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    Z 4 Hydroxytamoxifen
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    Millipore 4ht
    Z 4 Hydroxytamoxifen

    https://www.bioz.com/result/4ht/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    4ht - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) Product Images from "ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells"

    Article Title: ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells

    Journal: Scientific Data

    doi: 10.1038/sdata.2016.101

    ROCK kinase activation in PDAC cells. ( a ) ROCK functional domains and ROCK kinase expression constructs (CR, cysteine-rich; ER, estrogen receptor hormone binding domain; GFP, green fluorescent protein; PH, pleckstrin homology domain; RBD, Rho binding domain). ( b ) Conditional activation of ROCK kinase by 4-hydroxytamoxifen (4HT). ( c ) Overview of the ROCK signalling pathway. ( d ) PDAC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated with EtOH vehicle or 1 μM 4HT in the presence or absence of 10 μM Y27632 ROCK inhibitor. Immunoblotting shows ER-fusion proteins, endogenous ROCK1 and ROCK2, and phosphorylation status of ROCK targets LIMK1/2 (T508/T505) and regulatory myosin light chain (MLC2; T18S19). Total MLC2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.
    Figure Legend Snippet: ROCK kinase activation in PDAC cells. ( a ) ROCK functional domains and ROCK kinase expression constructs (CR, cysteine-rich; ER, estrogen receptor hormone binding domain; GFP, green fluorescent protein; PH, pleckstrin homology domain; RBD, Rho binding domain). ( b ) Conditional activation of ROCK kinase by 4-hydroxytamoxifen (4HT). ( c ) Overview of the ROCK signalling pathway. ( d ) PDAC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated with EtOH vehicle or 1 μM 4HT in the presence or absence of 10 μM Y27632 ROCK inhibitor. Immunoblotting shows ER-fusion proteins, endogenous ROCK1 and ROCK2, and phosphorylation status of ROCK targets LIMK1/2 (T508/T505) and regulatory myosin light chain (MLC2; T18S19). Total MLC2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.

    Techniques Used: Activation Assay, Functional Assay, Expressing, Construct, Binding Assay

    Quality control of RNA-Seq data. ( a ) Principal component plot of RNA-Seq (DESeq2) data indicating the clustering together of GFP:ER samples treated with vehicle control or 4HT, and ROCK1:ER plus ROCK2:ER samples treated with 4HT. ( b ) PDAC cells expressing GFP:ER, ROCK1:ER or ( c ) ROCK2:ER were treated with EtOH vehicle or 1 μM 4HT. Ptgs2 and Tff3 mRNA levels relative to Gapdh housekeeping gene were determined by qPCR. Means±s.e.m., unpaired t -test ( n =3). *** P
    Figure Legend Snippet: Quality control of RNA-Seq data. ( a ) Principal component plot of RNA-Seq (DESeq2) data indicating the clustering together of GFP:ER samples treated with vehicle control or 4HT, and ROCK1:ER plus ROCK2:ER samples treated with 4HT. ( b ) PDAC cells expressing GFP:ER, ROCK1:ER or ( c ) ROCK2:ER were treated with EtOH vehicle or 1 μM 4HT. Ptgs2 and Tff3 mRNA levels relative to Gapdh housekeeping gene were determined by qPCR. Means±s.e.m., unpaired t -test ( n =3). *** P

    Techniques Used: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth"

    Article Title: ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606743

    ROCK ‐induced collagen matrix invasion (related to Fig 2 ) H E‐stained sections of cell invasion into collagen matrix containing embedded fibroblasts after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with 1 μM 4HT. Means ± SEM ( n = 6), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. H E‐stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with EtOH vehicle, 1 μM 4HT, or 1 μM 4HT + 10 μM H1152. Means ± SEM ( n = 6; n = 5 for ROCK2:ER/4HT), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Quantification of cell number at the collagen matrix surface per 0.046 mm 2 field. Means ± SEM ( n = 24), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Ki67‐positive cell percentages at the surface. Means ± SEM ( n = 24), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test.
    Figure Legend Snippet: ROCK ‐induced collagen matrix invasion (related to Fig 2 ) H E‐stained sections of cell invasion into collagen matrix containing embedded fibroblasts after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with 1 μM 4HT. Means ± SEM ( n = 6), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. H E‐stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with EtOH vehicle, 1 μM 4HT, or 1 μM 4HT + 10 μM H1152. Means ± SEM ( n = 6; n = 5 for ROCK2:ER/4HT), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Quantification of cell number at the collagen matrix surface per 0.046 mm 2 field. Means ± SEM ( n = 24), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Ki67‐positive cell percentages at the surface. Means ± SEM ( n = 24), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test.

    Techniques Used: Staining, Immunofluorescence

    ROCK activation induces PDAC cell invasion H E‐stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPC cells in the presence or absence of 10 μM H1152 ROCK inhibitor. Means ± SEM ( n = 9 for untreated, n = 8 for H1152), P ‐value by unpaired t ‐test. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine‐rich). Conditionally activated human ROCK1, human ROCK2, and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone‐binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti‐GFP antibody. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 μM 4HT in the presence or absence of 1 μM or 10 μM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER‐fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were blotted as loading controls. H E‐stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with 1 μM 4HT. Means ± SEM ( n = 6), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Quantification of cell number at the collagen matrix surface per 0.046 mm 2 field. Means ± SEM ( n = 30), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. Ki67‐positive cell percentages at the surface and within the collagen matrix. Means ± SEM ( n = 30; n = 12 for GFP:ER/Matrix), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. Viable cell relative to starting cell numbers were determined after 24 or 48 h treatment with vehicle (−) or 4HT on uncoated plastic surfaces (H) or collagen1‐coated surfaces (I). Means ± SEM ( n = 3). Source data are available online for this figure.
    Figure Legend Snippet: ROCK activation induces PDAC cell invasion H E‐stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPC cells in the presence or absence of 10 μM H1152 ROCK inhibitor. Means ± SEM ( n = 9 for untreated, n = 8 for H1152), P ‐value by unpaired t ‐test. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine‐rich). Conditionally activated human ROCK1, human ROCK2, and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone‐binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti‐GFP antibody. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 μM 4HT in the presence or absence of 1 μM or 10 μM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER‐fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were blotted as loading controls. H E‐stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with 1 μM 4HT. Means ± SEM ( n = 6), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Quantification of cell number at the collagen matrix surface per 0.046 mm 2 field. Means ± SEM ( n = 30), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. Ki67‐positive cell percentages at the surface and within the collagen matrix. Means ± SEM ( n = 30; n = 12 for GFP:ER/Matrix), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Dunnett's multiple comparison test. Viable cell relative to starting cell numbers were determined after 24 or 48 h treatment with vehicle (−) or 4HT on uncoated plastic surfaces (H) or collagen1‐coated surfaces (I). Means ± SEM ( n = 3). Source data are available online for this figure.

    Techniques Used: Activation Assay, Staining, Binding Assay, Expressing, Immunofluorescence

    MMP proteins are efficiently released in response to ROCK activation Confocal microscope images of ROCK1:ER‐expressing cells co‐stained for F‐actin, MMP10 (left) or MMP13 (right), and DAPI following treatment with vehicle or 1 μM 4HT for 24 h. Multiple z‐planes were used to generate x − z and y − z images. Scale bar = 5 μm. Representative immunoblot of MMP13 and MMP10 in conditioned media, as well as MMP10 and GAPDH in cell lysates. Cells were treated with vehicle (−) or 1 μM 4HT for 48 h. Representative stained gel of ultracentrifuge‐enriched microvesicle protein from GFP:ER or ROCK1:ER‐expressing cell‐conditioned media following treatment with vehicle (−) or 1 μM 4HT for 24 h (top). Absolute arbitrary unit values for total microvesicle protein levels in stained gels (bottom). Means ± SEM ( n = 4), P ‐value by ratio paired t ‐test. Representative immunoblot of MMP10 and caveolin in ultracentrifuge‐enriched microvesicle proteins from cell‐conditioned media following treatment with vehicle (−) or 1 μM 4HT for 24 h as well as MMP10, caveolin, and GAPDH in cell lysates (top left). Transmission electron microscopy of ultracentrifuge‐enriched microvesicles (top center), and immunogold labeling of MMP10 in ROCK1:ER 4HT‐treated microvesicles, indicated by red arrows (top right). Absolute arbitrary unit values for MMP10 levels in Western blots of ultracentrifugation‐enriched microvesicle proteins (bottom left). Ratios of MMP10 to caveolin (bottom center) or to total microvesicle proteins (bottom right). Means ± SEM ( n = 4), P ‐value by ratio paired t ‐test. Absolute arbitrary unit values for caveolin levels in Western blots of ultracentrifugation‐enriched microvesicle proteins (left). Ratio of caveolin to total microvesicle proteins (right). Means ± SEM ( n = 4), P ‐value by ratio paired t ‐test. Ratios of MMP10 to GAPDH (left) and caveolin to GAPDH (right) in Western blots of whole‐cell lysates. Means ± SEM ( n = 3), P ‐value by ratio paired t ‐test. Source data are available online for this figure.
    Figure Legend Snippet: MMP proteins are efficiently released in response to ROCK activation Confocal microscope images of ROCK1:ER‐expressing cells co‐stained for F‐actin, MMP10 (left) or MMP13 (right), and DAPI following treatment with vehicle or 1 μM 4HT for 24 h. Multiple z‐planes were used to generate x − z and y − z images. Scale bar = 5 μm. Representative immunoblot of MMP13 and MMP10 in conditioned media, as well as MMP10 and GAPDH in cell lysates. Cells were treated with vehicle (−) or 1 μM 4HT for 48 h. Representative stained gel of ultracentrifuge‐enriched microvesicle protein from GFP:ER or ROCK1:ER‐expressing cell‐conditioned media following treatment with vehicle (−) or 1 μM 4HT for 24 h (top). Absolute arbitrary unit values for total microvesicle protein levels in stained gels (bottom). Means ± SEM ( n = 4), P ‐value by ratio paired t ‐test. Representative immunoblot of MMP10 and caveolin in ultracentrifuge‐enriched microvesicle proteins from cell‐conditioned media following treatment with vehicle (−) or 1 μM 4HT for 24 h as well as MMP10, caveolin, and GAPDH in cell lysates (top left). Transmission electron microscopy of ultracentrifuge‐enriched microvesicles (top center), and immunogold labeling of MMP10 in ROCK1:ER 4HT‐treated microvesicles, indicated by red arrows (top right). Absolute arbitrary unit values for MMP10 levels in Western blots of ultracentrifugation‐enriched microvesicle proteins (bottom left). Ratios of MMP10 to caveolin (bottom center) or to total microvesicle proteins (bottom right). Means ± SEM ( n = 4), P ‐value by ratio paired t ‐test. Absolute arbitrary unit values for caveolin levels in Western blots of ultracentrifugation‐enriched microvesicle proteins (left). Ratio of caveolin to total microvesicle proteins (right). Means ± SEM ( n = 4), P ‐value by ratio paired t ‐test. Ratios of MMP10 to GAPDH (left) and caveolin to GAPDH (right) in Western blots of whole‐cell lysates. Means ± SEM ( n = 3), P ‐value by ratio paired t ‐test. Source data are available online for this figure.

    Techniques Used: Activation Assay, Microscopy, Expressing, Staining, Transmission Assay, Electron Microscopy, Labeling, Western Blot

    ROCK activation promotes collagen degradation Representative fluorescence images of cells treated with 1 μM 4HT in the absence or presence of 10 μM H1152 for 18 h on Collagen1‐FITC (Col1‐FITC). Co‐staining for F‐actin, MMP10 (red), and DAPI (blue). Scale bar = 10 μm. Quantification of collagen degradation by cells treated with 1 μM 4HT or 4HT + 10 μM H1152 for 16 h. Means ± SEM ( n = 15), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Quantification of collagen degradation by cells treated with 1 μM 4HT, 4HT + DMSO vehicle, 4HT + 50 μM Blebbistatin or 4HT + 10 μM GM6001 for 16 h. Means ± SEM ( n = 15), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Immunofluorescence of collagen matrix sections co‐stained for MMP10 and DAPI. Scale bar = 20 μm. Mmp13 in situ hybridization‐stained sections of collagen matrices. Scale bar = 50 μm.
    Figure Legend Snippet: ROCK activation promotes collagen degradation Representative fluorescence images of cells treated with 1 μM 4HT in the absence or presence of 10 μM H1152 for 18 h on Collagen1‐FITC (Col1‐FITC). Co‐staining for F‐actin, MMP10 (red), and DAPI (blue). Scale bar = 10 μm. Quantification of collagen degradation by cells treated with 1 μM 4HT or 4HT + 10 μM H1152 for 16 h. Means ± SEM ( n = 15), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Quantification of collagen degradation by cells treated with 1 μM 4HT, 4HT + DMSO vehicle, 4HT + 50 μM Blebbistatin or 4HT + 10 μM GM6001 for 16 h. Means ± SEM ( n = 15), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Immunofluorescence of collagen matrix sections co‐stained for MMP10 and DAPI. Scale bar = 20 μm. Mmp13 in situ hybridization‐stained sections of collagen matrices. Scale bar = 50 μm.

    Techniques Used: Activation Assay, Fluorescence, Staining, Immunofluorescence, In Situ Hybridization

    Accumulation of MMP 10 and MMP 13 in bleblike protrusions (related to Fig 5 ) Confocal microscope images of KPflC cells expressing ROCK1:ER or ROCK2:ER as indicated co‐stained for F‐actin, MMP10 (A) or MMP13 (B), and DAPI following treatment with vehicle, 1 μM 4HT, or 1 μM 4HT + 1 μM H1152 for 24 h. Multiple z ‐planes were used to generate x − z and y − z images (bottom). Scale bar = 5 μm.
    Figure Legend Snippet: Accumulation of MMP 10 and MMP 13 in bleblike protrusions (related to Fig 5 ) Confocal microscope images of KPflC cells expressing ROCK1:ER or ROCK2:ER as indicated co‐stained for F‐actin, MMP10 (A) or MMP13 (B), and DAPI following treatment with vehicle, 1 μM 4HT, or 1 μM 4HT + 1 μM H1152 for 24 h. Multiple z ‐planes were used to generate x − z and y − z images (bottom). Scale bar = 5 μm.

    Techniques Used: Microscopy, Expressing, Staining

    ROCK activation promotes MMP ‐dependent invasive growth H E‐stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 μM GM6001 (bottom). Scale bar = 100 μm. Invasion index of KPflC cells. Means ± SEM ( n = 4), P ‐value by unpaired t ‐test. Quantification of cell number at the collagen matrix surface per 0.046 mm 2 field. Means ± SEM ( n = 20), P ‐value by unpaired t ‐test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Ki67‐positive cell percentages at the collagen matrix surface. Means ± SEM ( n = 20; n = 19 for GFP:ER/vehicle, n = 18 for GFP:ER/GM6001), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Ki67‐positive cell percentages in collagen matrix. Means ± SEM ( n = 20; n = 8 for GFP:ER/vehicle, n = 10 for GFP:ER/GM6001, n = 17 for ROCK1:ER/GM6001, n = 15 for ROCK2:ER/GM6001), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. The increase in viable cell numbers of cells plated on uncoated plastic surfaces (G) or collagen1 (Col1)‐coated surfaces (H) and treated with 1 μM 4HT for 24 h was not affected by 10 μM GM6001. Means ± SEM ( n = 3), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test.
    Figure Legend Snippet: ROCK activation promotes MMP ‐dependent invasive growth H E‐stained sections of cell invasion into collagen matrix after 8 days, in the absence (top) or presence of 10 μM GM6001 (bottom). Scale bar = 100 μm. Invasion index of KPflC cells. Means ± SEM ( n = 4), P ‐value by unpaired t ‐test. Quantification of cell number at the collagen matrix surface per 0.046 mm 2 field. Means ± SEM ( n = 20), P ‐value by unpaired t ‐test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Ki67‐positive cell percentages at the collagen matrix surface. Means ± SEM ( n = 20; n = 19 for GFP:ER/vehicle, n = 18 for GFP:ER/GM6001), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. Ki67‐positive cell percentages in collagen matrix. Means ± SEM ( n = 20; n = 8 for GFP:ER/vehicle, n = 10 for GFP:ER/GM6001, n = 17 for ROCK1:ER/GM6001, n = 15 for ROCK2:ER/GM6001), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test. The increase in viable cell numbers of cells plated on uncoated plastic surfaces (G) or collagen1 (Col1)‐coated surfaces (H) and treated with 1 μM 4HT for 24 h was not affected by 10 μM GM6001. Means ± SEM ( n = 3), one‐way ANOVA with multiplicity adjusted exact P ‐value by post hoc Tukey multiple comparison test.

    Techniques Used: Activation Assay, Staining, Immunofluorescence

    3) Product Images from "Epidermal ROCK2 induces AKT1/GSK3β/β-catenin, NFκB and dermal tenascin C; but enhanced differentiation and p53/p21 inhibit papilloma"

    Article Title: Epidermal ROCK2 induces AKT1/GSK3β/β-catenin, NFκB and dermal tenascin C; but enhanced differentiation and p53/p21 inhibit papilloma

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgz205

    Schematic of K14.creP/lslROCK er system and conformation of ROCK er activation. ( A ) K14 promoter drives expression of a Cre recombinase–progesterone ligand binding domain (PLB) fusion protein ( K14.creP ) and following RU486 treatment, CREP ablates a loxP -flanked stop cassette to express ROCK er from the CAGG promoter ( CAGGS-lslROCK er ). ( B ) PCR analysis of RU486-treated K14.creP/lslROCK er skin identifies K14.creP (~600 bp; lanes 1–3); primers 1/2 (arrows) identify the ablated stop codon band at ~500 bp and full-length product (~575bp) in untreated epidermis and non-epidermal tissue (lanes 1 and 2), untreated (lane 3*) or CAGGS-lslROCK2 er alone DNA (ethanol/lane 4*; RU486/lane 5). ( C ) Conformation of ROCK er activation . Primary K14creP.lslROCK er keratinocytes cultured in RU486 under proliferative low (L: 0.05 mM) or differentiating (H: 0.12 mM) calcium concentrations, with or without 1 nM 4HT (L+; H+) show ROCK er increases following 4HT-treatment. ROCK2 activity determined by phospho-MLC expression, increases on induction of differentiation and increases further following additional 4HT-mediated ROCK er activation. Total MLC served as a loading control.
    Figure Legend Snippet: Schematic of K14.creP/lslROCK er system and conformation of ROCK er activation. ( A ) K14 promoter drives expression of a Cre recombinase–progesterone ligand binding domain (PLB) fusion protein ( K14.creP ) and following RU486 treatment, CREP ablates a loxP -flanked stop cassette to express ROCK er from the CAGG promoter ( CAGGS-lslROCK er ). ( B ) PCR analysis of RU486-treated K14.creP/lslROCK er skin identifies K14.creP (~600 bp; lanes 1–3); primers 1/2 (arrows) identify the ablated stop codon band at ~500 bp and full-length product (~575bp) in untreated epidermis and non-epidermal tissue (lanes 1 and 2), untreated (lane 3*) or CAGGS-lslROCK2 er alone DNA (ethanol/lane 4*; RU486/lane 5). ( C ) Conformation of ROCK er activation . Primary K14creP.lslROCK er keratinocytes cultured in RU486 under proliferative low (L: 0.05 mM) or differentiating (H: 0.12 mM) calcium concentrations, with or without 1 nM 4HT (L+; H+) show ROCK er increases following 4HT-treatment. ROCK2 activity determined by phospho-MLC expression, increases on induction of differentiation and increases further following additional 4HT-mediated ROCK er activation. Total MLC served as a loading control.

    Techniques Used: Activation Assay, Expressing, Ligand Binding Assay, Polymerase Chain Reaction, Cell Culture, Activity Assay

    Analysis of β-catenin, p-GSK3β and p-AKT1 expression. ( A and B ) β-catenin expression in K14.creP/lslROCK er epidermis (4–6 weeks) at the junction between RU486 alone (left) and 4HT/RU486-treatment (right) shows expression increases in 4HT-treated areas [B: double-label versus K14 (red) counterstain]. ( C ) Older 4HT/RU486-treated K14.creP/lslROCK er hyperplasia (10 weeks) shows elevated β-catenin in basal and supra-basal layer keratinocytes. ( D ) Higher magnification of K14.creP/lslROCK er epidermis (10 weeks) shows detectable cytoplasmic/nuclear β-catenin expression in basal layer keratinocytes compared with ( E ) HK1.ras , available at Carcinogenesis Online). ( F ) p-GSK3β expression in (upper) 4HT-treated versus (lower) untreated K14.creP/lslROCK er ear skin shows stronger expression in 4HT-treated epidermis becoming stronger and more uniform with time in ( G ) older (12 weeks) 4HT/RU486-treated K14.creP/lslROCK er hyperplasia. Higher magnification ( H ) shows 4HT/RU486-treated K14.creP/lslROCK er hyperplasia maintains elevated p-GSK3β expression in both basal and suprabasal layers; whereas ( I ) HK1.ras , available at Carcinogenesis Online). ( J and K ) p-AKT1 analysis in 4HT/RU486-treated K14.creP/lslROCK er epidermis (4 weeks) shows elevated basal-layer expression compared with ( L ) sporadic, supra-basal p-AKT expression in normal adult skin; yet similar to ( M ) normal neonatal skin (24 h) that exhibits uniform, supra-basal p-AKT1. ( N ) Older (12 weeks) 4HT/RU486-treated K14.creP/lslROCK er epidermis retains elevated p-AKT1, but with areas of supra-basal expression; similar to neonates. ( O ) HK1.ras , available at Carcinogenesis Online). Bars: G, H and J ~120 µm; A, C, P and N ~80 µm; B, E, I, O and Q ~50 µm; Q, D, L and M ~30 µm).
    Figure Legend Snippet: Analysis of β-catenin, p-GSK3β and p-AKT1 expression. ( A and B ) β-catenin expression in K14.creP/lslROCK er epidermis (4–6 weeks) at the junction between RU486 alone (left) and 4HT/RU486-treatment (right) shows expression increases in 4HT-treated areas [B: double-label versus K14 (red) counterstain]. ( C ) Older 4HT/RU486-treated K14.creP/lslROCK er hyperplasia (10 weeks) shows elevated β-catenin in basal and supra-basal layer keratinocytes. ( D ) Higher magnification of K14.creP/lslROCK er epidermis (10 weeks) shows detectable cytoplasmic/nuclear β-catenin expression in basal layer keratinocytes compared with ( E ) HK1.ras , available at Carcinogenesis Online). ( F ) p-GSK3β expression in (upper) 4HT-treated versus (lower) untreated K14.creP/lslROCK er ear skin shows stronger expression in 4HT-treated epidermis becoming stronger and more uniform with time in ( G ) older (12 weeks) 4HT/RU486-treated K14.creP/lslROCK er hyperplasia. Higher magnification ( H ) shows 4HT/RU486-treated K14.creP/lslROCK er hyperplasia maintains elevated p-GSK3β expression in both basal and suprabasal layers; whereas ( I ) HK1.ras , available at Carcinogenesis Online). ( J and K ) p-AKT1 analysis in 4HT/RU486-treated K14.creP/lslROCK er epidermis (4 weeks) shows elevated basal-layer expression compared with ( L ) sporadic, supra-basal p-AKT expression in normal adult skin; yet similar to ( M ) normal neonatal skin (24 h) that exhibits uniform, supra-basal p-AKT1. ( N ) Older (12 weeks) 4HT/RU486-treated K14.creP/lslROCK er epidermis retains elevated p-AKT1, but with areas of supra-basal expression; similar to neonates. ( O ) HK1.ras , available at Carcinogenesis Online). Bars: G, H and J ~120 µm; A, C, P and N ~80 µm; B, E, I, O and Q ~50 µm; Q, D, L and M ~30 µm).

    Techniques Used: Expressing

    Analysis of p-Mypt1 and tenascin C expression in K14.creP/lslROCK er . ( A ) 4HT/RU486-treated K14.creP/lslROCK er epidermis at (12 weeks) shows ROCK er -activated p-Mypt1 expression in all layers is ( B ) maintained over time (24 weeks) compared with ( C ) equivalent 4HT/RU486-treated HK1.ras hyperplasia (** P
    Figure Legend Snippet: Analysis of p-Mypt1 and tenascin C expression in K14.creP/lslROCK er . ( A ) 4HT/RU486-treated K14.creP/lslROCK er epidermis at (12 weeks) shows ROCK er -activated p-Mypt1 expression in all layers is ( B ) maintained over time (24 weeks) compared with ( C ) equivalent 4HT/RU486-treated HK1.ras hyperplasia (** P

    Techniques Used: Expressing

    Analysis of differentiation marker expression. Western analysis of keratin K1/K6α expression: primary K14creP.lslROCK er or control lslcagROCK er keratinocytes are cultured in low or high calcium media (L = 0.05 mM; H = 0.12 mM) containing RU486 (5 nM) with/without 4HT (1 nM; L+/H+). ROCK er activation induces anomalous K1 expression in low calcium medium, which was absent in untreated or 4HT-treated lslROCK er controls (L+ versus L); with elevated K1 expression in high calcium similar to normal 4HT-treated lslROCK er controls keratinocytes. ROCK er activation also down-regulates K6α in hyperproliferative, low calcium K14creP.lslROCK er keratinocytes (L+) compared with normal4HT–treated lslROCK er , available at Carcinogenesis Online. Left: Immune fluorescence analysis of keratin K1 ( A – D ) and K6α ( E – H ) expression (green), counterstained for K14 (red) to delineate epidermis/dermis. (A and B) 4HT/RU486-treated K14.cre/lsl.ROCK er hyperplasia display a ragged/disordered, supra-basal K1 profile, as proliferative K14 +ve -basal layer cells differentiate and transit into supra-basal keratinocytes. (C) None 4HT/RU486-treated control K14.cre/lsl.ROCK er epidermis displays normal, supra-basal K1; while (D) HK1.ras hyperplasia exhibits an ordered K1 pattern at the K1/K14 transition border. (E) 4HT/RU486-treated K14.cre/lsl.ROCK er and (F) K14.ROCK er hyperplasia display patchy K6α expression compared with (G) normal epidermis with K6α restricted to hair follicles. (H) HK1.ras hyperplasia exhibits strong, uniform K6α. Bars: E, G and H, ~120 µm; A, D and F: ~70–80 µm; B and C: ~40 µm). Right panel: Filaggrin ( A – D ) and loricrin ( E – H ) expression in 4HT/RU486-treated K14.cre/lsl.ROCK er . (A and B) Elevated filaggrin expression increases with increasing hyperplasia (12 and 30 weeks); showing (B) premature filaggrin detectable below the granular layer compared with (C) granular layer filaggrin in normal epidermis. (D) HK1.ras displays reduced filaggrin as hyperplasia increases. (E and F) Loricrin expression in treated K14.cre/lsl.ROCK er hyperplasia (at 12 and 30 weeks) shows increased, premature expression becomes detectable in supra-basal layers. (G) Untreated K14.cre/lsl.ROCK er epidermis exhibits granular loricrin expression; while (H) HK1.ras hyperplasia expresses reduced, granular loricrin. Bars: ~70–80 µm.
    Figure Legend Snippet: Analysis of differentiation marker expression. Western analysis of keratin K1/K6α expression: primary K14creP.lslROCK er or control lslcagROCK er keratinocytes are cultured in low or high calcium media (L = 0.05 mM; H = 0.12 mM) containing RU486 (5 nM) with/without 4HT (1 nM; L+/H+). ROCK er activation induces anomalous K1 expression in low calcium medium, which was absent in untreated or 4HT-treated lslROCK er controls (L+ versus L); with elevated K1 expression in high calcium similar to normal 4HT-treated lslROCK er controls keratinocytes. ROCK er activation also down-regulates K6α in hyperproliferative, low calcium K14creP.lslROCK er keratinocytes (L+) compared with normal4HT–treated lslROCK er , available at Carcinogenesis Online. Left: Immune fluorescence analysis of keratin K1 ( A – D ) and K6α ( E – H ) expression (green), counterstained for K14 (red) to delineate epidermis/dermis. (A and B) 4HT/RU486-treated K14.cre/lsl.ROCK er hyperplasia display a ragged/disordered, supra-basal K1 profile, as proliferative K14 +ve -basal layer cells differentiate and transit into supra-basal keratinocytes. (C) None 4HT/RU486-treated control K14.cre/lsl.ROCK er epidermis displays normal, supra-basal K1; while (D) HK1.ras hyperplasia exhibits an ordered K1 pattern at the K1/K14 transition border. (E) 4HT/RU486-treated K14.cre/lsl.ROCK er and (F) K14.ROCK er hyperplasia display patchy K6α expression compared with (G) normal epidermis with K6α restricted to hair follicles. (H) HK1.ras hyperplasia exhibits strong, uniform K6α. Bars: E, G and H, ~120 µm; A, D and F: ~70–80 µm; B and C: ~40 µm). Right panel: Filaggrin ( A – D ) and loricrin ( E – H ) expression in 4HT/RU486-treated K14.cre/lsl.ROCK er . (A and B) Elevated filaggrin expression increases with increasing hyperplasia (12 and 30 weeks); showing (B) premature filaggrin detectable below the granular layer compared with (C) granular layer filaggrin in normal epidermis. (D) HK1.ras displays reduced filaggrin as hyperplasia increases. (E and F) Loricrin expression in treated K14.cre/lsl.ROCK er hyperplasia (at 12 and 30 weeks) shows increased, premature expression becomes detectable in supra-basal layers. (G) Untreated K14.cre/lsl.ROCK er epidermis exhibits granular loricrin expression; while (H) HK1.ras hyperplasia expresses reduced, granular loricrin. Bars: ~70–80 µm.

    Techniques Used: Marker, Expressing, Western Blot, Cell Culture, Activation Assay, Fluorescence

    Conformation of ROCK er expression in K14.creP/lslROCK er phenotypes. ( A ) 4HT/RU486-treated K14.creP/lslROCK er mice (20 weeks) exhibit mild keratosis but despite thrice-weekly treatments (up to 6 months), no papillomas appear. ( B ) 4HT/RU486-treated K14.creP/lslROCK er mice exhibit mild epidermal hyperplasia (12 weeks) compared with ( C ) normal RU486-alone control skin (20 weeks) that ( D ) increases over time (20 weeks) but remains hyperplastic. ( E – G ) GFP-tag analysis of K14.creP/lslROCK er skin confirmed ROCK er expression in (E) hyperplastic 4HT/RU486-treated and (F) normal RU486-alone skin, was absent in (G) untreated controls. ( H – J ) Analysis of ROCK2/ROCK er protein levels shows (H) hyperplastic K14.creP/lslROCK er epidermis exhibits elevated expression following 4HT/RU486 alongside (I) RU486-alone controls; compared with lower, suprabasal expression in (J) normal epidermis. Bars: B ~80 µm; C–J ~40 µm; E and H ~30 µm.
    Figure Legend Snippet: Conformation of ROCK er expression in K14.creP/lslROCK er phenotypes. ( A ) 4HT/RU486-treated K14.creP/lslROCK er mice (20 weeks) exhibit mild keratosis but despite thrice-weekly treatments (up to 6 months), no papillomas appear. ( B ) 4HT/RU486-treated K14.creP/lslROCK er mice exhibit mild epidermal hyperplasia (12 weeks) compared with ( C ) normal RU486-alone control skin (20 weeks) that ( D ) increases over time (20 weeks) but remains hyperplastic. ( E – G ) GFP-tag analysis of K14.creP/lslROCK er skin confirmed ROCK er expression in (E) hyperplastic 4HT/RU486-treated and (F) normal RU486-alone skin, was absent in (G) untreated controls. ( H – J ) Analysis of ROCK2/ROCK er protein levels shows (H) hyperplastic K14.creP/lslROCK er epidermis exhibits elevated expression following 4HT/RU486 alongside (I) RU486-alone controls; compared with lower, suprabasal expression in (J) normal epidermis. Bars: B ~80 µm; C–J ~40 µm; E and H ~30 µm.

    Techniques Used: Expressing, Mouse Assay

    Analysis of p53, p21 and NF-κB expression in K14.creP/lslROCK er and HK1.ras hyperplasia. ( A ) 4HT/RU486-treated K14.creP/lslROCK er epidermis (6 weeks) shows elevated p53 expression in basal layer keratinocyte cytoplasm/nuclei, becoming ( B ) expressed in all layers (12 weeks). In comparison ( C ) HK1.ras hyperplasia (6 weeks) displays weak p53 in sporadic keratinocytes (** P
    Figure Legend Snippet: Analysis of p53, p21 and NF-κB expression in K14.creP/lslROCK er and HK1.ras hyperplasia. ( A ) 4HT/RU486-treated K14.creP/lslROCK er epidermis (6 weeks) shows elevated p53 expression in basal layer keratinocyte cytoplasm/nuclei, becoming ( B ) expressed in all layers (12 weeks). In comparison ( C ) HK1.ras hyperplasia (6 weeks) displays weak p53 in sporadic keratinocytes (** P

    Techniques Used: Expressing

    4) Product Images from "Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia"

    Article Title: Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20101691

    T-ALL regression after MYC down-regulation occurs via mitochondrial apoptosis. (A) Immunohistochemical staining for the apoptotic marker activated caspase 3 was performed in rag2:MYC-ER transgenic zebrafish with T-ALL in the presence of 4HT (+4HT) and 4 d after 4HT removal (−4HT). Number of fish analyzed = 4 per condition. Bars, 10 µm. (B) One representative rag2:MYC-ER; rag2:EGFP-bcl2; rag2:dsRed2 triple-transgenic zebrafish is shown at the time of T-ALL onset and 3 wk after removal from 4HT. Bar, 1 mm. (C) Quantitation of T-ALL phenotypes 8 wk after MYC-ER inactivation, comparing rag2:EGFP-bcl2 transgenic zebrafish versus rag2:dsRed2 controls. Number of fish analyzed per condition: rag2:MYC-ER;rag2:dsRed2 , n = 12; rag2:MYC-ER; rag2:dsRed2; rag2:EGFP-bcl2 , n = 20.
    Figure Legend Snippet: T-ALL regression after MYC down-regulation occurs via mitochondrial apoptosis. (A) Immunohistochemical staining for the apoptotic marker activated caspase 3 was performed in rag2:MYC-ER transgenic zebrafish with T-ALL in the presence of 4HT (+4HT) and 4 d after 4HT removal (−4HT). Number of fish analyzed = 4 per condition. Bars, 10 µm. (B) One representative rag2:MYC-ER; rag2:EGFP-bcl2; rag2:dsRed2 triple-transgenic zebrafish is shown at the time of T-ALL onset and 3 wk after removal from 4HT. Bar, 1 mm. (C) Quantitation of T-ALL phenotypes 8 wk after MYC-ER inactivation, comparing rag2:EGFP-bcl2 transgenic zebrafish versus rag2:dsRed2 controls. Number of fish analyzed per condition: rag2:MYC-ER;rag2:dsRed2 , n = 12; rag2:MYC-ER; rag2:dsRed2; rag2:EGFP-bcl2 , n = 20.

    Techniques Used: Immunohistochemistry, Staining, Marker, Transgenic Assay, Fluorescence In Situ Hybridization, Quantitation Assay

    MYC represses pten. (A) Q-RT-PCR for ptenA and ptenB mRNA in T-ALL cells isolated from rag2:MYC-ER; rag2:EGFP-bcl2 transgenic zebrafish before and 4 d after 4HT removal, demonstrated that both zebrafish pten transcripts are repressed by MYC. β-Actin was used as the Q-RT-PCR control. Error bars represent standard error of the mean. Number of tumors analyzed per condition: 4HT+, n = 6; 4HT−, n = 7−. (B) Model to explain our findings.
    Figure Legend Snippet: MYC represses pten. (A) Q-RT-PCR for ptenA and ptenB mRNA in T-ALL cells isolated from rag2:MYC-ER; rag2:EGFP-bcl2 transgenic zebrafish before and 4 d after 4HT removal, demonstrated that both zebrafish pten transcripts are repressed by MYC. β-Actin was used as the Q-RT-PCR control. Error bars represent standard error of the mean. Number of tumors analyzed per condition: 4HT+, n = 6; 4HT−, n = 7−. (B) Model to explain our findings.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Transgenic Assay

    Constitutive Akt activation accelerates MYC-induced T-ALL and promotes loss of MYC transgene dependence. Germline rag2:MYC-ER heterozygous transgenic zebrafish were mated to wild-type fish, and all resultant offspring were injected at the one-cell stage with both rag2:GFP and rag2:myr-mAkt2 (15 pg of each DNA construct) or with rag2:GFP alone (30 pg). (A) Kaplan-Meier analysis of leukemia-free survival based on genotype. Number of GFP-fluorescent fish analyzed per genotype: rag2:MYC-ER negative, rag2:myr-mAkt2 negative, n = 10; rag2:MYC-ER negative, rag2:myr-mAkt2 positive; n = 18. rag2:MYC-ER positive, rag2:myr-mAkt2 negative, n = 12; rag2:MYC-ER positive, rag2:myr-mAkt2 positive, n = 21. (B) Representative rag2:MYC-ER germline transgenic zebrafish injected with rag2:myr-mAkt2 and rag2:GFP , shown at the time of T-ALL onset and 3 wk after removal from 4HT. Bar, 1 mm. (C) Quantitation of T-ALL phenotypes in rag2:MYC-ER –positive fish after 4HT removal, based on Akt genotype. Number of fish examined per group: rag2:myr-mAkt2, n = 21; rag2:GFP controls, n = 12.
    Figure Legend Snippet: Constitutive Akt activation accelerates MYC-induced T-ALL and promotes loss of MYC transgene dependence. Germline rag2:MYC-ER heterozygous transgenic zebrafish were mated to wild-type fish, and all resultant offspring were injected at the one-cell stage with both rag2:GFP and rag2:myr-mAkt2 (15 pg of each DNA construct) or with rag2:GFP alone (30 pg). (A) Kaplan-Meier analysis of leukemia-free survival based on genotype. Number of GFP-fluorescent fish analyzed per genotype: rag2:MYC-ER negative, rag2:myr-mAkt2 negative, n = 10; rag2:MYC-ER negative, rag2:myr-mAkt2 positive; n = 18. rag2:MYC-ER positive, rag2:myr-mAkt2 negative, n = 12; rag2:MYC-ER positive, rag2:myr-mAkt2 positive, n = 21. (B) Representative rag2:MYC-ER germline transgenic zebrafish injected with rag2:myr-mAkt2 and rag2:GFP , shown at the time of T-ALL onset and 3 wk after removal from 4HT. Bar, 1 mm. (C) Quantitation of T-ALL phenotypes in rag2:MYC-ER –positive fish after 4HT removal, based on Akt genotype. Number of fish examined per group: rag2:myr-mAkt2, n = 21; rag2:GFP controls, n = 12.

    Techniques Used: Activation Assay, Transgenic Assay, Fluorescence In Situ Hybridization, Injection, Construct, Quantitation Assay

    pten haploinsufficiency does not accelerate onset of MYC-induced T-ALL. (A) Experimental design to test the effect of pten haploinsufficiency on T-ALL onset upon MYC-ER activation and on tumor regression after 4HT removal. (B) Analysis of T-ALL onset in zebrafish from the experiment described in A. Number of fish analyzed per genotype: ptenA +/+, ptenB +/+, n = 39; ptenA +/−, ptenB +/+, n = 22; ptenA +/+, ptenB +/−, n = 12; ptenA +/−, ptenB +/−, n = 10.
    Figure Legend Snippet: pten haploinsufficiency does not accelerate onset of MYC-induced T-ALL. (A) Experimental design to test the effect of pten haploinsufficiency on T-ALL onset upon MYC-ER activation and on tumor regression after 4HT removal. (B) Analysis of T-ALL onset in zebrafish from the experiment described in A. Number of fish analyzed per genotype: ptenA +/+, ptenB +/+, n = 39; ptenA +/−, ptenB +/+, n = 22; ptenA +/+, ptenB +/−, n = 12; ptenA +/−, ptenB +/−, n = 10.

    Techniques Used: Activation Assay, Fluorescence In Situ Hybridization

    pten haploinsufficiency promotes loss of MYC transgene dependence. (A and C) One representative rag2:MYC-ER; rag2:GFP double-transgenic pten –wild-type zebrafish, shown at time of T-ALL onset (A) and 3 wk after removal from 4HT (C). (B and D) Representative rag2:MYC-ER; rag2:GFP double-transgenic zebrafish that harbored heterozygous mutations of both ptenA and ptenB , shown at time of T-ALL onset (B) and 3 wk after removal from 4HT (D). Bar, 1 mm. (E) Quantitation of T-ALL phenotypes after 4HT removal, based on pten genotype. Number of fish with T-ALL analyzed per genotype: ptenA +/+, ptenB +/+, n = 39; ptenA +/+, ptenB +/−, n = 12; ptenA +/−, ptenB +/+, n = 22; ptenA +/−, pten B +/−, n = 10. (F) Western blot analysis for phosphorylation of S6 ribosomal protein, a marker of Akt pathway activation, in sorted T-ALL cells from five different rag2:MYC-ER pten–wild-type zebrafish in which T-ALL progressed despite MYC down-regulation. Units for the molecular mass markers shown are in kD.
    Figure Legend Snippet: pten haploinsufficiency promotes loss of MYC transgene dependence. (A and C) One representative rag2:MYC-ER; rag2:GFP double-transgenic pten –wild-type zebrafish, shown at time of T-ALL onset (A) and 3 wk after removal from 4HT (C). (B and D) Representative rag2:MYC-ER; rag2:GFP double-transgenic zebrafish that harbored heterozygous mutations of both ptenA and ptenB , shown at time of T-ALL onset (B) and 3 wk after removal from 4HT (D). Bar, 1 mm. (E) Quantitation of T-ALL phenotypes after 4HT removal, based on pten genotype. Number of fish with T-ALL analyzed per genotype: ptenA +/+, ptenB +/+, n = 39; ptenA +/+, ptenB +/−, n = 12; ptenA +/−, ptenB +/+, n = 22; ptenA +/−, pten B +/−, n = 10. (F) Western blot analysis for phosphorylation of S6 ribosomal protein, a marker of Akt pathway activation, in sorted T-ALL cells from five different rag2:MYC-ER pten–wild-type zebrafish in which T-ALL progressed despite MYC down-regulation. Units for the molecular mass markers shown are in kD.

    Techniques Used: Transgenic Assay, Quantitation Assay, Fluorescence In Situ Hybridization, Western Blot, Marker, Activation Assay

    Conditional T-ALL development in rag2:MYC-ER transgenic zebrafish. (A and B) Thymic fluorescence in control MYC-ER–negative lck-EGFP transgenic (A) and rag2:dsRed2 transgenic (B) zebrafish raised in the absence of 4HT. (C and D) Thymic fluorescence in the absence of 4HT treatment in rag2:MYC-ER transgenic zebrafish that also expressed lck-EGFP (C) or rag2:dsRed2 (D). For A–D, one representative zebrafish is shown from a minimum of eight fish raised in each condition. (E and F) Fully penetrant T-ALL in rag2:MYC-ER transgenic zebrafish raised in 50 µg/liter (129 nM) 4HT. A representative triple-transgenic rag2:MYC-ER; lck:EGFP; rag2:dsRed2 zebrafish is shown at the time of disseminated T-ALL development, imaged in both green and red fluorescent channels. (G and H) Thymic fluorescence in the rag2:MYC-ER transgenic zebrafish from E and F, shown 4 wk after 4HT removal. In all triple-transgenic zebrafish in which regression occurred ( n = 6 of 8), T-ALL regression occurred simultaneously in both green and red fluorescent channels, with no evidence of residual EGFP-positive dsRed2-negative mature T cells, indicating that differentiation was not the primary mechanism of T-ALL regression. Bar, 1 mm. (I) After T-ALL development, zebrafish were either removed from 4HT to down-regulate the MYC transgene (−4HT) or kept in 4HT (+4HT), and tumor phenotype was assessed 8 wk after 4HT removal. Zebrafish that became moribund with leukemia before the 8-wk time point were euthanized and classified into the progression category. Number of fish analyzed per condition: −4HT, n = 8; +4HT, n = 13.
    Figure Legend Snippet: Conditional T-ALL development in rag2:MYC-ER transgenic zebrafish. (A and B) Thymic fluorescence in control MYC-ER–negative lck-EGFP transgenic (A) and rag2:dsRed2 transgenic (B) zebrafish raised in the absence of 4HT. (C and D) Thymic fluorescence in the absence of 4HT treatment in rag2:MYC-ER transgenic zebrafish that also expressed lck-EGFP (C) or rag2:dsRed2 (D). For A–D, one representative zebrafish is shown from a minimum of eight fish raised in each condition. (E and F) Fully penetrant T-ALL in rag2:MYC-ER transgenic zebrafish raised in 50 µg/liter (129 nM) 4HT. A representative triple-transgenic rag2:MYC-ER; lck:EGFP; rag2:dsRed2 zebrafish is shown at the time of disseminated T-ALL development, imaged in both green and red fluorescent channels. (G and H) Thymic fluorescence in the rag2:MYC-ER transgenic zebrafish from E and F, shown 4 wk after 4HT removal. In all triple-transgenic zebrafish in which regression occurred ( n = 6 of 8), T-ALL regression occurred simultaneously in both green and red fluorescent channels, with no evidence of residual EGFP-positive dsRed2-negative mature T cells, indicating that differentiation was not the primary mechanism of T-ALL regression. Bar, 1 mm. (I) After T-ALL development, zebrafish were either removed from 4HT to down-regulate the MYC transgene (−4HT) or kept in 4HT (+4HT), and tumor phenotype was assessed 8 wk after 4HT removal. Zebrafish that became moribund with leukemia before the 8-wk time point were euthanized and classified into the progression category. Number of fish analyzed per condition: −4HT, n = 8; +4HT, n = 13.

    Techniques Used: Transgenic Assay, Fluorescence, Fluorescence In Situ Hybridization

    Expression of activated Akt does not alter the transcriptional activity of MYC. (A) Whole mount RNA in situ hybridization for ddx18 , a known direct target of MYC in mammalian cells ( Grandori et al., 1996 ; O’Hagan et al., 2000 ), performed at the 24-h postfertilization developmental stage in zebrafish embryos that were injected with 100 pg mCherry (control) or Myc mRNA at the one-cell stage. One representative zebrafish is shown in each condition out of a minimum of 20 embryos analyzed per condition. (B) Q-RT-PCR for ddx18 expression, performed using RNA from T-ALL cells from rag2:MYC-ER;rag2:EGFP-bcl2 zebrafish which also expressed either a rag2:GFP or a rag2:myr-mAkt2 transgene. T-ALL cells were sorted from animals in 4HT (+4HT) or 4 d after 4HT removal (−4HT). β-Actin was used as the Q-RT-PCR control. Bcl2-transgenic T-ALL cells were used in all conditions to avoid comparing live versus dying cells after MYC-ER inactivation. Error bars represent standard error of the mean. Number of tumors analyzed per group: 4HT+, myr-mAkt2−, n = 6; 4HT−, myr-mAkt2−, n = 7; 4HT+, myr-mAkt2+, n = 5; 4HT−, myr-mAkt2+, n = 5.
    Figure Legend Snippet: Expression of activated Akt does not alter the transcriptional activity of MYC. (A) Whole mount RNA in situ hybridization for ddx18 , a known direct target of MYC in mammalian cells ( Grandori et al., 1996 ; O’Hagan et al., 2000 ), performed at the 24-h postfertilization developmental stage in zebrafish embryos that were injected with 100 pg mCherry (control) or Myc mRNA at the one-cell stage. One representative zebrafish is shown in each condition out of a minimum of 20 embryos analyzed per condition. (B) Q-RT-PCR for ddx18 expression, performed using RNA from T-ALL cells from rag2:MYC-ER;rag2:EGFP-bcl2 zebrafish which also expressed either a rag2:GFP or a rag2:myr-mAkt2 transgene. T-ALL cells were sorted from animals in 4HT (+4HT) or 4 d after 4HT removal (−4HT). β-Actin was used as the Q-RT-PCR control. Bcl2-transgenic T-ALL cells were used in all conditions to avoid comparing live versus dying cells after MYC-ER inactivation. Error bars represent standard error of the mean. Number of tumors analyzed per group: 4HT+, myr-mAkt2−, n = 6; 4HT−, myr-mAkt2−, n = 7; 4HT+, myr-mAkt2+, n = 5; 4HT−, myr-mAkt2+, n = 5.

    Techniques Used: Expressing, Activity Assay, RNA In Situ Hybridization, Injection, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay

    5) Product Images from "Suppression of type I interferon signaling overcomes oncogene-induced senescence and mediates melanoma development and progression"

    Article Title: Suppression of type I interferon signaling overcomes oncogene-induced senescence and mediates melanoma development and progression

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.03.006

    Suppression of IFN signaling is sufficient for Braf V600E ) A. Macroscopic appearance of tumor lesions and the sentinel lymph nodes developing in 4HT treated Braf CA/+ ; Ifnar1 −/− mice. B. H E staining of ear (left panel), lesions (middle panel) and immunohistochemistry staining of S100 in the lesions (right panel) from in Braf V600E ; Ifnar1 −/− mice. Magnification bar: 100 μm. C. Incidence of malignant melanomas (left panel) and Kaplan-Meier analysis of survival (right panel) of mice of indicated genotypes at indicated time after 4HT treatment. D. H E staining of lymph node and lung tissues from Braf V600E ; Ifnar1 −/− mice. Magnification bar: 100 μm.
    Figure Legend Snippet: Suppression of IFN signaling is sufficient for Braf V600E ) A. Macroscopic appearance of tumor lesions and the sentinel lymph nodes developing in 4HT treated Braf CA/+ ; Ifnar1 −/− mice. B. H E staining of ear (left panel), lesions (middle panel) and immunohistochemistry staining of S100 in the lesions (right panel) from in Braf V600E ; Ifnar1 −/− mice. Magnification bar: 100 μm. C. Incidence of malignant melanomas (left panel) and Kaplan-Meier analysis of survival (right panel) of mice of indicated genotypes at indicated time after 4HT treatment. D. H E staining of lymph node and lung tissues from Braf V600E ; Ifnar1 −/− mice. Magnification bar: 100 μm.

    Techniques Used: Mouse Assay, Staining, Immunohistochemistry

    6) Product Images from "Pharmacokinetics of endoxifen and tamoxifen in female mice: implications for comparative in vivo activity studies"

    Article Title: Pharmacokinetics of endoxifen and tamoxifen in female mice: implications for comparative in vivo activity studies

    Journal: Cancer chemotherapy and pharmacology

    doi: 10.1007/s00280-014-2605-7

    Plasma profiles for average ( n = 3) TAM ( closed circle ), 4HT ( open circle ), NDMT ( closed inverted triangle ) and ENDX ( open triangle ) following a s.c. and b oral administration of 500 μg (20 mg/kg) TAM
    Figure Legend Snippet: Plasma profiles for average ( n = 3) TAM ( closed circle ), 4HT ( open circle ), NDMT ( closed inverted triangle ) and ENDX ( open triangle ) following a s.c. and b oral administration of 500 μg (20 mg/kg) TAM

    Techniques Used:

    7) Product Images from "RIPK1- and RIPK3-induced cell death mode is determined by target availability"

    Article Title: RIPK1- and RIPK3-induced cell death mode is determined by target availability

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.70

    RIPK3 gyrase can cause cell death by a mechanism that requires neither caspase 8 nor RIPK1 (necroptosis). ( a ) Western blot analysis of lysates from caspase 8 −/− MEFs bearing the inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3, and reprobed with antibodies to β -actin as a loading control. ( b ) Caspase 8 −/− MEFs bearing the inducible FLAG-RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT and 800 nM C, with or without 10 μ M QVD, for 24 h. Viability of the cells was determined by PI exclusion. Mean±S.E.M. of five independently performed experiments are shown. Predictably, QVD was unable to prevent death of the caspase 8 −/− MEFs, indicating that RIPK3 was activating another death mechanism, presumably necroptosis. ( c ) Western blot of lysates of L929 cells bearing the inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3, and reprobed with antibodies to β -actin as a loading control. ( d ) L929 cells bearing the inducible FLAG-RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT, 800 nM C, 10 μ M QVD and 100 ng/ml TNF with or without 50 μ M nec-1 for 24 h, and cell viability was determined by PI exclusion. Mean±S.E.M. of five independently performed experiments are shown. Dimerized RIPK3 caused death of the L929 cells, and this was not inhibited by QVD, or by the same doses of nec-1 that were able to block necroptosis induced by TNF. ( e ) Western blot analysis of lysates from Fadd −/− MEFs bearing a 4HT-inducible RIPK3 gyrase construct and a doxycycline (dox)-inducible MLKL construct in the absence or presence (+) of 16 nM 4HT with or without 100 ng/ml dox for 6 h. The same blot was probed sequentially with antibodies to RIPK3, MLKL and β -actin as a loading control. Note that these FADD −/− MEFs do not express detectable levels of endogenous MLKL. ( f ) Fadd −/− MEFs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 100 ng/ml dox, with or without 10 μ M QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of six independently performed experiments are shown. Dimerized RIPK3 was not able to cause death of the cells unless MLKL was also induced, and the death that occurred was not inhibited by QVD, and was therefore presumably necroptosis. ( g ) Western blot analysis of lysates from a Ripk1 −/− MEF line (3.6) bearing a 4HT-inducible RIPK3 gyrase construct and a dox-inducible MLKL construct in the absence or presence (+) of 16 nM 4HT with or without 100 ng/ml dox for 6 h. The same blot was probed sequentially with antibodies to MLKL, RIPK3 and β- actin as a loading control. ( h ) Ripk1 −/− 3.6 MEFs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 100 ng/ml dox, with or without 10 μ M QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. Dimerized RIPK3 was able to cause some death of the Ripk1 −/− cells, whereas MLKL alone had no effect. When MLKL and RIPK3 dimers were induced together, more cell death occurred, and this was not blocked by QVD
    Figure Legend Snippet: RIPK3 gyrase can cause cell death by a mechanism that requires neither caspase 8 nor RIPK1 (necroptosis). ( a ) Western blot analysis of lysates from caspase 8 −/− MEFs bearing the inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3, and reprobed with antibodies to β -actin as a loading control. ( b ) Caspase 8 −/− MEFs bearing the inducible FLAG-RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT and 800 nM C, with or without 10 μ M QVD, for 24 h. Viability of the cells was determined by PI exclusion. Mean±S.E.M. of five independently performed experiments are shown. Predictably, QVD was unable to prevent death of the caspase 8 −/− MEFs, indicating that RIPK3 was activating another death mechanism, presumably necroptosis. ( c ) Western blot of lysates of L929 cells bearing the inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3, and reprobed with antibodies to β -actin as a loading control. ( d ) L929 cells bearing the inducible FLAG-RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT, 800 nM C, 10 μ M QVD and 100 ng/ml TNF with or without 50 μ M nec-1 for 24 h, and cell viability was determined by PI exclusion. Mean±S.E.M. of five independently performed experiments are shown. Dimerized RIPK3 caused death of the L929 cells, and this was not inhibited by QVD, or by the same doses of nec-1 that were able to block necroptosis induced by TNF. ( e ) Western blot analysis of lysates from Fadd −/− MEFs bearing a 4HT-inducible RIPK3 gyrase construct and a doxycycline (dox)-inducible MLKL construct in the absence or presence (+) of 16 nM 4HT with or without 100 ng/ml dox for 6 h. The same blot was probed sequentially with antibodies to RIPK3, MLKL and β -actin as a loading control. Note that these FADD −/− MEFs do not express detectable levels of endogenous MLKL. ( f ) Fadd −/− MEFs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 100 ng/ml dox, with or without 10 μ M QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of six independently performed experiments are shown. Dimerized RIPK3 was not able to cause death of the cells unless MLKL was also induced, and the death that occurred was not inhibited by QVD, and was therefore presumably necroptosis. ( g ) Western blot analysis of lysates from a Ripk1 −/− MEF line (3.6) bearing a 4HT-inducible RIPK3 gyrase construct and a dox-inducible MLKL construct in the absence or presence (+) of 16 nM 4HT with or without 100 ng/ml dox for 6 h. The same blot was probed sequentially with antibodies to MLKL, RIPK3 and β- actin as a loading control. ( h ) Ripk1 −/− 3.6 MEFs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 100 ng/ml dox, with or without 10 μ M QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. Dimerized RIPK3 was able to cause some death of the Ripk1 −/− cells, whereas MLKL alone had no effect. When MLKL and RIPK3 dimers were induced together, more cell death occurred, and this was not blocked by QVD

    Techniques Used: Western Blot, Construct, Blocking Assay

    Activation of RIPK3 can trigger caspase-dependent apoptosis as well as MLKL-dependent necroptosis. ( a ) Western blot analysis of lysates from WT and Ripk1 −/− 1.7 MEF lines bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3 and reprobed with antibodies to β -actin as a loading control. ( b ) WT (dark gray columns; data from three independent cell lines) and Ripk1 −/− MEFs (light gray columns; data combined from lines 1.7 and 2.6) were either untreated or treated with combinations of 16 nM 4HT, 800 nM C, 50 μ M nec-1 and/or 10 μ M QVD for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of 3–5 independently performed experiments are shown. RIPK3 was able to cause death of both WT and Ripk1 −/− cells, but it caused more cell death in the presence of RIPK1. In neither case was the death affected by nec-1, but it was reduced by QVD, indicating that RIPK3 can trigger a caspase-dependent death process, but it does so more efficiently in cells expressing RIPK1. ( c ) Western blot analysis of lysates from Mlkl −/− MEFs bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3 and reprobed with antibodies to β -actin as a loading control. ( d ) Mlkl −/− MEFs were either untreated or treated with combinations of 16 nM 4HT and 800 nM C, with or without 50 μ M nec-1 and/or 10 μ M QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of nine independently performed experiments are shown. RIPK3 was able to cause death of Mlkl −/− cells that was completely blocked by QVD, indicating that in the absence of MLKL, activated RIPK3 causes only caspase-dependent cell death. ( e ) WT and Ripk1 −/− MEFs bearing the RIPK3 gyrase construct were treated with 10 nM 4HT for 24 h and/or 700 nM C for 2 h. Lysates were run on replicate blots and were probed with antibodies to full-length caspase 8, processed caspase 8, processed caspase 3 and PARP. The full-length caspase 8 blots were washed and re-probed for β -actin to act as a loading control. ( f ) Western blot analysis of lysates from L929 cells and WT MEF lines bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT with or without 800 nM C for 5 h. The same blots were probed sequentially with antibodies to PARP, pro-caspase 8 and β -actin as a loading control. Dimerized RIPK3 gyrase can activate caspase 8 (but not detectably in L929 cells) and lead to processing of downstream caspases and PARP, and it does so more efficiently in the presence of RIPK1
    Figure Legend Snippet: Activation of RIPK3 can trigger caspase-dependent apoptosis as well as MLKL-dependent necroptosis. ( a ) Western blot analysis of lysates from WT and Ripk1 −/− 1.7 MEF lines bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3 and reprobed with antibodies to β -actin as a loading control. ( b ) WT (dark gray columns; data from three independent cell lines) and Ripk1 −/− MEFs (light gray columns; data combined from lines 1.7 and 2.6) were either untreated or treated with combinations of 16 nM 4HT, 800 nM C, 50 μ M nec-1 and/or 10 μ M QVD for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of 3–5 independently performed experiments are shown. RIPK3 was able to cause death of both WT and Ripk1 −/− cells, but it caused more cell death in the presence of RIPK1. In neither case was the death affected by nec-1, but it was reduced by QVD, indicating that RIPK3 can trigger a caspase-dependent death process, but it does so more efficiently in cells expressing RIPK1. ( c ) Western blot analysis of lysates from Mlkl −/− MEFs bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3 and reprobed with antibodies to β -actin as a loading control. ( d ) Mlkl −/− MEFs were either untreated or treated with combinations of 16 nM 4HT and 800 nM C, with or without 50 μ M nec-1 and/or 10 μ M QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of nine independently performed experiments are shown. RIPK3 was able to cause death of Mlkl −/− cells that was completely blocked by QVD, indicating that in the absence of MLKL, activated RIPK3 causes only caspase-dependent cell death. ( e ) WT and Ripk1 −/− MEFs bearing the RIPK3 gyrase construct were treated with 10 nM 4HT for 24 h and/or 700 nM C for 2 h. Lysates were run on replicate blots and were probed with antibodies to full-length caspase 8, processed caspase 8, processed caspase 3 and PARP. The full-length caspase 8 blots were washed and re-probed for β -actin to act as a loading control. ( f ) Western blot analysis of lysates from L929 cells and WT MEF lines bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT with or without 800 nM C for 5 h. The same blots were probed sequentially with antibodies to PARP, pro-caspase 8 and β -actin as a loading control. Dimerized RIPK3 gyrase can activate caspase 8 (but not detectably in L929 cells) and lead to processing of downstream caspases and PARP, and it does so more efficiently in the presence of RIPK1

    Techniques Used: Activation Assay, Western Blot, Construct, Expressing, Activated Clotting Time Assay

    MEF lines and L929 cells express varying amounts of cell death proteins. ( a ) Lysates were prepared from WT and gene-deleted MEF lines as well as L929 mouse fibroblastoid cells, and identical aliquots were run on three replicate gels and, after blotting, were probed with antibodies to the indicated proteins. The gene-deleted lines lacked expression of the corresponding proteins, but, in addition, the Fadd −/− MEFs expressed barely detectable levels of MLKL, the Ripk1 −/− line 3.6 expressed very low levels of MLKL, the Ripk3 −/− line had low levels of RIPK1 and the caspase 8 −/− line did not express detectable levels of RIPK3. ( b ) TNF alone causes death of L929 cells, strongly inhibited by nec-1 but unaffected by QVD. Death of Mlkl −/− MEFs caused by dimerization of RIPK3 gyrase is strongly inhibited by QVD but not by nec-1. L929 cells were treated with 100 ng/ml human Fc-TNF and Mlkl −/− cells were treated with 16 nM 4HT and 800 nM C for 24 h in the presence or absence of 50 μ M nec-1 or 10 μ M QVD. Viability was determined by PI exclusion on a flow cytometer. Triangles and circles indicate independently performed experiments, and error bars show S.E.M. values
    Figure Legend Snippet: MEF lines and L929 cells express varying amounts of cell death proteins. ( a ) Lysates were prepared from WT and gene-deleted MEF lines as well as L929 mouse fibroblastoid cells, and identical aliquots were run on three replicate gels and, after blotting, were probed with antibodies to the indicated proteins. The gene-deleted lines lacked expression of the corresponding proteins, but, in addition, the Fadd −/− MEFs expressed barely detectable levels of MLKL, the Ripk1 −/− line 3.6 expressed very low levels of MLKL, the Ripk3 −/− line had low levels of RIPK1 and the caspase 8 −/− line did not express detectable levels of RIPK3. ( b ) TNF alone causes death of L929 cells, strongly inhibited by nec-1 but unaffected by QVD. Death of Mlkl −/− MEFs caused by dimerization of RIPK3 gyrase is strongly inhibited by QVD but not by nec-1. L929 cells were treated with 100 ng/ml human Fc-TNF and Mlkl −/− cells were treated with 16 nM 4HT and 800 nM C for 24 h in the presence or absence of 50 μ M nec-1 or 10 μ M QVD. Viability was determined by PI exclusion on a flow cytometer. Triangles and circles indicate independently performed experiments, and error bars show S.E.M. values

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    K51A mutant of RIPK3 gyrase is catalytically inactive but apparently unstable. ( a ) WT and K51A RIPK3 gyrase were immunoprecipitated using anti-FLAG coupled beads from cells expressing the respective constructs, and used in in vitro kinase assays to show whether they were able to auto-phosphorylate. Washed beads were resuspended in 30 μ l of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MnCl 2 and 1 mM DTT), divided into two aliquots that were incubated in the presence versus absence of 250 μ M ATP at 30°C for 20 min and analyzed by electrophoresis followed by replicate western blots that were probed with anti-phosphothreonine and anti-RIPK3. ( b ) WT, Mlkl −/− , Ripk3 −/− , caspase 8 −/− MEFs and L929 cells bearing WT or K51A (kinase dead) RIPK3 gyrase constructs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 10 μ M QVD for 16 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. The kinase-mutant K51A RIPK3 was unable to cause statistically significant levels of cell death in the apoptosis-prone Mlkl −/− , or in caspase 8 −/− cells (that lack detectable RIPK3). It appeared to induce death of a small number of WT and Ripk3 −/− cells, but it was only able to cause convincing levels of cell death in L929 cells that express endogenous RIPK3 and show reduced activation of caspase 8. The death in L929 cells could not be blocked by QVD, and hence it is presumed to be necroptosis. Note the low levels of expression of K51A relative to WT in ( a ), ( c ) and track 6 of ( d ). ( c ) Western blots of lysates from MEFs bearing WT or K51A (kinase dead) RIPK3 gyrase constructs that were untreated or treated with 16 nM 4HT and 700 nM C for 6 h were probed sequentially with antibodies to caspase 8, PARP and RIPK3, followed by β -actin as loading controls. The asterisk indicates a nonspecific band. ( d ) Western blots of lysates from WT, Mlkl −/− , Ripk3 −/− , caspase 8 −/− MEFs and L929 cells bearing the K51A (kinase dead) RIPK3 gyrase construct, and WT and L929 cells bearing the WT RIPK3 gyrase were untreated or treated with 16 nM 4HT for 6 h and probed with antibodies to RIPK3 followed by β -actin as loading controls
    Figure Legend Snippet: K51A mutant of RIPK3 gyrase is catalytically inactive but apparently unstable. ( a ) WT and K51A RIPK3 gyrase were immunoprecipitated using anti-FLAG coupled beads from cells expressing the respective constructs, and used in in vitro kinase assays to show whether they were able to auto-phosphorylate. Washed beads were resuspended in 30 μ l of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MnCl 2 and 1 mM DTT), divided into two aliquots that were incubated in the presence versus absence of 250 μ M ATP at 30°C for 20 min and analyzed by electrophoresis followed by replicate western blots that were probed with anti-phosphothreonine and anti-RIPK3. ( b ) WT, Mlkl −/− , Ripk3 −/− , caspase 8 −/− MEFs and L929 cells bearing WT or K51A (kinase dead) RIPK3 gyrase constructs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 10 μ M QVD for 16 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. The kinase-mutant K51A RIPK3 was unable to cause statistically significant levels of cell death in the apoptosis-prone Mlkl −/− , or in caspase 8 −/− cells (that lack detectable RIPK3). It appeared to induce death of a small number of WT and Ripk3 −/− cells, but it was only able to cause convincing levels of cell death in L929 cells that express endogenous RIPK3 and show reduced activation of caspase 8. The death in L929 cells could not be blocked by QVD, and hence it is presumed to be necroptosis. Note the low levels of expression of K51A relative to WT in ( a ), ( c ) and track 6 of ( d ). ( c ) Western blots of lysates from MEFs bearing WT or K51A (kinase dead) RIPK3 gyrase constructs that were untreated or treated with 16 nM 4HT and 700 nM C for 6 h were probed sequentially with antibodies to caspase 8, PARP and RIPK3, followed by β -actin as loading controls. The asterisk indicates a nonspecific band. ( d ) Western blots of lysates from WT, Mlkl −/− , Ripk3 −/− , caspase 8 −/− MEFs and L929 cells bearing the K51A (kinase dead) RIPK3 gyrase construct, and WT and L929 cells bearing the WT RIPK3 gyrase were untreated or treated with 16 nM 4HT for 6 h and probed with antibodies to RIPK3 followed by β -actin as loading controls

    Techniques Used: Mutagenesis, Immunoprecipitation, Expressing, Construct, In Vitro, Incubation, Electrophoresis, Western Blot, Activation Assay

    The kinase activity of RIPK3 is needed for it to cause necrosis but not apoptosis. ( a and b ) Recombinant D143N and R142G mutant mRIPK3 kinase domains are catalytically defective. ( a ) A total volume of 1 μ g of wild-type (WT), D143N and R142G mRIPK3 kinase domain, which had been expressed in insect cells and purified, was resolved by reducing SDS-PAGE and stained with Coomassie Blue. Comparable intensity of Coomassie-stained bands confirmed reliability of concentration estimation by Absorbance at 280 nm. These concentration estimates were used subsequently to ensure that comparable quantities of WT and mutant RIPK3 were assayed in in vitro kinase assays. ( b ) In vitro whereas R142G mRIPK3 exhibits defective catalytic activity in autophosphorylation and MLKL phosphorylation. The shown experiment is representative of two independent experiments. BSA was added as a carrier protein (100 ng/ μ l). The Coomassie-stained image of the representative gel is shown on the left and the autoradiograph of the same gel on the right. Only 25 ng of each recombinant RIPK3 kinase domain was loaded in each experiment and is consequently not visible by Coomassie Blue staining. ( c ) Western blots of lysates from MEFs bearing WT, D143N (kinase dead) and R142G (kinase compromised) RIPK3 gyrase constructs that were untreated or treated with 16 nM 4HT and 800 nM C for 6 h were probed sequentially with antibodies to PARP and RIPK3, followed by β -actin as loading controls. The asterisks indicate nonspecific bands. Although a background level of cleaved PARP is detectable in all cell types in this experiment, the band intensity increases with induction of expression and dimerization of RIPK3 gyrase only in Ripk3 −/− and Mlkl −/− cells, in which caspase-dependent apoptosis occurs (see f and g ). ( d – g ) Kinase-inactive or -compromised RIPK3 gyrase can induce apoptosis, but cannot induce necroptosis without the presence of (endogenous) WT RIPK3. ( d ) Caspase 8 −/− cells bearing the inducible RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 10 μ M QVD. Mean±S.E.M. of four independently performed experiments are shown. ( e ) In the presence of WT RIPK3, kinase-inactive RIPK3 gyrase induces necroptosis. L929 cells bearing the inducible RIPK3-gyrase construct were either untreated or treated as in ( d ), or with the addition of nec-1. Mean±S.E.M. of four independently performed experiments are shown. The R142G mutant was as effective as the WT, and even the D143N mutant induced some cell death. ( f ) Apoptosis induced by dimerized RIPK3 is independent of its kinase activity. Mlkl −/− cells bearing the respective RIPK3 gyrase constructs were untreated or treated as in ( e ). Mean±S.E.M. of three independently performed experiments are shown. Both R142G and D143N mutants caused the death of substantial numbers of cells, almost equivalent to WT. ( g ) Apoptosis induced by dimerized RIPK3 is independent of bystander RIPK3. Ripk3 −/− MEFs bearing the respective RIPK3 gyrase constructs were untreated or treated as in ( e ). Mean±S.E.M. of three independently performed experiments are shown. Again, cell death was substantial in response to the WT and each of the mutants
    Figure Legend Snippet: The kinase activity of RIPK3 is needed for it to cause necrosis but not apoptosis. ( a and b ) Recombinant D143N and R142G mutant mRIPK3 kinase domains are catalytically defective. ( a ) A total volume of 1 μ g of wild-type (WT), D143N and R142G mRIPK3 kinase domain, which had been expressed in insect cells and purified, was resolved by reducing SDS-PAGE and stained with Coomassie Blue. Comparable intensity of Coomassie-stained bands confirmed reliability of concentration estimation by Absorbance at 280 nm. These concentration estimates were used subsequently to ensure that comparable quantities of WT and mutant RIPK3 were assayed in in vitro kinase assays. ( b ) In vitro whereas R142G mRIPK3 exhibits defective catalytic activity in autophosphorylation and MLKL phosphorylation. The shown experiment is representative of two independent experiments. BSA was added as a carrier protein (100 ng/ μ l). The Coomassie-stained image of the representative gel is shown on the left and the autoradiograph of the same gel on the right. Only 25 ng of each recombinant RIPK3 kinase domain was loaded in each experiment and is consequently not visible by Coomassie Blue staining. ( c ) Western blots of lysates from MEFs bearing WT, D143N (kinase dead) and R142G (kinase compromised) RIPK3 gyrase constructs that were untreated or treated with 16 nM 4HT and 800 nM C for 6 h were probed sequentially with antibodies to PARP and RIPK3, followed by β -actin as loading controls. The asterisks indicate nonspecific bands. Although a background level of cleaved PARP is detectable in all cell types in this experiment, the band intensity increases with induction of expression and dimerization of RIPK3 gyrase only in Ripk3 −/− and Mlkl −/− cells, in which caspase-dependent apoptosis occurs (see f and g ). ( d – g ) Kinase-inactive or -compromised RIPK3 gyrase can induce apoptosis, but cannot induce necroptosis without the presence of (endogenous) WT RIPK3. ( d ) Caspase 8 −/− cells bearing the inducible RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 10 μ M QVD. Mean±S.E.M. of four independently performed experiments are shown. ( e ) In the presence of WT RIPK3, kinase-inactive RIPK3 gyrase induces necroptosis. L929 cells bearing the inducible RIPK3-gyrase construct were either untreated or treated as in ( d ), or with the addition of nec-1. Mean±S.E.M. of four independently performed experiments are shown. The R142G mutant was as effective as the WT, and even the D143N mutant induced some cell death. ( f ) Apoptosis induced by dimerized RIPK3 is independent of its kinase activity. Mlkl −/− cells bearing the respective RIPK3 gyrase constructs were untreated or treated as in ( e ). Mean±S.E.M. of three independently performed experiments are shown. Both R142G and D143N mutants caused the death of substantial numbers of cells, almost equivalent to WT. ( g ) Apoptosis induced by dimerized RIPK3 is independent of bystander RIPK3. Ripk3 −/− MEFs bearing the respective RIPK3 gyrase constructs were untreated or treated as in ( e ). Mean±S.E.M. of three independently performed experiments are shown. Again, cell death was substantial in response to the WT and each of the mutants

    Techniques Used: Activity Assay, Recombinant, Mutagenesis, Purification, SDS Page, Staining, Concentration Assay, In Vitro, Autoradiography, Western Blot, Construct, Expressing

    Expression and dimerization of RIPK1 gyrase and RIPK3 gyrase is sufficient to cause death of MEFs. ( a The divalent antibiotic coumermycin (C) dimerizes the gyrase B domains. ( b ) Western blots of lysates from MEFs bearing the inducible RIPK1 gyrase or RIPK3 gyrase constructs in the absence or presence (+) of 16 nM 4HT for 6 h were probed with antibodies to RIPK1 or RIPK3. The same blots were probed with antibodies to β- actin as a loading control, either simultaneously, in the case of the RIPK1 blot, or sequentially in the case of the RIPK3 blot. Endogenous and induced fusion proteins are labeled. ( c ) Analysis of cell viability by propidium iodide exclusion in untreated cells, and those treated for 24 h with 800 nM C alone, 16 nM 4HT alone or 4HT+C. PI-negative (viable) cells are boxed, and their percentages are indicated. Although induction of RIPK1 gyrase alone caused some cell death (depending on expression levels), addition of coumermycin greatly increased the death of cells expressing either the RIPK1 gyrase or RIPK3 gyrase proteins
    Figure Legend Snippet: Expression and dimerization of RIPK1 gyrase and RIPK3 gyrase is sufficient to cause death of MEFs. ( a The divalent antibiotic coumermycin (C) dimerizes the gyrase B domains. ( b ) Western blots of lysates from MEFs bearing the inducible RIPK1 gyrase or RIPK3 gyrase constructs in the absence or presence (+) of 16 nM 4HT for 6 h were probed with antibodies to RIPK1 or RIPK3. The same blots were probed with antibodies to β- actin as a loading control, either simultaneously, in the case of the RIPK1 blot, or sequentially in the case of the RIPK3 blot. Endogenous and induced fusion proteins are labeled. ( c ) Analysis of cell viability by propidium iodide exclusion in untreated cells, and those treated for 24 h with 800 nM C alone, 16 nM 4HT alone or 4HT+C. PI-negative (viable) cells are boxed, and their percentages are indicated. Although induction of RIPK1 gyrase alone caused some cell death (depending on expression levels), addition of coumermycin greatly increased the death of cells expressing either the RIPK1 gyrase or RIPK3 gyrase proteins

    Techniques Used: Expressing, Western Blot, Construct, Labeling

    In the absence of both caspase 8 and MLKL, activated RIPK3 fails to induce death. ( a ) Western blot analysis of lysates from WT, caspase 8 −/− Ripk3 −/− and caspase 8 −/− Mlkl −/− mouse dermal fibroblasts (MDFs) either untreated or treated (+) with 10 nM 4HT for 24 h. The blots were probed for FLAG followed by β -actin as a loading control. ( b ) WT, caspase 8 −/− Ripk3 −/− and caspase 8 −/− Mlkl −/− MDFs were either untreated or treated with combinations of 10 nM 4HT, 700 nM C and 10 μ M QVD for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. Dimerized RIPK3 was able to cause some death of the WT cells, and this could be partly blocked by QVD. When both caspase 8 and MLKL were absent, no death was induced
    Figure Legend Snippet: In the absence of both caspase 8 and MLKL, activated RIPK3 fails to induce death. ( a ) Western blot analysis of lysates from WT, caspase 8 −/− Ripk3 −/− and caspase 8 −/− Mlkl −/− mouse dermal fibroblasts (MDFs) either untreated or treated (+) with 10 nM 4HT for 24 h. The blots were probed for FLAG followed by β -actin as a loading control. ( b ) WT, caspase 8 −/− Ripk3 −/− and caspase 8 −/− Mlkl −/− MDFs were either untreated or treated with combinations of 10 nM 4HT, 700 nM C and 10 μ M QVD for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. Dimerized RIPK3 was able to cause some death of the WT cells, and this could be partly blocked by QVD. When both caspase 8 and MLKL were absent, no death was induced

    Techniques Used: Western Blot

    8) Product Images from "Imprinted CDKN1C Is a Tumor Suppressor in Rhabdoid Tumor and Activated by Restoration of SMARCB1 and Histone Deacetylase Inhibitors"

    Article Title: Imprinted CDKN1C Is a Tumor Suppressor in Rhabdoid Tumor and Activated by Restoration of SMARCB1 and Histone Deacetylase Inhibitors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004482

    SMARCB1 induces CDKN1C expression in rhabdoid tumor cell lines. (A). Western blot showing induction of SMARCB1 protein in G401 clones F13 and F22, and in a transduced pool of STM91-01 cells, STMpc. Lanes show protein in un-induced (−) cells and in cells induced with 4HT (+). (B) Gene expression examined by RT-PCR for CDKN1C, CDKN1B, CDKN1A and the HPRT control gene in un-induced (−) and in 4HT-induced (+) cells. The -ve lane represents the PCR negative control, the RNA –ve control lane represents the negative control for reverse transcription and the RT-ve control represents a control for genomic contamination derived from the induced F22 sample. CDKN1C was amplified for 40 cycles and CDKN1B and CDKN1A for 28 cycles. All primers were located in separate exons. (C) CDKN1C expression normalized to the GUSB control gene, derived by real-time quantitative pcr in un-induced (−4HT) and in induced (+4HT) cultures of F22 cells (upper panel) and in cultures of STMpc cells (lower panel). The data represent the mean of three independent experiments and the error bars represent the standard error of the mean. (D) Western blot showing expression of endogenous CDKN1C protein pre and post SMARCB1 induction with 4HT. Immunoblotting was performed with the p57 Kip2 antibody from Cell Signaling Technologies with an exposure time of 2 minutes.
    Figure Legend Snippet: SMARCB1 induces CDKN1C expression in rhabdoid tumor cell lines. (A). Western blot showing induction of SMARCB1 protein in G401 clones F13 and F22, and in a transduced pool of STM91-01 cells, STMpc. Lanes show protein in un-induced (−) cells and in cells induced with 4HT (+). (B) Gene expression examined by RT-PCR for CDKN1C, CDKN1B, CDKN1A and the HPRT control gene in un-induced (−) and in 4HT-induced (+) cells. The -ve lane represents the PCR negative control, the RNA –ve control lane represents the negative control for reverse transcription and the RT-ve control represents a control for genomic contamination derived from the induced F22 sample. CDKN1C was amplified for 40 cycles and CDKN1B and CDKN1A for 28 cycles. All primers were located in separate exons. (C) CDKN1C expression normalized to the GUSB control gene, derived by real-time quantitative pcr in un-induced (−4HT) and in induced (+4HT) cultures of F22 cells (upper panel) and in cultures of STMpc cells (lower panel). The data represent the mean of three independent experiments and the error bars represent the standard error of the mean. (D) Western blot showing expression of endogenous CDKN1C protein pre and post SMARCB1 induction with 4HT. Immunoblotting was performed with the p57 Kip2 antibody from Cell Signaling Technologies with an exposure time of 2 minutes.

    Techniques Used: Expressing, Western Blot, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Derivative Assay, Amplification, Real-time Polymerase Chain Reaction

    SMARCB1 and HDACi have identical effects on allelic expression of CDKN1C. (A) CDKN1C allelic expression in un-induced and in induced cultures of G401, F22, F13, STM91-01 and in STMpc cells. Changes in the pattern of allelic expression can be seen in F22 and in F13 cells following induction of SMARCB1 expression with 1 uM 4HT. Wild-type G401 cells did not show any change in allelic expression following 4HT treatment. Biallelic CDKN1C expression persisted in STM91-01 cells and in STMpc cells. (B) CDKN1C allelic expression in un-induced cultures of F22 and F13 with 0.01% DMSO and 10 nM Romidepsin and in G401 cells. Changes in allelic expression can be seen with Romidepsin treatment in F13, F22 cells and in wild-type G401 cells. As with SMARCB1 induction in STMpc cells, allelic expression in STM91-01 cells was unchanged following Romidpesin treatment.
    Figure Legend Snippet: SMARCB1 and HDACi have identical effects on allelic expression of CDKN1C. (A) CDKN1C allelic expression in un-induced and in induced cultures of G401, F22, F13, STM91-01 and in STMpc cells. Changes in the pattern of allelic expression can be seen in F22 and in F13 cells following induction of SMARCB1 expression with 1 uM 4HT. Wild-type G401 cells did not show any change in allelic expression following 4HT treatment. Biallelic CDKN1C expression persisted in STM91-01 cells and in STMpc cells. (B) CDKN1C allelic expression in un-induced cultures of F22 and F13 with 0.01% DMSO and 10 nM Romidepsin and in G401 cells. Changes in allelic expression can be seen with Romidepsin treatment in F13, F22 cells and in wild-type G401 cells. As with SMARCB1 induction in STMpc cells, allelic expression in STM91-01 cells was unchanged following Romidpesin treatment.

    Techniques Used: Expressing

    9) Product Images from "Integrated Molecular Analysis of Tamoxifen-Resistant Invasive Lobular Breast Cancer Cells Identifies MAPK and GRM/mGluR Signaling as Therapeutic Vulnerabilities"

    Article Title: Integrated Molecular Analysis of Tamoxifen-Resistant Invasive Lobular Breast Cancer Cells Identifies MAPK and GRM/mGluR Signaling as Therapeutic Vulnerabilities

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2017.09.024

    Response to 4HT differs in SUM44 and LCCTam and suggests targetable pathways of TAM-resistance. A, SUM44 cells were treated with 500 nM 4HT for 24 hours and compared with controls while LCCTam maintained in 500 nM 4HT were compared with cells that were not treated with 4HT for 2 weeks. Affymetrix probe IDs for each comparison that had a fold change of ≥1.45 with a univariate p-value ≤0.05 were compared. B and C, Ingenuity Pathway Analysis (IPA) of Canonical Pathways for each cell line was completed and the top 20 affected pathways are shown; those discussed in the Results section are bolded. Red denotes pathways predicted to be downregulated in response to 4HT while green denotes upregulation.
    Figure Legend Snippet: Response to 4HT differs in SUM44 and LCCTam and suggests targetable pathways of TAM-resistance. A, SUM44 cells were treated with 500 nM 4HT for 24 hours and compared with controls while LCCTam maintained in 500 nM 4HT were compared with cells that were not treated with 4HT for 2 weeks. Affymetrix probe IDs for each comparison that had a fold change of ≥1.45 with a univariate p-value ≤0.05 were compared. B and C, Ingenuity Pathway Analysis (IPA) of Canonical Pathways for each cell line was completed and the top 20 affected pathways are shown; those discussed in the Results section are bolded. Red denotes pathways predicted to be downregulated in response to 4HT while green denotes upregulation.

    Techniques Used: Indirect Immunoperoxidase Assay

    MAPK1 amplification and FOXA1 gain in TAM-resistant ILC cells. A, Copy number plot for parental SUM44 cell line. Log2 copy number (CN) ratios (Y axis) are shown as colored points and the circular binary segmentation (CBS)-generated segments are shown as black lines. Gains and losses characteristic of ILC are shown. B, Copy number alterations in LCCTam vs. SUM44 cells. Genes within largest amplification and deletion as determined by CBS are shown as black squares. C, Table of selected chromosome locations, cytobands, and genes showing significant amplification, gain, or deletion in LCCTam cells as determined by ADM-2. *denotes chromosome 14 gain identified only by ADM-2, whereas others were identified by both CBS and ADM-2. D, Quantitative real-time polymerase chain reaction (RTPCR) validation of increased MAPK1 and FOXA1 mRNA expression in LCCTam cells. Data are presented as relative expression calculated by the 2 −ΔΔCt method and are the mean of 5 biological replicates ± standard deviation, with each biological replicate comprised of 3 technical replicates. Data were analyzed by Mann-Whitney U test. E, Western blot analysis of phosphorylated and total ERK1/2 ( MAPK1 gene = ERK2 protein), phosphorylated and total ER, and GAPDH as a loading control. LCCTam −4HT cells were cultured in the absence of 4HT for 48 hours. Data shown are from a single representative experiment that was performed independently twice.
    Figure Legend Snippet: MAPK1 amplification and FOXA1 gain in TAM-resistant ILC cells. A, Copy number plot for parental SUM44 cell line. Log2 copy number (CN) ratios (Y axis) are shown as colored points and the circular binary segmentation (CBS)-generated segments are shown as black lines. Gains and losses characteristic of ILC are shown. B, Copy number alterations in LCCTam vs. SUM44 cells. Genes within largest amplification and deletion as determined by CBS are shown as black squares. C, Table of selected chromosome locations, cytobands, and genes showing significant amplification, gain, or deletion in LCCTam cells as determined by ADM-2. *denotes chromosome 14 gain identified only by ADM-2, whereas others were identified by both CBS and ADM-2. D, Quantitative real-time polymerase chain reaction (RTPCR) validation of increased MAPK1 and FOXA1 mRNA expression in LCCTam cells. Data are presented as relative expression calculated by the 2 −ΔΔCt method and are the mean of 5 biological replicates ± standard deviation, with each biological replicate comprised of 3 technical replicates. Data were analyzed by Mann-Whitney U test. E, Western blot analysis of phosphorylated and total ERK1/2 ( MAPK1 gene = ERK2 protein), phosphorylated and total ER, and GAPDH as a loading control. LCCTam −4HT cells were cultured in the absence of 4HT for 48 hours. Data shown are from a single representative experiment that was performed independently twice.

    Techniques Used: Amplification, Generated, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Standard Deviation, MANN-WHITNEY, Western Blot, Cell Culture

    NF1 and multiple GRMs/mGluRs are mutated in TAM-resistant ILC cells. A, Distribution of mutations in LCCTam cells by chromosome. Number of mutations per chromosome were analyzed in relation to chromosome length in megabases (Mb) by linear regression analysis. Dashed lines represent the 90%, 95%, and 99% confidence intervals. Red points denote chromosomes (numbers shown) with a greater than expected number of mutations, while green points denote those with fewer than expected. B, Table of selected chromosome locations, exons, alterations, and predicted functional consequences for genes mutated in LCCTam cells. C, Quantitative RTPCR validation of NF1 mRNA expression in LCCTam cells. Data are presented as relative expression calculated by the 2 −ΔΔCt method and are the mean of 5 biological replicates ± standard deviation, with each biological replicate comprised of 3 technical replicates. Data were analyzed by Mann-Whitney U test. D, Lollipop plot showing location of GRM1 mutations in Luminal A ILC from the TCGA dataset, and the A229E mutation detected in LCCTam cells. Green denotes missense and black denotes nonsense mutations. Positions of the extracellular ligand (glutamate) binding domain (green rectangle) and 7-pass transmembrane domain (7Tm, blue rectangle) are shown. E, GRM1 protein expression in SUM44 and LCCTam cells by fluorescent immunocytochemistry. Negative staining control = no Ab (no primary antibody). Data shown are from a single representative experiment that was performed independently twice. F, Quantitative RTPCR validation of GRM2 mRNA expression in LCCTam cells. Data are presented as relative expression calculated by the 2 −ΔΔCt method and are the mean of 3 biological replicates ± standard deviation, with each biological replicate comprised of 3 technical replicates. Data were analyzed by Mann-Whitney U test. G, Multiple GRMs/mGluRs are upregulated in LCCTam cells. Affymetrix gene expression microarray data are presented as mean fold change (increase) in LCCTam −4HT vs. SUM44 cells for three biological replicates compared by unpaired t test.
    Figure Legend Snippet: NF1 and multiple GRMs/mGluRs are mutated in TAM-resistant ILC cells. A, Distribution of mutations in LCCTam cells by chromosome. Number of mutations per chromosome were analyzed in relation to chromosome length in megabases (Mb) by linear regression analysis. Dashed lines represent the 90%, 95%, and 99% confidence intervals. Red points denote chromosomes (numbers shown) with a greater than expected number of mutations, while green points denote those with fewer than expected. B, Table of selected chromosome locations, exons, alterations, and predicted functional consequences for genes mutated in LCCTam cells. C, Quantitative RTPCR validation of NF1 mRNA expression in LCCTam cells. Data are presented as relative expression calculated by the 2 −ΔΔCt method and are the mean of 5 biological replicates ± standard deviation, with each biological replicate comprised of 3 technical replicates. Data were analyzed by Mann-Whitney U test. D, Lollipop plot showing location of GRM1 mutations in Luminal A ILC from the TCGA dataset, and the A229E mutation detected in LCCTam cells. Green denotes missense and black denotes nonsense mutations. Positions of the extracellular ligand (glutamate) binding domain (green rectangle) and 7-pass transmembrane domain (7Tm, blue rectangle) are shown. E, GRM1 protein expression in SUM44 and LCCTam cells by fluorescent immunocytochemistry. Negative staining control = no Ab (no primary antibody). Data shown are from a single representative experiment that was performed independently twice. F, Quantitative RTPCR validation of GRM2 mRNA expression in LCCTam cells. Data are presented as relative expression calculated by the 2 −ΔΔCt method and are the mean of 3 biological replicates ± standard deviation, with each biological replicate comprised of 3 technical replicates. Data were analyzed by Mann-Whitney U test. G, Multiple GRMs/mGluRs are upregulated in LCCTam cells. Affymetrix gene expression microarray data are presented as mean fold change (increase) in LCCTam −4HT vs. SUM44 cells for three biological replicates compared by unpaired t test.

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Standard Deviation, MANN-WHITNEY, Mutagenesis, Binding Assay, Immunocytochemistry, Negative Staining, Microarray

    10) Product Images from "Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology"

    Article Title: Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004099

    Morphologies of AP-labeled neurons in the rostral half of an adult ChAT-IRES-CreER;Z/AP brain. The mouse received injections of 4HT on P8 (1 mg), P21 (2 mg), and P28 (2 mg). A,B, bright field images of AP-stained tissue corresponding to the boxed regions in J and E, respectively. C–M, AP-labeled processes traced from eleven serial 300 um coronal sections. Two adjacent neurons with cell bodies in the basal forebrain (black arrows in panels K and L) and their large axon arbors in the cerebral cortex are shown in red. All other processes are shown in green. Regions in which a high density of AP-labeled processes precluded accurate tracing are marked by semi-opaque grey zones. Scale bar: 0.5 mm in A, 0.2 mm in B.
    Figure Legend Snippet: Morphologies of AP-labeled neurons in the rostral half of an adult ChAT-IRES-CreER;Z/AP brain. The mouse received injections of 4HT on P8 (1 mg), P21 (2 mg), and P28 (2 mg). A,B, bright field images of AP-stained tissue corresponding to the boxed regions in J and E, respectively. C–M, AP-labeled processes traced from eleven serial 300 um coronal sections. Two adjacent neurons with cell bodies in the basal forebrain (black arrows in panels K and L) and their large axon arbors in the cerebral cortex are shown in red. All other processes are shown in green. Regions in which a high density of AP-labeled processes precluded accurate tracing are marked by semi-opaque grey zones. Scale bar: 0.5 mm in A, 0.2 mm in B.

    Techniques Used: Labeling, Staining

    Cell-type specificity of Cre recombinase activity in mouse lines with different IRES-CreER knock-ins. The cell-type specificity of each line was tested by crossing to the Z/AP reporter and inducing sparse recombination with 4HT. A, In the retina, the NFL-IRES-CreER line exhibits Cre-mediated recombination in retinal ganglion cells, each of which has a single axon projecting to the optic disc, and in horizontal cells; the image shown here shows only retinal ganglion cells. B, in the brain, the VAMP2-IRES-CreER line exhibits Cre-mediated recombination in both neurons and glia. C and D, in the retina, the ChAT-IRES-CreER line exhibits Cre-mediated recombination exclusively in cholinergic (starburst) amacrine cells. The region boxed in C is shown at higher magnification in D. Scale bars: 0.5 mm in A and B; 1 mm in C.
    Figure Legend Snippet: Cell-type specificity of Cre recombinase activity in mouse lines with different IRES-CreER knock-ins. The cell-type specificity of each line was tested by crossing to the Z/AP reporter and inducing sparse recombination with 4HT. A, In the retina, the NFL-IRES-CreER line exhibits Cre-mediated recombination in retinal ganglion cells, each of which has a single axon projecting to the optic disc, and in horizontal cells; the image shown here shows only retinal ganglion cells. B, in the brain, the VAMP2-IRES-CreER line exhibits Cre-mediated recombination in both neurons and glia. C and D, in the retina, the ChAT-IRES-CreER line exhibits Cre-mediated recombination exclusively in cholinergic (starburst) amacrine cells. The region boxed in C is shown at higher magnification in D. Scale bars: 0.5 mm in A and B; 1 mm in C.

    Techniques Used: Activity Assay

    Morphologies of cholinergic neurons in an adult ChAT-IRES-CreER;Z/AP brain. Different neurons are color-coded. The mouse received injections of 4HT on P8 (1 mg), P21 (2 mg), and P28 (2 mg). A–P, tracing of neuronal processes from 16 serial 300 um coronal sections. Q–S and Figure 4E show bright field images of the AP-stained tissue corresponding to the boxed and lettered regions in D and J. All of the AP-stained neurons within the 16 sections are shown. Regions in which a high density of AP-labeled processes precluded accurate tracing are indicated by a semi-opaque grey zone, as shown for the cell in panel R. Cells bodies are indicated by black arrows. Scale bars: 0.2 mm in Q and R, 0.5 mm in S.
    Figure Legend Snippet: Morphologies of cholinergic neurons in an adult ChAT-IRES-CreER;Z/AP brain. Different neurons are color-coded. The mouse received injections of 4HT on P8 (1 mg), P21 (2 mg), and P28 (2 mg). A–P, tracing of neuronal processes from 16 serial 300 um coronal sections. Q–S and Figure 4E show bright field images of the AP-stained tissue corresponding to the boxed and lettered regions in D and J. All of the AP-stained neurons within the 16 sections are shown. Regions in which a high density of AP-labeled processes precluded accurate tracing are indicated by a semi-opaque grey zone, as shown for the cell in panel R. Cells bodies are indicated by black arrows. Scale bars: 0.2 mm in Q and R, 0.5 mm in S.

    Techniques Used: Staining, Labeling

    Sparse recombination permits visualization of non-overlapping neuronal processes. A, 200 um vibratome section from the brain of an NFL-IRES-CreER;Z/AP mouse injected at P0 with 0.2 mg 4HT. In this section, three large neurons (or parts of neurons) are labeled in one hemisphere of the cerebral cortex. B–D, enlarged bright field images at one Z-plane for each of the three neurons. Scale bar: 0.5 mm.
    Figure Legend Snippet: Sparse recombination permits visualization of non-overlapping neuronal processes. A, 200 um vibratome section from the brain of an NFL-IRES-CreER;Z/AP mouse injected at P0 with 0.2 mg 4HT. In this section, three large neurons (or parts of neurons) are labeled in one hemisphere of the cerebral cortex. B–D, enlarged bright field images at one Z-plane for each of the three neurons. Scale bar: 0.5 mm.

    Techniques Used: Injection, Labeling

    11) Product Images from "CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation"

    Article Title: CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1702763114

    CK1α ablation in keratinocytes increased epidermal melanocyte number and eumelanin production. ( A and B ) Topical treatment with 4HT for 14 d induces skin hyperpigmentation on the ear. Fontana–Masson staining shows increased epidermal thickness and melanin content in the epidermis. ( C ) Topical treatment with 4HT for 14 d induces skin hyperpigmentation on the tail. TRP1 staining highlights the increased number of melanocytes in the tail epidermis. Quantitation of melanocyte staining shows that the number of dermal melanocytes was not significantly changed. ( D ) The amount of eumelanin is increased, but pheomelanin remains unchanged during CK1α inhibition. Data are shown as means ± SD; * P
    Figure Legend Snippet: CK1α ablation in keratinocytes increased epidermal melanocyte number and eumelanin production. ( A and B ) Topical treatment with 4HT for 14 d induces skin hyperpigmentation on the ear. Fontana–Masson staining shows increased epidermal thickness and melanin content in the epidermis. ( C ) Topical treatment with 4HT for 14 d induces skin hyperpigmentation on the tail. TRP1 staining highlights the increased number of melanocytes in the tail epidermis. Quantitation of melanocyte staining shows that the number of dermal melanocytes was not significantly changed. ( D ) The amount of eumelanin is increased, but pheomelanin remains unchanged during CK1α inhibition. Data are shown as means ± SD; * P

    Techniques Used: Staining, Quantitation Assay, Inhibition

    Comparison of signaling pathways between UV irradiation and CK1α ablation in keratinocytes. ( A ) Experimental design: WT mice under chronic UVB exposure and the topical 4HT induction of SKO and CK1α/p53-DKO mice. ( B ) RT-PCR analysis of epidermis at different time points (days 0, 7, and 14). Under chronic UV irradiation, Pomc is up-regulated eightfold on day 7. Pomc is not activated by CK1α ablation. Other genes, including Mc1r , Mitf , Edn1 , and Wnt7a , are up-regulated by UV irradiation much more intensely than in the CK1α-ablated condition on days 7 and 14. The CK1α ablation-induced KitL up-regulation is sustained on days 7 and 14. The Mitf up-regulation induced by CK1α ablation is delayed compared with the UV-induced Mitf up-regulation. The genes influenced in response to CK1α ablation are different from those affected by UV irradiation. Data are shown as means ± SD; * P
    Figure Legend Snippet: Comparison of signaling pathways between UV irradiation and CK1α ablation in keratinocytes. ( A ) Experimental design: WT mice under chronic UVB exposure and the topical 4HT induction of SKO and CK1α/p53-DKO mice. ( B ) RT-PCR analysis of epidermis at different time points (days 0, 7, and 14). Under chronic UV irradiation, Pomc is up-regulated eightfold on day 7. Pomc is not activated by CK1α ablation. Other genes, including Mc1r , Mitf , Edn1 , and Wnt7a , are up-regulated by UV irradiation much more intensely than in the CK1α-ablated condition on days 7 and 14. The CK1α ablation-induced KitL up-regulation is sustained on days 7 and 14. The Mitf up-regulation induced by CK1α ablation is delayed compared with the UV-induced Mitf up-regulation. The genes influenced in response to CK1α ablation are different from those affected by UV irradiation. Data are shown as means ± SD; * P

    Techniques Used: Irradiation, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Changes in melanocyte number after ACK2 intradermal injection in CK1α heterozygous (Het) mice 14 d after the 4HT administration. Dermal melanocytes were significantly reduced by ACK2 injection, whereas ACK2 did not affect epidermal melanocytes. Data are shown as means ± SD; * P
    Figure Legend Snippet: Changes in melanocyte number after ACK2 intradermal injection in CK1α heterozygous (Het) mice 14 d after the 4HT administration. Dermal melanocytes were significantly reduced by ACK2 injection, whereas ACK2 did not affect epidermal melanocytes. Data are shown as means ± SD; * P

    Techniques Used: Injection, Mouse Assay

    12) Product Images from "Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells"

    Article Title: Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells

    Journal: Oncogene

    doi: 10.1038/sj.onc.1204680

    MycER ™ modulates expression of endogenous c-Myc responsive genes, ( a ) CEM-C7-14, C7-MycER ™ mass culture or C7-MycER ™ #22 cells were treated with ethanol (C), 100 nM Dex (D), 250 nM 4HT (T) or Dex plus 4HT (T) for 32 h. Whole cell extracts (50 μ g protein) were resolved by 10% SDS–PAGE, transblotted on to PVDF membrane and probed with the monoclonal anti-p53 antibody. Membrane was stripped and reprobed with anti-CREB polyclonal antibody for normalization of loading, ( b ) CEM-C7-14, C7-MycER ™ mass culture or C7-MycER ™ #22 cells were treated with ethanol (C) or 1 μ M Dex (D) for 24 h. Whole cell extracts (50 μ g protein) were resolved by 10% SDS–PAGE and transblotted on to PVDF membrane and probed with a rabbit polyclonal antibody raised against p27 kip1 . Membrane was subsequently stripped and probed with a polyclonal antibody specific for CREB, to normalize for loading
    Figure Legend Snippet: MycER ™ modulates expression of endogenous c-Myc responsive genes, ( a ) CEM-C7-14, C7-MycER ™ mass culture or C7-MycER ™ #22 cells were treated with ethanol (C), 100 nM Dex (D), 250 nM 4HT (T) or Dex plus 4HT (T) for 32 h. Whole cell extracts (50 μ g protein) were resolved by 10% SDS–PAGE, transblotted on to PVDF membrane and probed with the monoclonal anti-p53 antibody. Membrane was stripped and reprobed with anti-CREB polyclonal antibody for normalization of loading, ( b ) CEM-C7-14, C7-MycER ™ mass culture or C7-MycER ™ #22 cells were treated with ethanol (C) or 1 μ M Dex (D) for 24 h. Whole cell extracts (50 μ g protein) were resolved by 10% SDS–PAGE and transblotted on to PVDF membrane and probed with a rabbit polyclonal antibody raised against p27 kip1 . Membrane was subsequently stripped and probed with a polyclonal antibody specific for CREB, to normalize for loading

    Techniques Used: Expressing, SDS Page

    Protection of C7-MycER ™ cells from Dex-evoked cell death, ( a ) CEM-C7-14 cells or the indicated individual C7-MycER ™ clones or the mass culture (M. Cult.) were seeded at a density of 1×10 5 cells/ml and treated with ethanol (solid symbols) or 100 nM Dex (open symbols) for 96 h. Viable cells were counted at 24 h intervals by Trypan blue exclusion. Data plotted are averages of duplicate treatments. ( b ) CEM-C7-14 or C7-MycER ™ clone #22 cells were treated in duplicate with ethanol or 100 nM Dex in the presence or absence of 100 nM or 1 μ M 4HT. Trypan blue excluding cells were counted after 72 h. Data are plotted as a percentage of untreated time matched control for each cell line. ( c ) CEM-C7-14 or C7-MycER ™ #22 cells were treated for 72 h with either ethanol, 100 nM staurosporine or 10 nM okadaic acid, and viable cell counts were determined at 24 h intervals. Data plotted are averages of duplicate treatments from a representative experiment
    Figure Legend Snippet: Protection of C7-MycER ™ cells from Dex-evoked cell death, ( a ) CEM-C7-14 cells or the indicated individual C7-MycER ™ clones or the mass culture (M. Cult.) were seeded at a density of 1×10 5 cells/ml and treated with ethanol (solid symbols) or 100 nM Dex (open symbols) for 96 h. Viable cells were counted at 24 h intervals by Trypan blue exclusion. Data plotted are averages of duplicate treatments. ( b ) CEM-C7-14 or C7-MycER ™ clone #22 cells were treated in duplicate with ethanol or 100 nM Dex in the presence or absence of 100 nM or 1 μ M 4HT. Trypan blue excluding cells were counted after 72 h. Data are plotted as a percentage of untreated time matched control for each cell line. ( c ) CEM-C7-14 or C7-MycER ™ #22 cells were treated for 72 h with either ethanol, 100 nM staurosporine or 10 nM okadaic acid, and viable cell counts were determined at 24 h intervals. Data plotted are averages of duplicate treatments from a representative experiment

    Techniques Used: Clone Assay

    Coimmunoprecipitation of Myc-ER ™ and endogenous c-Myc with anti-Max antibody. Whole cell extracts of CEM-C7-14 or C7-MycER ™ cells treated with ethanol (C), 1 μ M Dex (D), 250 nM 4HT (T) or Dex plus 4HT (DT) for 20 h were inmunoprecipitated with the polyclonal anti-Max antibody C-124. or non-specific antibody (P). Antibody bound proteins were purified on Protein A-Agarose beads and resolved by 10% SDS–PAGE and electroblotting. Whole cell extract (E) from C7-MycER ™ cells was run in one lane as a control. The membrane was probed with the c-Myc monoclonal antibody Mycl-9E10.2, and developed by ECL reaction
    Figure Legend Snippet: Coimmunoprecipitation of Myc-ER ™ and endogenous c-Myc with anti-Max antibody. Whole cell extracts of CEM-C7-14 or C7-MycER ™ cells treated with ethanol (C), 1 μ M Dex (D), 250 nM 4HT (T) or Dex plus 4HT (DT) for 20 h were inmunoprecipitated with the polyclonal anti-Max antibody C-124. or non-specific antibody (P). Antibody bound proteins were purified on Protein A-Agarose beads and resolved by 10% SDS–PAGE and electroblotting. Whole cell extract (E) from C7-MycER ™ cells was run in one lane as a control. The membrane was probed with the c-Myc monoclonal antibody Mycl-9E10.2, and developed by ECL reaction

    Techniques Used: Purification, SDS Page

    13) Product Images from "Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis"

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

    Journal: Genes & Development

    doi: 10.1101/gad.302497.117

    DLC1 promotes oligomerization and formation of high-MW Bim complexes. ( A ) Wild-type Bim EL forms high-MW complexes, while Bim EL AA forms mainly low-MW complexes. One percent digitonin lysates of mitochondria from Bax −/− Bak −/− MEFs overexpressing either murine V5-tagged wild-type Bim EL or the DLC1-binding mutant Bim EL AA were separated by BN-PAGE on a 6%–16.5% gradient gel and subjected to anti-V5 Western blotting. Data are representative of four independent experiments. ( B , C ) BN-PAGE was performed with the mitochondrial lysates of wild-type or Bim EL AA-overexpressing Bax −/− Bak −/− MEFs. After blotting on PVDF membrane, membranes were probed for DLC1 or DIC. Results are representative of three independent experiments. ( D ) RNAi against DLC1 reduces the amount of high-MW Bim complexes. Mitochondrial lysates from Bax −/− Bak −/− MEFs overexpressing murine V5-tagged Bim EL were prepared 28 h after transfection of the cells with siRNA against DLC1 or control siRNA. Proteins were separated on a 6%–16.5% gradient gel, and Bim was detected as in A . Data are representative of three independent experiments. ( E ) Bim complexes as assessed by gel filtration. Mitochondrial lysates from Bax −/− Bak −/− MEFs overexpressing either murine V5-tagged Bim EL or Bim EL AA were separated by gel filtration on a Superdex200 10/300 GL column. Individual fractions were collected and tested for the presence of Bim EL by Western blotting. Sizes of marker proteins are indicated. Data are representative of two independent experiments. ( F ) HA-tagged Bim EL copurifies with untagged Bim EL . Anti-HA immunoprecipitation was performed with whole-cell lysates (1% Triton X-100) from Bax −/− Bak −/− MEFs overexpressing either untagged or 3xHA-tagged murine Bim EL . Note the precipitation of endogenous Bim EL with HA-Bim EL . Data are representative of five independent experiments. An asterisk indicates endogenous Bim EL signal. ( G ) Bim EL and Bim L but not Bim S form high-MW complexes. Western blots of BN-PAGE (6%–16.5% gradient gel) from mitochondrial fractions (1% digitonin lysates) of Bax −/− Bak −/− MEFs stably overexpressing the indicated murine Bim splice variants. Data are representative of two independent experiments. ( H ) An inactivating mutation of the DLC1-binding site in Bim EL abolishes its ability to form Bim–Bim complexes. Anti-HA immunoprecipitation was conducted with lysates of Bax −/− Bak −/− MEFs overexpressing murine V5-tagged wild-type Bim EL or mutant Bim EL (Bim EL AA) and carrying an inducible murine 3xHA-tagged Bim EL construct. 3xHA-Bim EL was induced for 48 h with 100 nM tamoxifen (4-hydroxytamoxifen [4HT]) and whole-cell lysates were prepared with 1% Triton X-100. Membranes were reprobed with anti-HA-antibodies. Data are representative of three independent experiments. The asterisk indicates anti-V5 signal from the first blot. ( I ) In situ identification of complexes containing at least two Bim EL molecules by proximity ligation assay (PLA). Bax −/− Bak −/− MEFs expressing murine V5-tagged Bim EL and carrying a 4HT-inducible murine 3xHA-tagged Bim EL construct were fixed and permeabilized directly ( left ) or upon induction of 3xHA-tagged Bim EL by treatment with 100 nM 4HT for 24 h ( right ). Images were acquired under identical conditions and exposure times and are representative of three independent experiments. Bars, 50 µm. ( J ) Bax −/− Bak −/− MEFs overexpressing 3xHA-Bim EL were labeled with HA-specific primary and Alexa 647 secondary antibodies, and three-dimensional (3D) stochastic optical reconstruction microscopy (STORM) imaging was performed. The rectangular boxes indicate regions of the zoom-ins (shown in the right panel). The individual localizations are colored according to their z -positions (see the color bar). Bim was visualized by the HA labeling and exhibits structures that might suggest the localization of Bim in complexes. Bars: left images, 2 µm; right images, 500 nm. ( K ) Endogenous Bim in wild-type HeLa cells was detected with anti-Bim primary and Alexa 647 secondary antibodies, and 3D STORM imaging was performed similar to J . The individual localizations are colored as in J . Bar, 1 µm. ( L ) Confocal microscopy was performed with HeLa cells after staining the mitochondria with MitoTracker (red) and endogenous Bim with anti-Bim primary and Alexa 488 (green) secondary antibody. The overlay shows the mitochondrial localization of Bim in HeLa cells. The rectangular box depicts the region of a zoom-in (overlay). Bars, 10 µm. ( M ) Endogenous Bim forms high-MW complexes on mitochondria. BN-PAGE was performed with solubilized mitochondrial fractions (1% digitonin) of the indicated cell lines (except for MEFs, cell lines are human tumor lines). Proteins were transferred to PVDF membrane and were detected by Western blotting as indicated. In order to achieve good detection, the amounts of proteins loaded and exposure times were sometimes different for different cell lines. Data are representative of at least three independent experiments with each cell line.
    Figure Legend Snippet: DLC1 promotes oligomerization and formation of high-MW Bim complexes. ( A ) Wild-type Bim EL forms high-MW complexes, while Bim EL AA forms mainly low-MW complexes. One percent digitonin lysates of mitochondria from Bax −/− Bak −/− MEFs overexpressing either murine V5-tagged wild-type Bim EL or the DLC1-binding mutant Bim EL AA were separated by BN-PAGE on a 6%–16.5% gradient gel and subjected to anti-V5 Western blotting. Data are representative of four independent experiments. ( B , C ) BN-PAGE was performed with the mitochondrial lysates of wild-type or Bim EL AA-overexpressing Bax −/− Bak −/− MEFs. After blotting on PVDF membrane, membranes were probed for DLC1 or DIC. Results are representative of three independent experiments. ( D ) RNAi against DLC1 reduces the amount of high-MW Bim complexes. Mitochondrial lysates from Bax −/− Bak −/− MEFs overexpressing murine V5-tagged Bim EL were prepared 28 h after transfection of the cells with siRNA against DLC1 or control siRNA. Proteins were separated on a 6%–16.5% gradient gel, and Bim was detected as in A . Data are representative of three independent experiments. ( E ) Bim complexes as assessed by gel filtration. Mitochondrial lysates from Bax −/− Bak −/− MEFs overexpressing either murine V5-tagged Bim EL or Bim EL AA were separated by gel filtration on a Superdex200 10/300 GL column. Individual fractions were collected and tested for the presence of Bim EL by Western blotting. Sizes of marker proteins are indicated. Data are representative of two independent experiments. ( F ) HA-tagged Bim EL copurifies with untagged Bim EL . Anti-HA immunoprecipitation was performed with whole-cell lysates (1% Triton X-100) from Bax −/− Bak −/− MEFs overexpressing either untagged or 3xHA-tagged murine Bim EL . Note the precipitation of endogenous Bim EL with HA-Bim EL . Data are representative of five independent experiments. An asterisk indicates endogenous Bim EL signal. ( G ) Bim EL and Bim L but not Bim S form high-MW complexes. Western blots of BN-PAGE (6%–16.5% gradient gel) from mitochondrial fractions (1% digitonin lysates) of Bax −/− Bak −/− MEFs stably overexpressing the indicated murine Bim splice variants. Data are representative of two independent experiments. ( H ) An inactivating mutation of the DLC1-binding site in Bim EL abolishes its ability to form Bim–Bim complexes. Anti-HA immunoprecipitation was conducted with lysates of Bax −/− Bak −/− MEFs overexpressing murine V5-tagged wild-type Bim EL or mutant Bim EL (Bim EL AA) and carrying an inducible murine 3xHA-tagged Bim EL construct. 3xHA-Bim EL was induced for 48 h with 100 nM tamoxifen (4-hydroxytamoxifen [4HT]) and whole-cell lysates were prepared with 1% Triton X-100. Membranes were reprobed with anti-HA-antibodies. Data are representative of three independent experiments. The asterisk indicates anti-V5 signal from the first blot. ( I ) In situ identification of complexes containing at least two Bim EL molecules by proximity ligation assay (PLA). Bax −/− Bak −/− MEFs expressing murine V5-tagged Bim EL and carrying a 4HT-inducible murine 3xHA-tagged Bim EL construct were fixed and permeabilized directly ( left ) or upon induction of 3xHA-tagged Bim EL by treatment with 100 nM 4HT for 24 h ( right ). Images were acquired under identical conditions and exposure times and are representative of three independent experiments. Bars, 50 µm. ( J ) Bax −/− Bak −/− MEFs overexpressing 3xHA-Bim EL were labeled with HA-specific primary and Alexa 647 secondary antibodies, and three-dimensional (3D) stochastic optical reconstruction microscopy (STORM) imaging was performed. The rectangular boxes indicate regions of the zoom-ins (shown in the right panel). The individual localizations are colored according to their z -positions (see the color bar). Bim was visualized by the HA labeling and exhibits structures that might suggest the localization of Bim in complexes. Bars: left images, 2 µm; right images, 500 nm. ( K ) Endogenous Bim in wild-type HeLa cells was detected with anti-Bim primary and Alexa 647 secondary antibodies, and 3D STORM imaging was performed similar to J . The individual localizations are colored as in J . Bar, 1 µm. ( L ) Confocal microscopy was performed with HeLa cells after staining the mitochondria with MitoTracker (red) and endogenous Bim with anti-Bim primary and Alexa 488 (green) secondary antibody. The overlay shows the mitochondrial localization of Bim in HeLa cells. The rectangular box depicts the region of a zoom-in (overlay). Bars, 10 µm. ( M ) Endogenous Bim forms high-MW complexes on mitochondria. BN-PAGE was performed with solubilized mitochondrial fractions (1% digitonin) of the indicated cell lines (except for MEFs, cell lines are human tumor lines). Proteins were transferred to PVDF membrane and were detected by Western blotting as indicated. In order to achieve good detection, the amounts of proteins loaded and exposure times were sometimes different for different cell lines. Data are representative of at least three independent experiments with each cell line.

    Techniques Used: Binding Assay, Mutagenesis, Polyacrylamide Gel Electrophoresis, Western Blot, Transfection, Filtration, Marker, Immunoprecipitation, Stable Transfection, Construct, In Situ, Proximity Ligation Assay, Expressing, Labeling, Microscopy, Imaging, Confocal Microscopy, Staining

    Loss of Bim EL –DLC1 binding enhances the proapoptotic activity of Bim EL . ( A ) RNAi specific for DLC1 sensitizes MEFs for apoptosis induced by thapsigargin in a predominantly Bim-dependent manner. Cells were transfected with control siRNA or siRNA specific for DLC1 16 h prior to treatment with 200 nM thapsigargin for 24 h. QVD-OPh (10 µM) was added to control samples together with thapsigargin to test for caspase dependency. Data show means/SEM of three independent experiments. (*) P = 0.015 wild type; (*) P = 0.005 Bim −/− , two-tailed paired t -test. ( B ) Bim EL AA has higher proapoptotic activity than wild-type Bim EL when expressed in MEFs. MEFs carrying a 4HT-inducible construct for the expression of either wild-type Bim EL or Bim EL AA were treated with 100 nM 4HT for 24 h. Apoptosis was measured as the percentage of cells positive for active caspase-3. Data show means/SEM of four independent experiments. (**) P = 0.010, two-tailed paired t -test. Both Bim EL and Bim EL ). ( C , D ) RNAi specific for DLC1 can sensitize MEFs to the induction of wild-type Bim EL but not Bim EL AA. MEFs carrying a 4HT-inducible construct for the expression of either wild-type Bim EL ( C ) or Bim EL AA ( D ) were transfected with control or DLC1-specific siRNA 16 h prior to Bim induction with 100 nM 4HT for 24 h. Cells with active caspase-3 were detected by flow cytometry as above. Data show means/SEM of four independent experiments. (*) P = 0.014 ( C ); (ns) nonsignificant ( P = 0.679) ( D ), two-tailed paired t-test.
    Figure Legend Snippet: Loss of Bim EL –DLC1 binding enhances the proapoptotic activity of Bim EL . ( A ) RNAi specific for DLC1 sensitizes MEFs for apoptosis induced by thapsigargin in a predominantly Bim-dependent manner. Cells were transfected with control siRNA or siRNA specific for DLC1 16 h prior to treatment with 200 nM thapsigargin for 24 h. QVD-OPh (10 µM) was added to control samples together with thapsigargin to test for caspase dependency. Data show means/SEM of three independent experiments. (*) P = 0.015 wild type; (*) P = 0.005 Bim −/− , two-tailed paired t -test. ( B ) Bim EL AA has higher proapoptotic activity than wild-type Bim EL when expressed in MEFs. MEFs carrying a 4HT-inducible construct for the expression of either wild-type Bim EL or Bim EL AA were treated with 100 nM 4HT for 24 h. Apoptosis was measured as the percentage of cells positive for active caspase-3. Data show means/SEM of four independent experiments. (**) P = 0.010, two-tailed paired t -test. Both Bim EL and Bim EL ). ( C , D ) RNAi specific for DLC1 can sensitize MEFs to the induction of wild-type Bim EL but not Bim EL AA. MEFs carrying a 4HT-inducible construct for the expression of either wild-type Bim EL ( C ) or Bim EL AA ( D ) were transfected with control or DLC1-specific siRNA 16 h prior to Bim induction with 100 nM 4HT for 24 h. Cells with active caspase-3 were detected by flow cytometry as above. Data show means/SEM of four independent experiments. (*) P = 0.014 ( C ); (ns) nonsignificant ( P = 0.679) ( D ), two-tailed paired t-test.

    Techniques Used: Binding Assay, Activity Assay, Transfection, Two Tailed Test, Construct, Expressing, Flow Cytometry, Cytometry

    14) Product Images from "Selective Estrogen Receptor Modulators 4-Hydroxytamoxifen and Raloxifene Impact the Stability and Function of SRC-1 and SRC-3 Coactivator Proteins"

    Article Title: Selective Estrogen Receptor Modulators 4-Hydroxytamoxifen and Raloxifene Impact the Stability and Function of SRC-1 and SRC-3 Coactivator Proteins

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.1.14-24.2004

    (A) 4HT can elevate the steady-state level of SRC-1A-LUC and SRC-3-LUC in HeLa cells. HeLa cells were transfected with pERE-E1b-CAT and expression vectors for the indicated coactivators along with either pCR3.1 hERα (+) or its empty vector, pCR3.1 (−). Twenty-four hours thereafter, cells were treated with ethanol vehicle (−), E2 (E), or 4HT (T) for 24 h and harvested for luciferase activity. (B) The AF-2 of ERα is dispensable for 4HT-induced elevation of SRC-1A-LUC and SRC-3-LUC protein levels. HeLa cells were transfected in a similar manner as described above, with the addition of an AF-2-defective mutant for ERα (L539A). Cells were treated with hormone as described above with the addition of raloxifene (R). (C) The AF-1 and DBD of ERα are required for 4HT-induced elevation in coactivator expression. HeLa cells were transfected and treated as described above with the cotransfection of expression vectors for the DBD mutant (C201H/C205H) or ERα with AF-1 deleted (179C ΔAF-1) instead of the wild-type receptor. (D) Deletion of amino acid residues 1138 to 1216 of SRC-1 blocks 4HT-induced elevation of the coactivator. Expression vectors for ERα and the wild-type FLAG-tagged SRC-1 or SRC-1 deletion mutants Δ913-979 and Δ1138-1216 were transfected into HeLa cells. Twenty-four hours later, cells were treated with E2, 4HT, or their ethanol vehicle for an additional 24 h and harvested for Western analysis.
    Figure Legend Snippet: (A) 4HT can elevate the steady-state level of SRC-1A-LUC and SRC-3-LUC in HeLa cells. HeLa cells were transfected with pERE-E1b-CAT and expression vectors for the indicated coactivators along with either pCR3.1 hERα (+) or its empty vector, pCR3.1 (−). Twenty-four hours thereafter, cells were treated with ethanol vehicle (−), E2 (E), or 4HT (T) for 24 h and harvested for luciferase activity. (B) The AF-2 of ERα is dispensable for 4HT-induced elevation of SRC-1A-LUC and SRC-3-LUC protein levels. HeLa cells were transfected in a similar manner as described above, with the addition of an AF-2-defective mutant for ERα (L539A). Cells were treated with hormone as described above with the addition of raloxifene (R). (C) The AF-1 and DBD of ERα are required for 4HT-induced elevation in coactivator expression. HeLa cells were transfected and treated as described above with the cotransfection of expression vectors for the DBD mutant (C201H/C205H) or ERα with AF-1 deleted (179C ΔAF-1) instead of the wild-type receptor. (D) Deletion of amino acid residues 1138 to 1216 of SRC-1 blocks 4HT-induced elevation of the coactivator. Expression vectors for ERα and the wild-type FLAG-tagged SRC-1 or SRC-1 deletion mutants Δ913-979 and Δ1138-1216 were transfected into HeLa cells. Twenty-four hours later, cells were treated with E2, 4HT, or their ethanol vehicle for an additional 24 h and harvested for Western analysis.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Cotransfection, Western Blot

    SERMs can potentiate the transcriptional activity of other nuclear hormone receptors. (A) HeLa cells were transfected with a progesterone-responsive reporter (pGRE-E1b-LUC), pCR3.1 hERα L539A, or its vector backbone, pCR3.1 (−), and a PR expression vector (pCR3.1 hPR-B). Twenty-four hours thereafter, the cells were treated with ethanol vehicle (−), E2 (E), 4HT (T), or P4 (P). (B) Cells were transfected with a GR-responsive reporter (pGRE-E1b-LUC) and ERα and then treated with 10 −7 M dexamethasone (Dex) and with the ERα ligands listed above. (C) SERM-mediated potentiation of GR-mediated transcription in MCF-7 cells. Untransfected MCF-7 cells were treated with ligands as described above and harvested for total RNA 12 h thereafter. The mRNA for the GR-inducible SGK1 gene was quantitated by real-time quantitative PCR normalized against 18S RNA. (D) Treatment with 4HT promotes increased interaction between PR and SRC-3-LUC in the HeLa cell line. HeLa cells were transfected with pERE-E1b-CAT and expression vectors for ERα, PR-B and SRC-3-LUC. Twenty-four hours thereafter, the cells were treated with E2 or 4HT as indicated. After an additional 24 h, the cells were treated with progesterone (P) for 1 h and then cells were harvested and subjected to immunoprecipitation with an anti-PR antibody. The antibody-associated luciferase (LUC) or SRC-3-LUC protein was detected with a standard luciferase assay.
    Figure Legend Snippet: SERMs can potentiate the transcriptional activity of other nuclear hormone receptors. (A) HeLa cells were transfected with a progesterone-responsive reporter (pGRE-E1b-LUC), pCR3.1 hERα L539A, or its vector backbone, pCR3.1 (−), and a PR expression vector (pCR3.1 hPR-B). Twenty-four hours thereafter, the cells were treated with ethanol vehicle (−), E2 (E), 4HT (T), or P4 (P). (B) Cells were transfected with a GR-responsive reporter (pGRE-E1b-LUC) and ERα and then treated with 10 −7 M dexamethasone (Dex) and with the ERα ligands listed above. (C) SERM-mediated potentiation of GR-mediated transcription in MCF-7 cells. Untransfected MCF-7 cells were treated with ligands as described above and harvested for total RNA 12 h thereafter. The mRNA for the GR-inducible SGK1 gene was quantitated by real-time quantitative PCR normalized against 18S RNA. (D) Treatment with 4HT promotes increased interaction between PR and SRC-3-LUC in the HeLa cell line. HeLa cells were transfected with pERE-E1b-CAT and expression vectors for ERα, PR-B and SRC-3-LUC. Twenty-four hours thereafter, the cells were treated with E2 or 4HT as indicated. After an additional 24 h, the cells were treated with progesterone (P) for 1 h and then cells were harvested and subjected to immunoprecipitation with an anti-PR antibody. The antibody-associated luciferase (LUC) or SRC-3-LUC protein was detected with a standard luciferase assay.

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Luciferase

    4HT can elevate endogenous SRC-1 and SRC-3 in HeLa cells transiently transfected with ERα and in MCF-7 cells. (A) HeLa cells were transfected with pERE-E1b-CAT and pCR3.1 hERα, incubated 24 h, and then treated with ethanol vehicle (−), E2, or 4HT for 24 h and harvested for Western analysis. (B) 4HT is able to promote elevation of endogenous SRC-3 in MCF-7 but not T-47D or ZR-75-1 ERα-positive breast cancer cell lines. Untransfected MCF-7, T-47D, and ZR-75-1 cell lines were treated with ligands and harvested for Western analysis as described for panel A. (C) 4HT treatment has no effect on SRC-1 or SRC-3 mRNA levels in HeLa cells transfected with ERα. HeLa cells were transfected with pERE-E1b-CAT and pCR3.1 hERα and then treated for 24 h with ligands as described above. Cells were harvested for total RNA, and mRNA for SRC-1 and SRC-3 was quantitated by real-time PCR. (D) In MG132-treated HeLa cells, SRC-1A-LUC protein is not further elevated when cotreated with 4HT. HeLa cells were transfected with pERE-E1b-CAT, pCR3.1hERα, or pCR3.1 SRC-1A-LUC and treated with ethanol vehicle (−), E2, or 4HT with or without MG132 for 8 h and harvested for luciferase activity.
    Figure Legend Snippet: 4HT can elevate endogenous SRC-1 and SRC-3 in HeLa cells transiently transfected with ERα and in MCF-7 cells. (A) HeLa cells were transfected with pERE-E1b-CAT and pCR3.1 hERα, incubated 24 h, and then treated with ethanol vehicle (−), E2, or 4HT for 24 h and harvested for Western analysis. (B) 4HT is able to promote elevation of endogenous SRC-3 in MCF-7 but not T-47D or ZR-75-1 ERα-positive breast cancer cell lines. Untransfected MCF-7, T-47D, and ZR-75-1 cell lines were treated with ligands and harvested for Western analysis as described for panel A. (C) 4HT treatment has no effect on SRC-1 or SRC-3 mRNA levels in HeLa cells transfected with ERα. HeLa cells were transfected with pERE-E1b-CAT and pCR3.1 hERα and then treated for 24 h with ligands as described above. Cells were harvested for total RNA, and mRNA for SRC-1 and SRC-3 was quantitated by real-time PCR. (D) In MG132-treated HeLa cells, SRC-1A-LUC protein is not further elevated when cotreated with 4HT. HeLa cells were transfected with pERE-E1b-CAT, pCR3.1hERα, or pCR3.1 SRC-1A-LUC and treated with ethanol vehicle (−), E2, or 4HT with or without MG132 for 8 h and harvested for luciferase activity.

    Techniques Used: Transfection, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

    15) Product Images from "ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells"

    Article Title: ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells

    Journal: Scientific Data

    doi: 10.1038/sdata.2016.101

    ROCK kinase activation in PDAC cells. ( a ) ROCK functional domains and ROCK kinase expression constructs (CR, cysteine-rich; ER, estrogen receptor hormone binding domain; GFP, green fluorescent protein; PH, pleckstrin homology domain; RBD, Rho binding domain). ( b ) Conditional activation of ROCK kinase by 4-hydroxytamoxifen (4HT). ( c ) Overview of the ROCK signalling pathway. ( d ) PDAC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated with EtOH vehicle or 1 μM 4HT in the presence or absence of 10 μM Y27632 ROCK inhibitor. Immunoblotting shows ER-fusion proteins, endogenous ROCK1 and ROCK2, and phosphorylation status of ROCK targets LIMK1/2 (T508/T505) and regulatory myosin light chain (MLC2; T18S19). Total MLC2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.
    Figure Legend Snippet: ROCK kinase activation in PDAC cells. ( a ) ROCK functional domains and ROCK kinase expression constructs (CR, cysteine-rich; ER, estrogen receptor hormone binding domain; GFP, green fluorescent protein; PH, pleckstrin homology domain; RBD, Rho binding domain). ( b ) Conditional activation of ROCK kinase by 4-hydroxytamoxifen (4HT). ( c ) Overview of the ROCK signalling pathway. ( d ) PDAC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated with EtOH vehicle or 1 μM 4HT in the presence or absence of 10 μM Y27632 ROCK inhibitor. Immunoblotting shows ER-fusion proteins, endogenous ROCK1 and ROCK2, and phosphorylation status of ROCK targets LIMK1/2 (T508/T505) and regulatory myosin light chain (MLC2; T18S19). Total MLC2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.

    Techniques Used: Activation Assay, Functional Assay, Expressing, Construct, Binding Assay

    Quality control of RNA-Seq data. ( a ) Principal component plot of RNA-Seq (DESeq2) data indicating the clustering together of GFP:ER samples treated with vehicle control or 4HT, and ROCK1:ER plus ROCK2:ER samples treated with 4HT. ( b ) PDAC cells expressing GFP:ER, ROCK1:ER or ( c ) ROCK2:ER were treated with EtOH vehicle or 1 μM 4HT. Ptgs2 and Tff3 mRNA levels relative to Gapdh housekeeping gene were determined by qPCR. Means±s.e.m., unpaired t -test ( n =3). *** P
    Figure Legend Snippet: Quality control of RNA-Seq data. ( a ) Principal component plot of RNA-Seq (DESeq2) data indicating the clustering together of GFP:ER samples treated with vehicle control or 4HT, and ROCK1:ER plus ROCK2:ER samples treated with 4HT. ( b ) PDAC cells expressing GFP:ER, ROCK1:ER or ( c ) ROCK2:ER were treated with EtOH vehicle or 1 μM 4HT. Ptgs2 and Tff3 mRNA levels relative to Gapdh housekeeping gene were determined by qPCR. Means±s.e.m., unpaired t -test ( n =3). *** P

    Techniques Used: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction

    16) Product Images from "New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling"

    Article Title: New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007859

    Dose and timing of doxycycline and 4HT exposure on Cre-mediated recombination efficiency using R26rtTACreER . (A) Individual experiments are numbered, the doxycycline and 4HT regimens are shown schematically, and the number of mice with high/medium, sparse, or no AP+ labeling of RGCs is shown in the color-coded histograms at right. Experiments 1–21 and 22–33 were performed with R26rtTACreER;Brn3a CKOAP/+ and R26rtTACreER;Brn3b CKOAP/+ mice, respectively. (B–F) Representative retina flat mounts (B,C,E,F) and a 200 µm coronal brain section at the level of the superior colliculus (D) were obtained from the numbered experiments indicated in the lower right corner of each panel. A single IP injection was used to deliver 4HT either to the mother for in utero exposure or to individual pups for postnatal exposure.
    Figure Legend Snippet: Dose and timing of doxycycline and 4HT exposure on Cre-mediated recombination efficiency using R26rtTACreER . (A) Individual experiments are numbered, the doxycycline and 4HT regimens are shown schematically, and the number of mice with high/medium, sparse, or no AP+ labeling of RGCs is shown in the color-coded histograms at right. Experiments 1–21 and 22–33 were performed with R26rtTACreER;Brn3a CKOAP/+ and R26rtTACreER;Brn3b CKOAP/+ mice, respectively. (B–F) Representative retina flat mounts (B,C,E,F) and a 200 µm coronal brain section at the level of the superior colliculus (D) were obtained from the numbered experiments indicated in the lower right corner of each panel. A single IP injection was used to deliver 4HT either to the mother for in utero exposure or to individual pups for postnatal exposure.

    Techniques Used: Mouse Assay, Labeling, Injection, In Utero

    Morphologies of individual AP+ type 1 DA cells from the retinas of TH-IRES-CreER;R26IAP mice. A single 100–300 µg 4HT injection was administered IP between P3 and P8; retinas were analyzed ∼6 weeks later. Retina flat mounts were processed for AP histochemistry and clarified in BBBA. Axon-like processes (green) and dendrites (blue) were traced with Neuromantic software. The soma is indicated by a red arrow. The outline of the flattened retina and the location of the optic disc are shown in red. (A) Twelve retinas, each of which had a single type 1 DA cell. (B) A retina with two type 1 DA cells. (C) Image of the boxed region of the retina shown in (B). The dendrites (lower right) are uniformly thicker and more darkly stained than the axon-like processes. (D) A putative type 2 DA cell. Scale bars in B and D, 500 µm. Scale bar in B applies to panels A and B. BBBA causes ∼30% tissue shrinkage.
    Figure Legend Snippet: Morphologies of individual AP+ type 1 DA cells from the retinas of TH-IRES-CreER;R26IAP mice. A single 100–300 µg 4HT injection was administered IP between P3 and P8; retinas were analyzed ∼6 weeks later. Retina flat mounts were processed for AP histochemistry and clarified in BBBA. Axon-like processes (green) and dendrites (blue) were traced with Neuromantic software. The soma is indicated by a red arrow. The outline of the flattened retina and the location of the optic disc are shown in red. (A) Twelve retinas, each of which had a single type 1 DA cell. (B) A retina with two type 1 DA cells. (C) Image of the boxed region of the retina shown in (B). The dendrites (lower right) are uniformly thicker and more darkly stained than the axon-like processes. (D) A putative type 2 DA cell. Scale bars in B and D, 500 µm. Scale bar in B applies to panels A and B. BBBA causes ∼30% tissue shrinkage.

    Techniques Used: Mouse Assay, Injection, Software, Staining

    Survey of cholinergic neuronal morphologies and projections in the brain and retina of R26IAP;ChAT-IRES-CreER mice. Animals were injected with a single IP injection of 200 µg 4HT at P8. (A,C,F,I,K) 300 µm coronal sections at Bregma positions 1.2, 0, −2.5, −5.0, and −7.8, respectively, as shown schematically in (N). (B,D,E,G,H,J,L) enlargements of boxed regions. (M) In a flat mounted retina, AP-expression is present exclusively in starburst amacrine cells. In the absence of 4HT, retinas were devoid of AP+ cells. BNA, basolateral nucleus of the amygdala; DMN(VN), dorsal motor nucleus of the vagus nerve; LDTN, laterodorsal tegmental nucleus; MS, Medial septum; PPTN; pedunculopontine tegmental nucleus; SI, substantia innominata; St, striatum.
    Figure Legend Snippet: Survey of cholinergic neuronal morphologies and projections in the brain and retina of R26IAP;ChAT-IRES-CreER mice. Animals were injected with a single IP injection of 200 µg 4HT at P8. (A,C,F,I,K) 300 µm coronal sections at Bregma positions 1.2, 0, −2.5, −5.0, and −7.8, respectively, as shown schematically in (N). (B,D,E,G,H,J,L) enlargements of boxed regions. (M) In a flat mounted retina, AP-expression is present exclusively in starburst amacrine cells. In the absence of 4HT, retinas were devoid of AP+ cells. BNA, basolateral nucleus of the amygdala; DMN(VN), dorsal motor nucleus of the vagus nerve; LDTN, laterodorsal tegmental nucleus; MS, Medial septum; PPTN; pedunculopontine tegmental nucleus; SI, substantia innominata; St, striatum.

    Techniques Used: Mouse Assay, Injection, Expressing, Mass Spectrometry

    Survey of catecholaminergic neuronal morphologies and projections in the brain of TH-IRES-CreER;R26IAP mice. Animals were injected with 100 µg 4HT at P3. (A–E,G,I,K,L) 300 µm coronal sections at Bregma positions 1.7, 1.1, 0, −1.6, −2.2, −3.2, −4.7, −5.5, −8.2, respectively, as shown schematically in (N). (F,H,J,M) enlargements of boxed regions. A1, A1 noradrenergic cell group; A2, A2 noradrenergic cell group; AcN, accumbens nucleus; AN, arcuate nucleus; CNA, central nucleus of the amygdala; DRN, dorsal raphe nucleus; LC, locus coeruleus; ME, median eminence; PPTN, pedunculopontine tegmental nucleus; SN(PR), substantia nigra (pars reticulata); St, striatum; VP, ventral pallidum; VTA, ventral tegmental area; ZI, zona incerta.
    Figure Legend Snippet: Survey of catecholaminergic neuronal morphologies and projections in the brain of TH-IRES-CreER;R26IAP mice. Animals were injected with 100 µg 4HT at P3. (A–E,G,I,K,L) 300 µm coronal sections at Bregma positions 1.7, 1.1, 0, −1.6, −2.2, −3.2, −4.7, −5.5, −8.2, respectively, as shown schematically in (N). (F,H,J,M) enlargements of boxed regions. A1, A1 noradrenergic cell group; A2, A2 noradrenergic cell group; AcN, accumbens nucleus; AN, arcuate nucleus; CNA, central nucleus of the amygdala; DRN, dorsal raphe nucleus; LC, locus coeruleus; ME, median eminence; PPTN, pedunculopontine tegmental nucleus; SN(PR), substantia nigra (pars reticulata); St, striatum; VP, ventral pallidum; VTA, ventral tegmental area; ZI, zona incerta.

    Techniques Used: Mouse Assay, Injection

    R26IAP is a sensitive reporter for Cre-mediated recombination in individual cells. (A–D) R26CreER;R26IAP mice either in the absence (A,B) or following intra-ocular injection (C,D) of 4HT show widespread Cre-mediated recombination in diverse cell types. A, brain; B, retina, C, lens, D, cornea. AP+ lens fiber and lens epithelial cells are seen in C. The overall frequency of AP-expressing cells in the brain and retina in the absence of 4HT is ∼0.01%. (E–G) R26IAP mice that received an IP injection of ∼184 µg of purified His-NLS-Cre protein show scattered Cre-mediated recombination only in epithelial cells lining the peritoneal cavity or in the muscle fibers of the abdominal wall immediately beneath the peritoneal surface. E, liver; F, abdominal wall; G, stomach. (H) R26IAP mice that received an intraocular injection of ∼7 µg of purified His-NLS-Cre protein at ∼P5 show scattered Cre-mediated in RGCs and in occasional astrocytes. RGC axons are converging on the optic nerve head at the top of the panel. (I,J) Retina (I) and cornea (J) following an intra-ocular injection of a replication defective adenovirus expressing Cre. Cre-mediated recombination and AP expression are confined to tissues near the injection site. In the retina, the compact appearance of the AP+ cells and the absence of AP+ axons suggests that most of the labeled cells are astrocytes and/or Muller glia. (K–O) Tissues from R26IAP mice in the absence (K) or following IV injection (L–O) of a replication defective adenovirus expressing Cre (Ad-Cre). K–N, kidney; O, liver. The boxed region of the renal pelvis in L is enlarged in M. N shows a region of renal cortex from a different mouse that received an IV injection of Ad-Cre in which Cre-mediated recombination was also observed in a subset of cortical tubules. Scale bars are: 1 mm for E and O; and 200 µm for D,F,G,H,J,M, and N.
    Figure Legend Snippet: R26IAP is a sensitive reporter for Cre-mediated recombination in individual cells. (A–D) R26CreER;R26IAP mice either in the absence (A,B) or following intra-ocular injection (C,D) of 4HT show widespread Cre-mediated recombination in diverse cell types. A, brain; B, retina, C, lens, D, cornea. AP+ lens fiber and lens epithelial cells are seen in C. The overall frequency of AP-expressing cells in the brain and retina in the absence of 4HT is ∼0.01%. (E–G) R26IAP mice that received an IP injection of ∼184 µg of purified His-NLS-Cre protein show scattered Cre-mediated recombination only in epithelial cells lining the peritoneal cavity or in the muscle fibers of the abdominal wall immediately beneath the peritoneal surface. E, liver; F, abdominal wall; G, stomach. (H) R26IAP mice that received an intraocular injection of ∼7 µg of purified His-NLS-Cre protein at ∼P5 show scattered Cre-mediated in RGCs and in occasional astrocytes. RGC axons are converging on the optic nerve head at the top of the panel. (I,J) Retina (I) and cornea (J) following an intra-ocular injection of a replication defective adenovirus expressing Cre. Cre-mediated recombination and AP expression are confined to tissues near the injection site. In the retina, the compact appearance of the AP+ cells and the absence of AP+ axons suggests that most of the labeled cells are astrocytes and/or Muller glia. (K–O) Tissues from R26IAP mice in the absence (K) or following IV injection (L–O) of a replication defective adenovirus expressing Cre (Ad-Cre). K–N, kidney; O, liver. The boxed region of the renal pelvis in L is enlarged in M. N shows a region of renal cortex from a different mouse that received an IV injection of Ad-Cre in which Cre-mediated recombination was also observed in a subset of cortical tubules. Scale bars are: 1 mm for E and O; and 200 µm for D,F,G,H,J,M, and N.

    Techniques Used: Mouse Assay, Injection, Expressing, Purification, Labeling, IV Injection

    17) Product Images from "STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis"

    Article Title: STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E10-02-0112

    STAT1 is down-regulated in activated Raf:ER cells. (A) Protein levels of STAT1 were assayed in either 48-h HGF-treated MDCK or 4HT-treated Raf:ER cysts by immunoblot. (B) Raf:ER cysts treated with the combination of 4HT and HGF were lysed and blotted for STAT1. GAPDH was used as a loading control. The relative decrease of STAT1 is shown (mean ± SEM, n = 3). *p
    Figure Legend Snippet: STAT1 is down-regulated in activated Raf:ER cells. (A) Protein levels of STAT1 were assayed in either 48-h HGF-treated MDCK or 4HT-treated Raf:ER cysts by immunoblot. (B) Raf:ER cysts treated with the combination of 4HT and HGF were lysed and blotted for STAT1. GAPDH was used as a loading control. The relative decrease of STAT1 is shown (mean ± SEM, n = 3). *p

    Techniques Used:

    Overexpression of constitutively active form of STAT1 restores redifferentiation of Raf:ER cells in the presence of 4HT. (A) Raf:ER cysts (left) and Raf:ER cysts overexpressing CA-STAT1 were treated with 4HT for 72 h. CA-STAT1 overexpression promotes redifferentiation in activated Raf:ER cysts. Bar, 10 μm. (B) Quantification of cords plus tubules in the presence of 4HT. One hundred GFP-positive structures per sample were counted (mean ± SEM, n = 3). *p
    Figure Legend Snippet: Overexpression of constitutively active form of STAT1 restores redifferentiation of Raf:ER cells in the presence of 4HT. (A) Raf:ER cysts (left) and Raf:ER cysts overexpressing CA-STAT1 were treated with 4HT for 72 h. CA-STAT1 overexpression promotes redifferentiation in activated Raf:ER cysts. Bar, 10 μm. (B) Quantification of cords plus tubules in the presence of 4HT. One hundred GFP-positive structures per sample were counted (mean ± SEM, n = 3). *p

    Techniques Used: Over Expression

    STAT1 mRNA is down-regulated in activated Raf:ER cells. (A) MDCK or Raf:ER cysts grown in collagen were treated with either HGF or 4HT. (B) Raf:ER cysts were treated with both HGF and 4HT for 72 h. STAT1 mRNA levels were analyzed by RT-PCR using GAPDH as an internal control (mean ± SEM, n = 3). *p
    Figure Legend Snippet: STAT1 mRNA is down-regulated in activated Raf:ER cells. (A) MDCK or Raf:ER cysts grown in collagen were treated with either HGF or 4HT. (B) Raf:ER cysts were treated with both HGF and 4HT for 72 h. STAT1 mRNA levels were analyzed by RT-PCR using GAPDH as an internal control (mean ± SEM, n = 3). *p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Redifferentiation is not promoted by (additional) HGF treatment in activated Raf:ER cells. Raf:ER cysts were treated with 4HT alone for 48 h, and HGF was added or not for additional 24 h. (A) In the absence of 4HT, HGF-induced cysts formed tubules with mature lumens. (B) Cysts treated with 4HT alone for 72 h exhibited cell dispersion. GFP fluorescence reflects Raf:ER induction. (C) Treatment with HGF plus 4HT failed to complete tubule development. Quantification of cysts with tubule(s) (bottom) (mean ± SEM, n = 3). Bar, 10 μm.
    Figure Legend Snippet: Redifferentiation is not promoted by (additional) HGF treatment in activated Raf:ER cells. Raf:ER cysts were treated with 4HT alone for 48 h, and HGF was added or not for additional 24 h. (A) In the absence of 4HT, HGF-induced cysts formed tubules with mature lumens. (B) Cysts treated with 4HT alone for 72 h exhibited cell dispersion. GFP fluorescence reflects Raf:ER induction. (C) Treatment with HGF plus 4HT failed to complete tubule development. Quantification of cysts with tubule(s) (bottom) (mean ± SEM, n = 3). Bar, 10 μm.

    Techniques Used: Fluorescence

    Activation of Raf-1 represses the association between Raf-1 and STAT1. (A) CA-STAT1 overexpressing or not Raf:ER cells were cultured either in the presence or absence 4HT for 48 h. Cell lysates were coimmunoprecipitated with the antibody to Raf-1 and then blotted for STAT1 or Raf-1 or FLAG tag on CA-STAT1 (a and b). (c) Raf:ER cell lysates in the absence of 4-HT were immunoprecipitated with Raf-1 or mouse IgG and analyzed by immunoblotting with STAT1 (lanes 1 and 2). Total lysates were immunoblotted for STAT1 as loading controls (lane 3 and 4). (B) Quantification of ERK activation. The ratios of phospho-ERK (green) to total ERK (red) were quantified (bottom) (mean ± SEM, n = 3). Activation of Raf-1 by 4HT induces ERK activation, yielding green and yellow bands. but CA-STAT1 overexpression has no effect on it.
    Figure Legend Snippet: Activation of Raf-1 represses the association between Raf-1 and STAT1. (A) CA-STAT1 overexpressing or not Raf:ER cells were cultured either in the presence or absence 4HT for 48 h. Cell lysates were coimmunoprecipitated with the antibody to Raf-1 and then blotted for STAT1 or Raf-1 or FLAG tag on CA-STAT1 (a and b). (c) Raf:ER cell lysates in the absence of 4-HT were immunoprecipitated with Raf-1 or mouse IgG and analyzed by immunoblotting with STAT1 (lanes 1 and 2). Total lysates were immunoblotted for STAT1 as loading controls (lane 3 and 4). (B) Quantification of ERK activation. The ratios of phospho-ERK (green) to total ERK (red) were quantified (bottom) (mean ± SEM, n = 3). Activation of Raf-1 by 4HT induces ERK activation, yielding green and yellow bands. but CA-STAT1 overexpression has no effect on it.

    Techniques Used: Activation Assay, Cell Culture, FLAG-tag, Immunoprecipitation, Over Expression

    Overexpression of tyrosine phosphorylation defective STAT1 partially rescues cord formation of activated Raf:ER cysts. (A) Raf:ER cysts (left) and three clones overexpressing Y701F-STAT1 were treated with 4HT for 72 h. Bar, 10 μm. (B) Total lysate from Raf:ER cells and three Y701F-STAT1 transfectants were immunoblotted for FLAG tag on Y701F-STAT1. (C) Quantification of cords in the presence of 4HT (mean ± SEM, n = 3). *p
    Figure Legend Snippet: Overexpression of tyrosine phosphorylation defective STAT1 partially rescues cord formation of activated Raf:ER cysts. (A) Raf:ER cysts (left) and three clones overexpressing Y701F-STAT1 were treated with 4HT for 72 h. Bar, 10 μm. (B) Total lysate from Raf:ER cells and three Y701F-STAT1 transfectants were immunoblotted for FLAG tag on Y701F-STAT1. (C) Quantification of cords in the presence of 4HT (mean ± SEM, n = 3). *p

    Techniques Used: Over Expression, Clone Assay, FLAG-tag

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    In Vivo Imaging:

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    Activation Assay:

    Article Title: Oncogenic ALKF1174L drives tumorigenesis in cutaneous squamous cell carcinoma
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    Article Title: Activation of Cre Recombinase Alone Can Induce Complete Tumor Regression
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    Mouse Assay:

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    Cell Culture:

    Article Title: JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis
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    Article Title: RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair
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    In Vitro:

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    Plasmid Preparation:

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    Injection:

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    Millipore 4ht delivery doxycycline
    Dose and timing of doxycycline and <t>4HT</t> exposure on Cre-mediated recombination efficiency using R26rtTACreER . (A) Individual experiments are numbered, the doxycycline and 4HT regimens are shown schematically, and the number of mice with high/medium, sparse, or no AP+ labeling of RGCs is shown in the color-coded histograms at right. Experiments 1–21 and 22–33 were performed with R26rtTACreER;Brn3a CKOAP/+ and R26rtTACreER;Brn3b CKOAP/+ mice, respectively. (B–F) Representative retina flat mounts (B,C,E,F) and a 200 µm coronal brain section at the level of the superior colliculus (D) were obtained from the numbered experiments indicated in the lower right corner of each panel. A single IP injection was used to deliver 4HT either to the mother for in utero exposure or to individual pups for postnatal exposure.
    4ht Delivery Doxycycline, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Increased nuclear pore density in OIS is necessary for SAHF formation. ( A ) Schematic showing the depletion experiment for B – E . ( B ) MAB414 (antibody recognizing several nucleoporins) immunostaining in ER:Stop cells treated with <t>4HT</t> after 2 d of knockdown with scramble (Scr) or POM121 siRNAs. ( Left ) The bottom plane of the nucleus imaged by SIM. ( Right ) Enlargement of the insets . Bars, 2 µm. ( C ) Mean (±SEM) nuclear pore density (pores per square micrometer) in 4HT-treated ER:Stop cells after scramble (Scr) or POM121 siRNA knockdown, as assayed by TPR staining in three biological replicates, (*) P
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    Epidermal melanoma cells invade the dermis and undergo phenotypic switch to a neuronal-like state. a Schematic showing <t>TAM</t> treatment and skin implantation onto nude mice for b – h . b , c Immunofluorescence for GFP at 10 days b and 17 days c after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. Arrowheads point to GFP + melanoma cells that invade into the dermis. d Immunofluorescence for GFP (green) and E-cadherin, MCAM (red) at 17 days after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. e Immunofluorescence for Sox10 (red) and E-cadherin, MCAM (green) in human melanoma specimen during the invasive radial growth phase. f , g Immunofluorescence images for GFP (green) and Dct, MITF, Nestin, GFAP, Tubb3 (red) and brightfield images in areas of dermal melanoma at 22 days f and 30 days g after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. h Dot plot showing percentage of pigment, Dct, Nestin, GFAP and Tubb3 in GFP + dermal melanoma cells (mean ± s.d.; 3 independent tumors from 3 mice were analyzed in each group, n = the number of randomly selected tumor areas analyzed. At least 1500 GFP + tumor cells were analyzed in each group). i Schematic model illustration showing that upon oncogenic induction, McSCs first give rise to epidermal melanoma. Then melanoma cells in the epithelial compartments invade into the dermis. Finally, dermal melanoma cells undergo a phenotypic switch to acquire neuronal signatures. j Schematic showing <t>4-hydroxytamoxifen</t> (4HT-TAM) treatment and analysis regimen. k Immunofluorescence images for Tomato (red) and Sox10, Dct, Tubb3, Nestin (green) and brightfield images in c-Kit-CreER:Braf:Pten:Tomato mouse skin at 47 days. l Immunofluorescence images for Tomato (red), Dct (green) and brightfield images in lymph node of c-Kit-CreER:Braf:Pten:Tomato mouse at 47 days. m Survival graph showing the days of survival of indicated mice ( n = the number of mice). Dashed line outlines the boundary of epithelium and dermis. Scale bars, 50 μm. Source data are provided as a Source Data file
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    Dose and timing of doxycycline and 4HT exposure on Cre-mediated recombination efficiency using R26rtTACreER . (A) Individual experiments are numbered, the doxycycline and 4HT regimens are shown schematically, and the number of mice with high/medium, sparse, or no AP+ labeling of RGCs is shown in the color-coded histograms at right. Experiments 1–21 and 22–33 were performed with R26rtTACreER;Brn3a CKOAP/+ and R26rtTACreER;Brn3b CKOAP/+ mice, respectively. (B–F) Representative retina flat mounts (B,C,E,F) and a 200 µm coronal brain section at the level of the superior colliculus (D) were obtained from the numbered experiments indicated in the lower right corner of each panel. A single IP injection was used to deliver 4HT either to the mother for in utero exposure or to individual pups for postnatal exposure.

    Journal: PLoS ONE

    Article Title: New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

    doi: 10.1371/journal.pone.0007859

    Figure Lengend Snippet: Dose and timing of doxycycline and 4HT exposure on Cre-mediated recombination efficiency using R26rtTACreER . (A) Individual experiments are numbered, the doxycycline and 4HT regimens are shown schematically, and the number of mice with high/medium, sparse, or no AP+ labeling of RGCs is shown in the color-coded histograms at right. Experiments 1–21 and 22–33 were performed with R26rtTACreER;Brn3a CKOAP/+ and R26rtTACreER;Brn3b CKOAP/+ mice, respectively. (B–F) Representative retina flat mounts (B,C,E,F) and a 200 µm coronal brain section at the level of the superior colliculus (D) were obtained from the numbered experiments indicated in the lower right corner of each panel. A single IP injection was used to deliver 4HT either to the mother for in utero exposure or to individual pups for postnatal exposure.

    Article Snippet: Doxycycline and 4HT Delivery Doxycycline (Sigma, St. Louis, MO) was mixed with drinking water and/or delivered as premixed food pellets (Research Diets, Inc., New Brunswick, NJ).

    Techniques: Mouse Assay, Labeling, Injection, In Utero

    Morphologies of individual AP+ type 1 DA cells from the retinas of TH-IRES-CreER;R26IAP mice. A single 100–300 µg 4HT injection was administered IP between P3 and P8; retinas were analyzed ∼6 weeks later. Retina flat mounts were processed for AP histochemistry and clarified in BBBA. Axon-like processes (green) and dendrites (blue) were traced with Neuromantic software. The soma is indicated by a red arrow. The outline of the flattened retina and the location of the optic disc are shown in red. (A) Twelve retinas, each of which had a single type 1 DA cell. (B) A retina with two type 1 DA cells. (C) Image of the boxed region of the retina shown in (B). The dendrites (lower right) are uniformly thicker and more darkly stained than the axon-like processes. (D) A putative type 2 DA cell. Scale bars in B and D, 500 µm. Scale bar in B applies to panels A and B. BBBA causes ∼30% tissue shrinkage.

    Journal: PLoS ONE

    Article Title: New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

    doi: 10.1371/journal.pone.0007859

    Figure Lengend Snippet: Morphologies of individual AP+ type 1 DA cells from the retinas of TH-IRES-CreER;R26IAP mice. A single 100–300 µg 4HT injection was administered IP between P3 and P8; retinas were analyzed ∼6 weeks later. Retina flat mounts were processed for AP histochemistry and clarified in BBBA. Axon-like processes (green) and dendrites (blue) were traced with Neuromantic software. The soma is indicated by a red arrow. The outline of the flattened retina and the location of the optic disc are shown in red. (A) Twelve retinas, each of which had a single type 1 DA cell. (B) A retina with two type 1 DA cells. (C) Image of the boxed region of the retina shown in (B). The dendrites (lower right) are uniformly thicker and more darkly stained than the axon-like processes. (D) A putative type 2 DA cell. Scale bars in B and D, 500 µm. Scale bar in B applies to panels A and B. BBBA causes ∼30% tissue shrinkage.

    Article Snippet: Doxycycline and 4HT Delivery Doxycycline (Sigma, St. Louis, MO) was mixed with drinking water and/or delivered as premixed food pellets (Research Diets, Inc., New Brunswick, NJ).

    Techniques: Mouse Assay, Injection, Software, Staining

    Survey of cholinergic neuronal morphologies and projections in the brain and retina of R26IAP;ChAT-IRES-CreER mice. Animals were injected with a single IP injection of 200 µg 4HT at P8. (A,C,F,I,K) 300 µm coronal sections at Bregma positions 1.2, 0, −2.5, −5.0, and −7.8, respectively, as shown schematically in (N). (B,D,E,G,H,J,L) enlargements of boxed regions. (M) In a flat mounted retina, AP-expression is present exclusively in starburst amacrine cells. In the absence of 4HT, retinas were devoid of AP+ cells. BNA, basolateral nucleus of the amygdala; DMN(VN), dorsal motor nucleus of the vagus nerve; LDTN, laterodorsal tegmental nucleus; MS, Medial septum; PPTN; pedunculopontine tegmental nucleus; SI, substantia innominata; St, striatum.

    Journal: PLoS ONE

    Article Title: New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

    doi: 10.1371/journal.pone.0007859

    Figure Lengend Snippet: Survey of cholinergic neuronal morphologies and projections in the brain and retina of R26IAP;ChAT-IRES-CreER mice. Animals were injected with a single IP injection of 200 µg 4HT at P8. (A,C,F,I,K) 300 µm coronal sections at Bregma positions 1.2, 0, −2.5, −5.0, and −7.8, respectively, as shown schematically in (N). (B,D,E,G,H,J,L) enlargements of boxed regions. (M) In a flat mounted retina, AP-expression is present exclusively in starburst amacrine cells. In the absence of 4HT, retinas were devoid of AP+ cells. BNA, basolateral nucleus of the amygdala; DMN(VN), dorsal motor nucleus of the vagus nerve; LDTN, laterodorsal tegmental nucleus; MS, Medial septum; PPTN; pedunculopontine tegmental nucleus; SI, substantia innominata; St, striatum.

    Article Snippet: Doxycycline and 4HT Delivery Doxycycline (Sigma, St. Louis, MO) was mixed with drinking water and/or delivered as premixed food pellets (Research Diets, Inc., New Brunswick, NJ).

    Techniques: Mouse Assay, Injection, Expressing, Mass Spectrometry

    Survey of catecholaminergic neuronal morphologies and projections in the brain of TH-IRES-CreER;R26IAP mice. Animals were injected with 100 µg 4HT at P3. (A–E,G,I,K,L) 300 µm coronal sections at Bregma positions 1.7, 1.1, 0, −1.6, −2.2, −3.2, −4.7, −5.5, −8.2, respectively, as shown schematically in (N). (F,H,J,M) enlargements of boxed regions. A1, A1 noradrenergic cell group; A2, A2 noradrenergic cell group; AcN, accumbens nucleus; AN, arcuate nucleus; CNA, central nucleus of the amygdala; DRN, dorsal raphe nucleus; LC, locus coeruleus; ME, median eminence; PPTN, pedunculopontine tegmental nucleus; SN(PR), substantia nigra (pars reticulata); St, striatum; VP, ventral pallidum; VTA, ventral tegmental area; ZI, zona incerta.

    Journal: PLoS ONE

    Article Title: New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

    doi: 10.1371/journal.pone.0007859

    Figure Lengend Snippet: Survey of catecholaminergic neuronal morphologies and projections in the brain of TH-IRES-CreER;R26IAP mice. Animals were injected with 100 µg 4HT at P3. (A–E,G,I,K,L) 300 µm coronal sections at Bregma positions 1.7, 1.1, 0, −1.6, −2.2, −3.2, −4.7, −5.5, −8.2, respectively, as shown schematically in (N). (F,H,J,M) enlargements of boxed regions. A1, A1 noradrenergic cell group; A2, A2 noradrenergic cell group; AcN, accumbens nucleus; AN, arcuate nucleus; CNA, central nucleus of the amygdala; DRN, dorsal raphe nucleus; LC, locus coeruleus; ME, median eminence; PPTN, pedunculopontine tegmental nucleus; SN(PR), substantia nigra (pars reticulata); St, striatum; VP, ventral pallidum; VTA, ventral tegmental area; ZI, zona incerta.

    Article Snippet: Doxycycline and 4HT Delivery Doxycycline (Sigma, St. Louis, MO) was mixed with drinking water and/or delivered as premixed food pellets (Research Diets, Inc., New Brunswick, NJ).

    Techniques: Mouse Assay, Injection

    R26IAP is a sensitive reporter for Cre-mediated recombination in individual cells. (A–D) R26CreER;R26IAP mice either in the absence (A,B) or following intra-ocular injection (C,D) of 4HT show widespread Cre-mediated recombination in diverse cell types. A, brain; B, retina, C, lens, D, cornea. AP+ lens fiber and lens epithelial cells are seen in C. The overall frequency of AP-expressing cells in the brain and retina in the absence of 4HT is ∼0.01%. (E–G) R26IAP mice that received an IP injection of ∼184 µg of purified His-NLS-Cre protein show scattered Cre-mediated recombination only in epithelial cells lining the peritoneal cavity or in the muscle fibers of the abdominal wall immediately beneath the peritoneal surface. E, liver; F, abdominal wall; G, stomach. (H) R26IAP mice that received an intraocular injection of ∼7 µg of purified His-NLS-Cre protein at ∼P5 show scattered Cre-mediated in RGCs and in occasional astrocytes. RGC axons are converging on the optic nerve head at the top of the panel. (I,J) Retina (I) and cornea (J) following an intra-ocular injection of a replication defective adenovirus expressing Cre. Cre-mediated recombination and AP expression are confined to tissues near the injection site. In the retina, the compact appearance of the AP+ cells and the absence of AP+ axons suggests that most of the labeled cells are astrocytes and/or Muller glia. (K–O) Tissues from R26IAP mice in the absence (K) or following IV injection (L–O) of a replication defective adenovirus expressing Cre (Ad-Cre). K–N, kidney; O, liver. The boxed region of the renal pelvis in L is enlarged in M. N shows a region of renal cortex from a different mouse that received an IV injection of Ad-Cre in which Cre-mediated recombination was also observed in a subset of cortical tubules. Scale bars are: 1 mm for E and O; and 200 µm for D,F,G,H,J,M, and N.

    Journal: PLoS ONE

    Article Title: New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling

    doi: 10.1371/journal.pone.0007859

    Figure Lengend Snippet: R26IAP is a sensitive reporter for Cre-mediated recombination in individual cells. (A–D) R26CreER;R26IAP mice either in the absence (A,B) or following intra-ocular injection (C,D) of 4HT show widespread Cre-mediated recombination in diverse cell types. A, brain; B, retina, C, lens, D, cornea. AP+ lens fiber and lens epithelial cells are seen in C. The overall frequency of AP-expressing cells in the brain and retina in the absence of 4HT is ∼0.01%. (E–G) R26IAP mice that received an IP injection of ∼184 µg of purified His-NLS-Cre protein show scattered Cre-mediated recombination only in epithelial cells lining the peritoneal cavity or in the muscle fibers of the abdominal wall immediately beneath the peritoneal surface. E, liver; F, abdominal wall; G, stomach. (H) R26IAP mice that received an intraocular injection of ∼7 µg of purified His-NLS-Cre protein at ∼P5 show scattered Cre-mediated in RGCs and in occasional astrocytes. RGC axons are converging on the optic nerve head at the top of the panel. (I,J) Retina (I) and cornea (J) following an intra-ocular injection of a replication defective adenovirus expressing Cre. Cre-mediated recombination and AP expression are confined to tissues near the injection site. In the retina, the compact appearance of the AP+ cells and the absence of AP+ axons suggests that most of the labeled cells are astrocytes and/or Muller glia. (K–O) Tissues from R26IAP mice in the absence (K) or following IV injection (L–O) of a replication defective adenovirus expressing Cre (Ad-Cre). K–N, kidney; O, liver. The boxed region of the renal pelvis in L is enlarged in M. N shows a region of renal cortex from a different mouse that received an IV injection of Ad-Cre in which Cre-mediated recombination was also observed in a subset of cortical tubules. Scale bars are: 1 mm for E and O; and 200 µm for D,F,G,H,J,M, and N.

    Article Snippet: Doxycycline and 4HT Delivery Doxycycline (Sigma, St. Louis, MO) was mixed with drinking water and/or delivered as premixed food pellets (Research Diets, Inc., New Brunswick, NJ).

    Techniques: Mouse Assay, Injection, Expressing, Purification, Labeling, IV Injection

    Increased nuclear pore density in OIS is necessary for SAHF formation. ( A ) Schematic showing the depletion experiment for B – E . ( B ) MAB414 (antibody recognizing several nucleoporins) immunostaining in ER:Stop cells treated with 4HT after 2 d of knockdown with scramble (Scr) or POM121 siRNAs. ( Left ) The bottom plane of the nucleus imaged by SIM. ( Right ) Enlargement of the insets . Bars, 2 µm. ( C ) Mean (±SEM) nuclear pore density (pores per square micrometer) in 4HT-treated ER:Stop cells after scramble (Scr) or POM121 siRNA knockdown, as assayed by TPR staining in three biological replicates, (*) P

    Journal: Genes & Development

    Article Title: Nuclear pore density controls heterochromatin reorganization during senescence

    doi: 10.1101/gad.321117.118

    Figure Lengend Snippet: Increased nuclear pore density in OIS is necessary for SAHF formation. ( A ) Schematic showing the depletion experiment for B – E . ( B ) MAB414 (antibody recognizing several nucleoporins) immunostaining in ER:Stop cells treated with 4HT after 2 d of knockdown with scramble (Scr) or POM121 siRNAs. ( Left ) The bottom plane of the nucleus imaged by SIM. ( Right ) Enlargement of the insets . Bars, 2 µm. ( C ) Mean (±SEM) nuclear pore density (pores per square micrometer) in 4HT-treated ER:Stop cells after scramble (Scr) or POM121 siRNA knockdown, as assayed by TPR staining in three biological replicates, (*) P

    Article Snippet: Ras translocation to the nucleus was induced by addition of 4HT (Sigma) diluted in DMSO to 100 nM.

    Techniques: Immunostaining, Staining

    TPR is necessary for the SASP. ( A ) Mean (±SEM) mRNA level measured by quantitative RT–PCR (qRT–PCR) for SASP genes ( IL1A , IL1B , IL6 , and IL8 ) in 4HT-treated ER:Stop and ER:Ras cells after knockdown with scramble (Scr) siRNAs and in 4HT-treated ER:Ras cells with TPR siRNAs. Expression is relative to ER:Ras cells transfected with Scr siRNAs. Data are from three experiments. (h.s.) Highly significant. ( B ) Mean (±SEM) percentage of cells positive by immunostaining for SASP cytokines (IL1α, IL1β, IL6, and IL8) in 4HT-treated ER:Stop and ER:Ras cells after siRNA knockdown as in A . Data are from three experiments. (**) P

    Journal: Genes & Development

    Article Title: Nuclear pore density controls heterochromatin reorganization during senescence

    doi: 10.1101/gad.321117.118

    Figure Lengend Snippet: TPR is necessary for the SASP. ( A ) Mean (±SEM) mRNA level measured by quantitative RT–PCR (qRT–PCR) for SASP genes ( IL1A , IL1B , IL6 , and IL8 ) in 4HT-treated ER:Stop and ER:Ras cells after knockdown with scramble (Scr) siRNAs and in 4HT-treated ER:Ras cells with TPR siRNAs. Expression is relative to ER:Ras cells transfected with Scr siRNAs. Data are from three experiments. (h.s.) Highly significant. ( B ) Mean (±SEM) percentage of cells positive by immunostaining for SASP cytokines (IL1α, IL1β, IL6, and IL8) in 4HT-treated ER:Stop and ER:Ras cells after siRNA knockdown as in A . Data are from three experiments. (**) P

    Article Snippet: Ras translocation to the nucleus was induced by addition of 4HT (Sigma) diluted in DMSO to 100 nM.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Immunostaining

    Nuclear pore density increases in OIS. ( A ) Model of the NPC showing the position of TPR, NUP153, and POM121. ( B ) Schematic showing the balance of forces attracting heterochromatin to the NL and repelling heterochromatin from nuclear pores. ( C ) Schematic of OIS induction in ER:Ras cells by 4-hydroxy-tamoxifen (4HT) and continued proliferation in ER:Stop cells. ( D ) Western blot showing POM121 ( left panel) and TPR ( right panel) levels in 4HT-treated ER:Stop and ER:Ras cells. ( E ) TPR immunostaining in ER:Stop and ER:Ras cells treated with 4HT. ( Left ) The bottom plane of a nucleus imaged by structured illuminated microscopy (SIM). ( Right ) Enlargement of the insets . Bars, 2 µm. ( F ) Mean (±SEM) nuclear pore density (pores per square micrometer) in 4HT-treated ER:Stop and ER:Ras cells as counted by TPR staining in three biological replicates. (***) P = 0.0001. ( G ) As in F , but for Pom121 staining. (h.s.) Highly significant ( P = 1.3 × 10 −06 ).

    Journal: Genes & Development

    Article Title: Nuclear pore density controls heterochromatin reorganization during senescence

    doi: 10.1101/gad.321117.118

    Figure Lengend Snippet: Nuclear pore density increases in OIS. ( A ) Model of the NPC showing the position of TPR, NUP153, and POM121. ( B ) Schematic showing the balance of forces attracting heterochromatin to the NL and repelling heterochromatin from nuclear pores. ( C ) Schematic of OIS induction in ER:Ras cells by 4-hydroxy-tamoxifen (4HT) and continued proliferation in ER:Stop cells. ( D ) Western blot showing POM121 ( left panel) and TPR ( right panel) levels in 4HT-treated ER:Stop and ER:Ras cells. ( E ) TPR immunostaining in ER:Stop and ER:Ras cells treated with 4HT. ( Left ) The bottom plane of a nucleus imaged by structured illuminated microscopy (SIM). ( Right ) Enlargement of the insets . Bars, 2 µm. ( F ) Mean (±SEM) nuclear pore density (pores per square micrometer) in 4HT-treated ER:Stop and ER:Ras cells as counted by TPR staining in three biological replicates. (***) P = 0.0001. ( G ) As in F , but for Pom121 staining. (h.s.) Highly significant ( P = 1.3 × 10 −06 ).

    Article Snippet: Ras translocation to the nucleus was induced by addition of 4HT (Sigma) diluted in DMSO to 100 nM.

    Techniques: Western Blot, Immunostaining, Microscopy, Staining

    Chromatin reorganization seems necessary for the SASP. ( A ) Mean (±SEM) ASF1a mRNA level established by qRT–PCR in 4HT-treated ER:Stop and ER:Ras cells after knockdown with scramble (Scr) or ASF1a siRNAs. Expression is shown relative to ER:Stop cells transfected with Scr siRNAs. Data are from three experiments. (*) P

    Journal: Genes & Development

    Article Title: Nuclear pore density controls heterochromatin reorganization during senescence

    doi: 10.1101/gad.321117.118

    Figure Lengend Snippet: Chromatin reorganization seems necessary for the SASP. ( A ) Mean (±SEM) ASF1a mRNA level established by qRT–PCR in 4HT-treated ER:Stop and ER:Ras cells after knockdown with scramble (Scr) or ASF1a siRNAs. Expression is shown relative to ER:Stop cells transfected with Scr siRNAs. Data are from three experiments. (*) P

    Article Snippet: Ras translocation to the nucleus was induced by addition of 4HT (Sigma) diluted in DMSO to 100 nM.

    Techniques: Quantitative RT-PCR, Expressing, Transfection

    TPR is necessary for SAHF formation and maintenance. ( A ) DAPI staining of nonsenescent 4HT-treated ER:Stop and OIS (ER:Ras) cells after control scramble (Scr) siRNA and upon TPR depletion (siTPR). Bars, 10 µm. ( B ) Mean (±SEM) percentage of cells containing SAHFs in 4HT-treated ER:Stop and ER:Ras cells after knockdown siRNAs as in A . Data are from three experiments. (**) P

    Journal: Genes & Development

    Article Title: Nuclear pore density controls heterochromatin reorganization during senescence

    doi: 10.1101/gad.321117.118

    Figure Lengend Snippet: TPR is necessary for SAHF formation and maintenance. ( A ) DAPI staining of nonsenescent 4HT-treated ER:Stop and OIS (ER:Ras) cells after control scramble (Scr) siRNA and upon TPR depletion (siTPR). Bars, 10 µm. ( B ) Mean (±SEM) percentage of cells containing SAHFs in 4HT-treated ER:Stop and ER:Ras cells after knockdown siRNAs as in A . Data are from three experiments. (**) P

    Article Snippet: Ras translocation to the nucleus was induced by addition of 4HT (Sigma) diluted in DMSO to 100 nM.

    Techniques: Staining

    Stabilizing beta-catenin signaling suppresses EC fenestrae and reduces vascular permeability. ( A–F ) Transmission electron micrographs from control mice (left) and from age-matched mice with EC-specific deletion of Ctnnb1 exon 3 (right) showing representative sections of choriocapillaris ( A,B ), posterior pituitary ( C,D ), and choroid plexus ( E,F ). RPE, retinal pigment epithelium. Vertical arrows mark fenestrae. Neurohormone secretory granules are seen in the posterior pituitary parenchyma. Scale bar, 500 nm. ( G ) Quantification of the density of capillary EC fenestrae in age-matched and 4HT-treated control ( Ctnnb1 flex3/+ ) vs. Ctnnb1 exon 3 stabilized ( Ctnnb1 flex3/+ ;Pdgfb-CreER ) mice for the choriocapillaris, posterior pituitary, choroid plexus, and ciliary body. Each data-point represents all of the vascular wall length within a single 10 μm x 10 μm TEM image. Bars show mean ± S.D. Each cluster of data points represents a different mouse. For each location and genotype, 3–5 mice were analyzed. For each of the four anatomic locations, the p-values are calculated for all of the Ctnnb1 flex3/+ vs. all of the Ctnnb1 flex3/+ ;Pdgfb-CreER data-points. ( H,I ) Sagittal brain sections show vascular markers GLUT1 and PLVAP and perivascular accumulation of Sulfo-NHS-biotin (following IP injection) in the SFO and adjacent choroid plexus ( H ) and in the area postrema and adjacent choroid plexus ( I ) from ~P30 WT vs. Ctnnb1 flex3/+ ;Pdgfb-CreER mice. AP, area postrema; CP, choroid plexus. A, anterior; P, posterior. Scale bars in ( H ) and ( I ), 200 μm.

    Journal: eLife

    Article Title: Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures

    doi: 10.7554/eLife.43257

    Figure Lengend Snippet: Stabilizing beta-catenin signaling suppresses EC fenestrae and reduces vascular permeability. ( A–F ) Transmission electron micrographs from control mice (left) and from age-matched mice with EC-specific deletion of Ctnnb1 exon 3 (right) showing representative sections of choriocapillaris ( A,B ), posterior pituitary ( C,D ), and choroid plexus ( E,F ). RPE, retinal pigment epithelium. Vertical arrows mark fenestrae. Neurohormone secretory granules are seen in the posterior pituitary parenchyma. Scale bar, 500 nm. ( G ) Quantification of the density of capillary EC fenestrae in age-matched and 4HT-treated control ( Ctnnb1 flex3/+ ) vs. Ctnnb1 exon 3 stabilized ( Ctnnb1 flex3/+ ;Pdgfb-CreER ) mice for the choriocapillaris, posterior pituitary, choroid plexus, and ciliary body. Each data-point represents all of the vascular wall length within a single 10 μm x 10 μm TEM image. Bars show mean ± S.D. Each cluster of data points represents a different mouse. For each location and genotype, 3–5 mice were analyzed. For each of the four anatomic locations, the p-values are calculated for all of the Ctnnb1 flex3/+ vs. all of the Ctnnb1 flex3/+ ;Pdgfb-CreER data-points. ( H,I ) Sagittal brain sections show vascular markers GLUT1 and PLVAP and perivascular accumulation of Sulfo-NHS-biotin (following IP injection) in the SFO and adjacent choroid plexus ( H ) and in the area postrema and adjacent choroid plexus ( I ) from ~P30 WT vs. Ctnnb1 flex3/+ ;Pdgfb-CreER mice. AP, area postrema; CP, choroid plexus. A, anterior; P, posterior. Scale bars in ( H ) and ( I ), 200 μm.

    Article Snippet: 4HT preparation and administration Solid 4HT (Sigma-Aldrich H7904) was dissolved at 20 mg/ml in ethanol by extensive vortexing.

    Techniques: Permeability, Transmission Assay, Mouse Assay, Transmission Electron Microscopy, Injection

    Assessing the specificity and sensitivity of the R26-8xTCF/LEF-LSL-H2B-GFP canonical Wnt reporter in the retinal vasculature by assessing its response to decreased or increased beta-catenin signaling. Flatmounted retinas with EC-specific Cre recombination were stained with GS lectin to visualize ECs and macrophages, and immunostained with anti-myc antibodies for H2B-GFP-6xmyc. In developing retinas [P6-P8]; panels ( A ), ( B ), ( D ), and ( E ), the LSL cassette has been removed in all ECs using Tie2-Cre . ECs in R26-8xTCF/LEF-LSL-H2B-GFP;Ndp KO ;Tie2-Cre ( A ) and R26-8xTCF/LEF-LSL-H2B-GFP;Fz4 CKO/- ;Tie2-Cre ( B ) retinas have little or no beta-catenin signaling and show a nearly complete loss of expression of the nuclear-localized H2B-GFP reporter. [ Ndp is X-linked, and ‘ Ndp KO ’ refers to both male Ndp -/Y males and Ndp -/- females.] Littermate controls were heterozygous for Ndp or Fz4 ( D, E ) and show reporter expression at approximately WT levels. In mature retinas [P21]; panels ( C ) and ( F ), the LSL cassette was removed in nearly all ECs using Pdgfb-CreER and 200 ug 4HT at P4. ECs in the R26-8xTCF/LEF-LSL-H2B-GFP;Ctnnb1 flex3/+ ;Pdgfb-CreER retina ( C ) have high levels of beta-catenin signaling and show a higher level of accumulation of the nuclear-localized H2B-GFP reporter compared to the 8xTCF/LEF-LSL-H2B-GFP;Pdgfb-CreER control retina ( F ) that lacks Ctnnb1 flex3 . Paired experimental and control retinas were processed in parallel and imaged with identical microscope settings, and the resulting images were processed identically. Scale bar, 200 μm.

    Journal: eLife

    Article Title: Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures

    doi: 10.7554/eLife.43257

    Figure Lengend Snippet: Assessing the specificity and sensitivity of the R26-8xTCF/LEF-LSL-H2B-GFP canonical Wnt reporter in the retinal vasculature by assessing its response to decreased or increased beta-catenin signaling. Flatmounted retinas with EC-specific Cre recombination were stained with GS lectin to visualize ECs and macrophages, and immunostained with anti-myc antibodies for H2B-GFP-6xmyc. In developing retinas [P6-P8]; panels ( A ), ( B ), ( D ), and ( E ), the LSL cassette has been removed in all ECs using Tie2-Cre . ECs in R26-8xTCF/LEF-LSL-H2B-GFP;Ndp KO ;Tie2-Cre ( A ) and R26-8xTCF/LEF-LSL-H2B-GFP;Fz4 CKO/- ;Tie2-Cre ( B ) retinas have little or no beta-catenin signaling and show a nearly complete loss of expression of the nuclear-localized H2B-GFP reporter. [ Ndp is X-linked, and ‘ Ndp KO ’ refers to both male Ndp -/Y males and Ndp -/- females.] Littermate controls were heterozygous for Ndp or Fz4 ( D, E ) and show reporter expression at approximately WT levels. In mature retinas [P21]; panels ( C ) and ( F ), the LSL cassette was removed in nearly all ECs using Pdgfb-CreER and 200 ug 4HT at P4. ECs in the R26-8xTCF/LEF-LSL-H2B-GFP;Ctnnb1 flex3/+ ;Pdgfb-CreER retina ( C ) have high levels of beta-catenin signaling and show a higher level of accumulation of the nuclear-localized H2B-GFP reporter compared to the 8xTCF/LEF-LSL-H2B-GFP;Pdgfb-CreER control retina ( F ) that lacks Ctnnb1 flex3 . Paired experimental and control retinas were processed in parallel and imaged with identical microscope settings, and the resulting images were processed identically. Scale bar, 200 μm.

    Article Snippet: 4HT preparation and administration Solid 4HT (Sigma-Aldrich H7904) was dissolved at 20 mg/ml in ethanol by extensive vortexing.

    Techniques: Staining, Expressing, Microscopy

    Epidermal melanoma cells invade the dermis and undergo phenotypic switch to a neuronal-like state. a Schematic showing TAM treatment and skin implantation onto nude mice for b – h . b , c Immunofluorescence for GFP at 10 days b and 17 days c after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. Arrowheads point to GFP + melanoma cells that invade into the dermis. d Immunofluorescence for GFP (green) and E-cadherin, MCAM (red) at 17 days after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. e Immunofluorescence for Sox10 (red) and E-cadherin, MCAM (green) in human melanoma specimen during the invasive radial growth phase. f , g Immunofluorescence images for GFP (green) and Dct, MITF, Nestin, GFAP, Tubb3 (red) and brightfield images in areas of dermal melanoma at 22 days f and 30 days g after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. h Dot plot showing percentage of pigment, Dct, Nestin, GFAP and Tubb3 in GFP + dermal melanoma cells (mean ± s.d.; 3 independent tumors from 3 mice were analyzed in each group, n = the number of randomly selected tumor areas analyzed. At least 1500 GFP + tumor cells were analyzed in each group). i Schematic model illustration showing that upon oncogenic induction, McSCs first give rise to epidermal melanoma. Then melanoma cells in the epithelial compartments invade into the dermis. Finally, dermal melanoma cells undergo a phenotypic switch to acquire neuronal signatures. j Schematic showing 4-hydroxytamoxifen (4HT-TAM) treatment and analysis regimen. k Immunofluorescence images for Tomato (red) and Sox10, Dct, Tubb3, Nestin (green) and brightfield images in c-Kit-CreER:Braf:Pten:Tomato mouse skin at 47 days. l Immunofluorescence images for Tomato (red), Dct (green) and brightfield images in lymph node of c-Kit-CreER:Braf:Pten:Tomato mouse at 47 days. m Survival graph showing the days of survival of indicated mice ( n = the number of mice). Dashed line outlines the boundary of epithelium and dermis. Scale bars, 50 μm. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease

    doi: 10.1038/s41467-019-12733-1

    Figure Lengend Snippet: Epidermal melanoma cells invade the dermis and undergo phenotypic switch to a neuronal-like state. a Schematic showing TAM treatment and skin implantation onto nude mice for b – h . b , c Immunofluorescence for GFP at 10 days b and 17 days c after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. Arrowheads point to GFP + melanoma cells that invade into the dermis. d Immunofluorescence for GFP (green) and E-cadherin, MCAM (red) at 17 days after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. e Immunofluorescence for Sox10 (red) and E-cadherin, MCAM (green) in human melanoma specimen during the invasive radial growth phase. f , g Immunofluorescence images for GFP (green) and Dct, MITF, Nestin, GFAP, Tubb3 (red) and brightfield images in areas of dermal melanoma at 22 days f and 30 days g after skin implantation in c-Kit-CreER:Braf:Pten:GFP mice. h Dot plot showing percentage of pigment, Dct, Nestin, GFAP and Tubb3 in GFP + dermal melanoma cells (mean ± s.d.; 3 independent tumors from 3 mice were analyzed in each group, n = the number of randomly selected tumor areas analyzed. At least 1500 GFP + tumor cells were analyzed in each group). i Schematic model illustration showing that upon oncogenic induction, McSCs first give rise to epidermal melanoma. Then melanoma cells in the epithelial compartments invade into the dermis. Finally, dermal melanoma cells undergo a phenotypic switch to acquire neuronal signatures. j Schematic showing 4-hydroxytamoxifen (4HT-TAM) treatment and analysis regimen. k Immunofluorescence images for Tomato (red) and Sox10, Dct, Tubb3, Nestin (green) and brightfield images in c-Kit-CreER:Braf:Pten:Tomato mouse skin at 47 days. l Immunofluorescence images for Tomato (red), Dct (green) and brightfield images in lymph node of c-Kit-CreER:Braf:Pten:Tomato mouse at 47 days. m Survival graph showing the days of survival of indicated mice ( n = the number of mice). Dashed line outlines the boundary of epithelium and dermis. Scale bars, 50 μm. Source data are provided as a Source Data file

    Article Snippet: To induce Cre recombination, Tamoxifen (TAM) (Sigma-Aldrich) treatment was performed by intraperitoneal (i.p.) injection (0.1 mg/g body weight) of a 20 mg/ml solution in corn oil per day except for one experiment (Fig. ) in which mice were topically treated with 4-hydroxytamoxifen (4HT-TAM) (Sigma-Aldrich).

    Techniques: Mouse Assay, Immunofluorescence