pearson’s correlation test for RPL23 vs. c-Myc: r = 0.703, p < 0.000. (e) Immunohistochemical staining for RPL23, c-Myc and Miz-1 in BM trephine tissue of lower/higher-risk MDS patients and healthy donors. RPL23, c-Myc and Miz-1 expression is indicated by yellowish-brown cytoplasmic staining. Original magnification: 400×. Clearly positively stained cells are indicated with arrows (scale bar: 2 μm). The mean densities of the positive sections were calculated for measuring in vivo protein expression and are displayed in scatter plots in panels (f), (g) and (h). Unpaired Student’s t tests were used to calculate all p values shown throughout the figure. The data are expressed as the means ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following primary antibodies were used: a mouse mAb against c-Myc (5605, CST, 1:800; cytoplasmic staining), a rabbit pAb against Miz-1 (sc-22837, Santa Cruz, 1:50; cytoplasmic staining) and a rabbit pAb against RPL23 (16086-1-AP, ProteinTech, 1:50; cytoplasmic staining).

Techniques: Expressing, cDNA Synthesis, Quantitative RT-PCR, Immunohistochemical staining, Staining, In Vivo

Journal: Scientific reports

Article Title: Ribosomal protein L23 negatively regulates cellular apoptosis via the RPL23/Miz-1/c-Myc circuit in higher-risk myelodysplastic syndrome.

doi: 10.1038/s41598-017-02403-x

Figure Lengend Snippet: Figure 6. Schematic showing the potential role of RPL23 in the RPL23/Miz-1/c-Myc circuit. Our study suggests a regulatory feedback loop through which RPL23 reinforces the Myc-dependent oncogenic functions. Elevated expression of RPL23 would decrease the Miz-1-dependent expression of p21Cip1 and p15Ink4b, thereby increasing the ability of c-Myc to promote cell cycle progression. In turn, the activation of c-Myc function in the context of RPL23 overexpression might further stimulate RPL23 expression, representing a positive feedback mechanism that couples efficient RPL23 expression with c-Myc’s function to suppress Miz-1-induced Cdk inhibitors and thereby promote cell cycle progression and induce apoptotic resistance.

Article Snippet: The following primary antibodies were used: a mouse mAb against c-Myc (5605, CST, 1:800; cytoplasmic staining), a rabbit pAb against Miz-1 (sc-22837, Santa Cruz, 1:50; cytoplasmic staining) and a rabbit pAb against RPL23 (16086-1-AP, ProteinTech, 1:50; cytoplasmic staining).

Techniques: Expressing, Activation Assay, Over Expression

Journal: Cell metabolism

Article Title: Glutamate/metabotropic glutamate receptor-5 signaling in hepatic stellate cells drives endocannabinoid-mediated alcoholic steatosis

doi: 10.1016/j.cmet.2019.08.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Endogenous peroxidases were blocked with 0.03% H 2 O 2 for 30 min and unspecific binding was blocked with 2.5% normal horse serum for 1 h. Tissues were incubated with primary antibody for 30 min at room temperature and further incubated with ImmPRESS HRP Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratories) according to the manufacturer’s recommendation.

Techniques: Recombinant, Modification, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot, SYBR Green Assay, Expressing, Microarray, Knock-Out, shRNA, Software

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: ( A ) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also – . ( B ) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice ( n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also – . ( C ) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment ( n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also – . ( D ) Lung sections of GFP;CCSP.CRE mice ( n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also – . CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Labeling, Marker, Staining

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: TUBA1A, Tubulin alpha 1a or acetylated tubulin; KRT5, Keratin 5; FOXJ1, Forkhead box J1; CCSP, Secretoglobin, family 1A, member 1 (uteroglobin) or Clara cell secretory protein or Clara cell 10 KDa protein; SFTPC, Surfactant protein C; LYZ2, Lysozyme 2; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques:

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: CRE, causes recombination; TOMATO, tdTomato; GFP, green fluorescent protein; CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; SOX2, sex determining region Y (SRY)-box 2; VAV, Vav Guanine Nucleotide Exchange Factor 1; NES, nestin; JAX, Jackson Laboratories; EMMA, European Mutant Mouse Archive; MGI, Mouse Genome Informatics; AEC, airway epithelial cells; BASC, bronchoalveolar stem cells; ATII, alveolar type II cells or type II pneumocytes; AMΦ, alveolar macrophages; ΒM, bone marrow (myeloid) cells. Symbols indicate: - (white), no genetic labeling; + (magenta), complete genetic labeling; ± (blue), partial genetic labeling.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Mutagenesis, Labeling

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage-labeled mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; TUBA1A, acetylated tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Marker, Staining, Labeling, Expressing

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: Representative photographs (top row) and green epifluorescence images (second row), as well as merged fluorescent microscopic images of lung sections for nuclear Hoechst33258 stain, endogenous TOMATO, and endogenous GFP (bottom three rows), of tumor-bearing lungs from genetically marked mice employed in these studies (described in detail in ) at six months after initiation of ten weekly intraperitoneal urethane injections ( n = 30, 22, 18, and 20/strain, respectively). b, bronchi. Top two rows: arrows indicate lung tumors. Bottom three rows: white arrows indicate GFP-labeled cells in apparently non-affected alveolar areas of GFP;CCSP.CRE mice; green arrow indicates rare GFP+ cell in non-affected central airway of GFP;LYZ2.CRE mouse. Note the absence of GFP-labeling of lung tumors in TOMATO mice, the complete GFP-labeling in GFP;CCSP.CRE and GFP;SFTPC.CRE mice, and the partial GFP-labeling in GFP;LYZ2.CRE mice.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Staining, Labeling

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: Lineage marker protein-stained lung adenocarcinomas (dashed outlines) from genetically marked mice ( n = 10/group). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Marker, Staining, Labeling, Expressing

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: Representative merged fluorescent microscopic images from lineage marker-stained lung sections of 6-week-old lineage reporter mice ( n = 5/group). Arrows indicate cells expressing the respective marker protein with (green) or without (red) genetic lineage-labeling. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2; b, bronchi; a, alveoli.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Marker, Staining, Expressing, Labeling

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: Representative merged fluorescent microscopic images of lineage marker protein-stained lung tumors (dashed outlines) from genetically marked mice (FVB background) at six months after a single intraperitoneal urethane injection ( n ≥ 10/strain). Note the genetic GFP-labeled tumor cells of GFP;CCSP.CRE mice that have lost CCSP and have acquired SFTPC with or without LYZ2 protein marker expression. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Marker, Staining, Injection, Labeling, Expressing

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: ( A ) DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Summary of duplexed digital droplet PCR (ddPCR) results using primers and probes specific for the Rosa mT and the Kras WT sequences. Note that all cell types equally suffer initial Kras Q61R mutations, but only GFP-labeled cells of GFP;CCSP.CRE mice (i.e. airway cells) maintain the Kras Q61R mutation after two weeks. See also . Data are shown as violin plot. P , overall probability, two-way ANOVA. ***: p<0.001 compared with all other groups, Bonferroni post-tests. ( B ) Representative high-resolution micro-computed tomography (μCT) lung sections (top) and three-dimensional reconstructions (bottom) from urethane-treated FVB mice six months after treatment ( n = 10). Note lung tumors attached to (green arrows) or contained within (blue arrows) the airways, as well as lung tumors with no obvious link to a bronchus (red arrows). ( C ) Summary of results from μCT (data from ) and pathology (data from ) shown as violin plot. P , probability, two-way ANOVA.*, ***, and ****: p<0.05, p<0.001, and p<0.0001, respectively, compared with airway-attached tumors, Bonferroni post-tests. Shown are also Spearman’s correlation coefficient ( ρ ) and probability ( P ) for correlation of μCT and pathology results. 10.7554/eLife.45571.031 Figure 2—source data 1. Quantification of Kras mutant droplets in duplexed digital droplet PCR (ddPCR). 10.7554/eLife.45571.032 Figure 2—source data 2. Quantification of tumor airway link.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Labeling, Mutagenesis, Micro-CT

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment ( n = 5/group). Representative gating strategy of digital droplet PCR (ddPCR) using primers and probes specific for the Rosa mT and the Kras WT sequences. Dashed outlines indicated GFP+ Kras Q61R + droplet gates.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques:

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: ( A ) Non-neoplastic alveolar regions from lung sections of aging GFP;CCSP.CRE mice (bottom right section is also SFTPC-immunostained) show increasing numbers of alveolar GFP-labeled cells with age (arrows). Green arrows: genetically GFP-labeled, SFTPC-immunoreactive airway cells in alveolus of 15-month-old GFP;CCSP.CRE mouse. ( B ) Data summary ( n = 5 mice/time-point) from ( A ) shown as violin plot. Color-coded boxes indicate time windows of experiments in ( C-H ). P , probability, one-way ANOVA. ns, ***, and ****: p>0.05, p<0.001, and p<0.0001, respectively, for comparison with time-point zero by Bonferroni post-tests. ( C ) SFTPC-immunostained lung sections of GFP;CCSP.CRE mice show accelerated increase of alveolar GFP-labeled SFTPC-immunoreactive airway cells after bleomycin treatment (arrows). See also and . ( D ) Data summary from ( C ) shown as violin plots ( n = 4 mice/time-point). P , probabilities, one-way ANOVA. ns, *, **, ***, and ****: p>0.05, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, for comparison with day zero by Bonferroni post-tests. ( E ) SFTPC-stained lung sections of GFP;CCSP.CRE mice at two months after perinatal exposure to 98% O 2 show enlarged alveoli (evident by increased mean linear intercept) enriched in GFP-labeled SFTPC-immunoreactive airway cells (arrows) compared with 21% O 2 . ( F ) Data summary from ( E ) shown as violin plots ( n = 6 mice/group). P , probabilities, t-test. ( G ) Lung sections (top) of GFP;CCSP.CRE mice ( n = 5 mice/group) show enrichment of alveoli in GFP-labeled cells post-naphthalene treatment (arrows). Lung sections (bottom) of GFP;LYZ2.CRE mice ( n = 5 mice/group) at six weeks post-naphthalene show no bronchial ( b ) GFP-labeled cells. See also and . ( H ) Data summary from ( G ) shown as violin plot ( n = 5 mice/time-point). P , probability, two-way ANOVA. ns and ****: p>0.05 and p<0.0001, respectively, for comparison with corn oil by Bonferroni post-tests. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.047 Figure 4—source data 1. Quantification of alveolar GFP+ cells in GFP;CCSP.CRE mice during aging. 10.7554/eLife.45571.048 Figure 4—source data 2. Quantification of SFTPC+ and GFP+ cells in GFP;CCSP.CRE mice after bleomycin treatment. 10.7554/eLife.45571.049 Figure 4—source data 3. Data of mean linear intercept and GFP+/SFTPC+cells in GFP;CCSP.CRE mice after hyperoxia treatment. 10.7554/eLife.45571.050 Figure 4—source data 4. Data of GFP+/SFTPC+ cells in GFP;CCSP.CRE and GFP;LYZ2.CRE mice after naphthalene treatment.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Labeling, Staining

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: ( A ) Lineage marker-immunostained lung sections of 12-week-old GFP;CCSP.CRE;DTA and GFP;LYZ2.CRE;DTA mice ( n = 6/group) show increased bronchial and alveolar size and flat CCSP+ SFTPC+ LYZ2+ cells in the airways of GFP;CCSP.CRE;DTA mice (green arrows), and CCSP-SFTPC-LYZ2+ alveolar macrophages in the airspaces of GFP;LYZ2.CRE;DTA mice (blue arrows). See also and . ( B ) Hematoxylin and eosin-stained lung sections ( n = 6/group) from 12-week-old DTA (controls), CCSP.CRE;DTA (airway epithelial suicide model), and LYZ2.CRE;DTA (alveolar epithelial suicide model) mice. ( C ) Data summaries of mean linear intercept, bronchoalveolar lavage (BAL) myeloid cells, pressure-volume curves, airway resistance, and static compliance ( n = 6–10/group) from 12-week-old DTA, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice shown as violin plots. P , probabilities, one-way ANOVA. ns, **, and ***: p>0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Bonferroni post-tests. ( D ) Lung photographs of control, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice at six months into treatment with urethane started at six weeks of age. ( E ) Incidence table and data summaries of lung tumors from ( D ) (violin plots; n is given in table). P , probabilities, χ -test (table) and one-way ANOVA (graphs). ns, *, **, and ***: p>0.05, p<0.05, p<0.01, and p<0.001, respectively, for the indicated comparisons, Fischer’s exact tests (table) or Bonferroni post-tests (graphs). a, alveoli; b, bronchi; ps, pleural space; v, vessel. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. 10.7554/eLife.45571.056 Figure 5—source data 1. Quantifications of data shown in . 10.7554/eLife.45571.057 Figure 5—source data 2. Quantifications of data shown in .

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Marker, Staining

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: Representative lung sections of 12-week-old GFP;CCSP.CRE, GFP;LYZ2.CRE, GFP;CCSP.CRE;DTA, and GFP;LYZ2.CRE;DTA mice ( n = 6/group). Shown are merges of Hoechst 33258-stained endogenous TOMATO- and GFP-labeling. Note increased bronchial ( b ) and alveolar ( a ) size, complete airway epithelial denudement, and prominent distortion of bronchial and alveolar structure of GFP;CCSP.CRE;DTA mice compared with other strains, mimicking chronic obstructive pulmonary disease. Note also the presence of some GFP-labeled alveolar macrophages in GFP;LYZ2.CRE;DTA mice (arrows). a, alveoli; b, bronchi.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Staining, Labeling

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet: ( A ) Our evidence supports the existence of distinct developmental ancestries for airway epithelial (AEC) and alveolar type II (ATII) cells, notwithstanding their common descent from an early (possibly Sftpc +) lung epithelial progenitor. The developmental airway lineage ( Scgb1a1+ Sftpc ±; green) gives rise to all types of airway cells, including club, ciliated, goblet, basal, and other cells, while the developmental ATII lineage ( Sftpc+ Lyz2 ±; red) gives rise to ATII cells before birth. These lineages appear to be segregated in the growing unaffected lung of the mouse till the age of six weeks, which roughly corresponds to a human age of six years, where cellular proliferation in the human lungs ceases. Thereafter, and likely due to the continuous exposure of the lungs to inhaled noxious agents, gradual expansion of Scgb1a1+ Sftpc ± marked cells ensues. Upon lung injury, this process is accelerated. Similarly, during carcinogenesis caused by chemical tobacco smoke carcinogens, Scgb1a1+ Sftpc ± marked cells expand and are ubiquitously present in peripheral lung adenocarcinomas. ( B ) Proposed neonatal proportions and postnatal dynamics of pulmonary epithelial cells during adulthood. Estimated proportions of lineage-marked cells at birth, based on flow cytometry and co-localization of proteinaceous and genetic cell marking. Lung lineages appear to be segregated in the growing lung till the age of full lung development (six weeks in mice and 6–8 years in humans) or till lung injury ensues. Schematic of proposed postnatal redistribution of marked cells in the adult lung. Upon injury, during multi-stage field carcinogenesis, or even during unchallenged aging, Scgb1a1 + marked cells appear in the distal alveolar regions, thereby maintaining lung structure and function. Bubble size indicates relative marked cell abundance. CCSP, Clara cell secretory protein; FOXJ1, forkhead box J1; KRT5, keratin 5; LYZ2, lysozyme 2; SFTPC, surfactant protein C; TUB1A1, acetylated α-tubulin.

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Flow Cytometry

Journal: eLife

Article Title: Club cells form lung adenocarcinomas and maintain the alveoli of adult mice

doi: 10.7554/eLife.45571

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: rabbit anti-proliferating cell nuclear antigen (PCNA, 1:3000 dilution, ab2426, Abcam, London, UK), rabbit anti-LYZ2 (1:50 dilution, ab108508, Abcam), rabbit anti-KRT5 (1:200 dilution, ab53121, Abcam), rabbit anti-SFTPC (1:200 dilution, sc-13979, Santa Cruz, Dallas, TX), rabbit anti-CCSP (1:200 dilution, sc-25555, Santa Cruz), goat anti-CCSP (1:1000 dilution, sc-9772, Santa Cruz), mouse anti-acetylated α-tubulin (1:2000 dilution, T7451, Sigma-Aldrich, St. Lewis, MO), rabbit anti-SFTPC (1:500 dilution, AB3786, Merck-Millipore, Burlington, MA), and mouse anti-KRT5 (1:200 dilution, MA5-17057, Thermo Fisher Scientific, Waltham, MA).

Techniques: Mutagenesis, Derivative Assay, Sequencing, Real-time Polymerase Chain Reaction, Software, Microarray, Expressing