dmba  (Worthington Biochemical)


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    Name:
    Neutral Protease Dispase Purified
    Description:
    Animal Origin Free Chromatographically purified A lyophilized powder
    Catalog Number:
    ls02100
    Price:
    70
    Size:
    10 mg
    Source:
    Bacillus polymyxa
    Cas Number:
    42613.33.2
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    Structured Review

    Worthington Biochemical dmba
    The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, <t>TPA-treated</t> skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the <t>DMBA-TPA</t> treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P
    Animal Origin Free Chromatographically purified A lyophilized powder
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    dmba - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors"

    Article Title: Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2016.05.002

    The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, TPA-treated skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the DMBA-TPA treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P
    Figure Legend Snippet: The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, TPA-treated skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the DMBA-TPA treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P

    Techniques Used: Over Expression, Papanicolaou Stain, FACS, Mouse Assay

    DMBA-TPA-induced skin carcinogenesis in VEGF-D knockout mice. Tumor incidence (A) and cumulative multiplicity (B) in the KO males (N = 26) and the WT males (N = 27) did not show statistically significant differences between the genotypes. ( C-D ) Expression of VEGF-D in mouse skin tumors. Representative immunohistochemical stainings showing VEGF-D expression in mouse cSCCs. (C) Consecutive sections of the WT cSCC stained with antibodies against VEGF-D and F4/80 demonstrate that VEGF-D is expressed by F4/80-positive macrophages (arrowheads) within the WT cSCC stroma. Asterisks indicate vessels that facilitate the identification of co-stained cells in the consecutive sections. (D) VEGF-D expression by invasive carcinoma cells (arrowheads) within the WT cSCC. The KO cSCC is used as a negative control for VEGF-D staining. (E) The quantification of lymphatic vessel density in the untreated (UT) skin, TPA-treated skin, and papillomas (PAP) and cSCCs collected at different time points of skin carcinogenesis. The KO tumors showed a trend for reduced lymphangiogenesis. In the UT skin, and in cSCCs at week 29, the difference between the genotypes was significant. (F) No significant differences in blood vessel densities between the KO and WT males in any type of sample. Scale bars: 50 μm (C) and 100 μm (D): Error bars: SD. *: P
    Figure Legend Snippet: DMBA-TPA-induced skin carcinogenesis in VEGF-D knockout mice. Tumor incidence (A) and cumulative multiplicity (B) in the KO males (N = 26) and the WT males (N = 27) did not show statistically significant differences between the genotypes. ( C-D ) Expression of VEGF-D in mouse skin tumors. Representative immunohistochemical stainings showing VEGF-D expression in mouse cSCCs. (C) Consecutive sections of the WT cSCC stained with antibodies against VEGF-D and F4/80 demonstrate that VEGF-D is expressed by F4/80-positive macrophages (arrowheads) within the WT cSCC stroma. Asterisks indicate vessels that facilitate the identification of co-stained cells in the consecutive sections. (D) VEGF-D expression by invasive carcinoma cells (arrowheads) within the WT cSCC. The KO cSCC is used as a negative control for VEGF-D staining. (E) The quantification of lymphatic vessel density in the untreated (UT) skin, TPA-treated skin, and papillomas (PAP) and cSCCs collected at different time points of skin carcinogenesis. The KO tumors showed a trend for reduced lymphangiogenesis. In the UT skin, and in cSCCs at week 29, the difference between the genotypes was significant. (F) No significant differences in blood vessel densities between the KO and WT males in any type of sample. Scale bars: 50 μm (C) and 100 μm (D): Error bars: SD. *: P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Immunohistochemistry, Staining, Negative Control

    Impaired skin tumor formation in TG K14-mVEGF-D mice in a chemical skin carcinogenesis model. Tumors were induced in the mouse skin using a multistage DMBA-TPA protocol and their growth was monitored for up to 30 weeks (Wk). Data from two separate experimental groups of males (WT N = 23, TG N = 25) are combined. (A) Representative photographs of the WT and K14-mVEGF-D TG male mice at different time points. Black arrowheads indicate exophytic papillomas and white arrowheads regressing papillomas. (B) Tumor incidence showed a significant delay in the TG mice. (C) The TG mice developed markedly less tumors than the WT mice. Cumulative tumor multiplicity is shown. (D) The percentage of regressing papillomas (the black portion of the column) was higher in the TG mice (red) than in the WT mice (green). (E) Smaller tumor size in the TG mice than in the WT mice. Tumor sizes are shown as a percentage of tumors in three different size categories. Statistical significance for each size category is shown in between the column pairs. (F) At weeks 20–30 the percentage of TG male mice with clinically apparent cSCC was lower than that of WT control males. (G) A trend towards less cSCCs per mouse was observed in TG males. N = number of mice. Error bars: SD.*: P
    Figure Legend Snippet: Impaired skin tumor formation in TG K14-mVEGF-D mice in a chemical skin carcinogenesis model. Tumors were induced in the mouse skin using a multistage DMBA-TPA protocol and their growth was monitored for up to 30 weeks (Wk). Data from two separate experimental groups of males (WT N = 23, TG N = 25) are combined. (A) Representative photographs of the WT and K14-mVEGF-D TG male mice at different time points. Black arrowheads indicate exophytic papillomas and white arrowheads regressing papillomas. (B) Tumor incidence showed a significant delay in the TG mice. (C) The TG mice developed markedly less tumors than the WT mice. Cumulative tumor multiplicity is shown. (D) The percentage of regressing papillomas (the black portion of the column) was higher in the TG mice (red) than in the WT mice (green). (E) Smaller tumor size in the TG mice than in the WT mice. Tumor sizes are shown as a percentage of tumors in three different size categories. Statistical significance for each size category is shown in between the column pairs. (F) At weeks 20–30 the percentage of TG male mice with clinically apparent cSCC was lower than that of WT control males. (G) A trend towards less cSCCs per mouse was observed in TG males. N = number of mice. Error bars: SD.*: P

    Techniques Used: Mouse Assay

    2) Product Images from "Antioxidant Peroxiredoxin 6 protein rescues toxicity due to oxidative stress and cellular hypoxia in vitro, and attenuates prion-related pathology in vivo"

    Article Title: Antioxidant Peroxiredoxin 6 protein rescues toxicity due to oxidative stress and cellular hypoxia in vitro, and attenuates prion-related pathology in vivo

    Journal: Neurochemistry international

    doi: 10.1016/j.neuint.2015.08.006

    Prdx6 expression in nonTg, Prdx6 Tg and Prdx6 KO animals. ( A ) Western blot analysis to confirm Prdx6 expression. Primary neurons, primary astrocytes and brain homogenates from naïve wild type nonTg, Prdx6 Tg and Prdx6 KO animals were probed for
    Figure Legend Snippet: Prdx6 expression in nonTg, Prdx6 Tg and Prdx6 KO animals. ( A ) Western blot analysis to confirm Prdx6 expression. Primary neurons, primary astrocytes and brain homogenates from naïve wild type nonTg, Prdx6 Tg and Prdx6 KO animals were probed for

    Techniques Used: Expressing, Western Blot

    ( A ) MTT assay showing that TAT-Prdx6 (5ug/ml) attenuates H 2 O 2 and CoCl 2 mediated cytotoxicity in SK-N-SH human neuroblastoma cells. Cells were cultured and treated for 48 h with indicated concentrations of H 2 O 2 and CoCl 2 . The capacity of the cells to
    Figure Legend Snippet: ( A ) MTT assay showing that TAT-Prdx6 (5ug/ml) attenuates H 2 O 2 and CoCl 2 mediated cytotoxicity in SK-N-SH human neuroblastoma cells. Cells were cultured and treated for 48 h with indicated concentrations of H 2 O 2 and CoCl 2 . The capacity of the cells to

    Techniques Used: MTT Assay, Cell Culture

    Representative western blots showing change in prion related biochemistry representative of n=6 per group. We observed a significant increase in PrP immunoreactivity (IR) (PrP C /PrP Sc ) as detected by anti-PrP 6D11 mAb in nonTg, Prdx6 KO and Prdx6 Tg-ME7
    Figure Legend Snippet: Representative western blots showing change in prion related biochemistry representative of n=6 per group. We observed a significant increase in PrP immunoreactivity (IR) (PrP C /PrP Sc ) as detected by anti-PrP 6D11 mAb in nonTg, Prdx6 KO and Prdx6 Tg-ME7

    Techniques Used: Western Blot

    3) Product Images from "Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis"

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.09.018

    TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). (E) Time-lapse IVM images ( Video S1 ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). (G) IVM images ( Video S2 ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p
    Figure Legend Snippet: TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). (E) Time-lapse IVM images ( Video S1 ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). (G) IVM images ( Video S2 ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p

    Techniques Used: Mouse Assay, In Vivo, Labeling, Blocking Assay, Injection, Transmission Electron Microscopy

    Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. (C) Time-lapse IVM images ( Video S3 ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). (H) Time-lapse confocal IVM images ( Video S4 ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.
    Figure Legend Snippet: Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. (C) Time-lapse IVM images ( Video S3 ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). (H) Time-lapse confocal IVM images ( Video S4 ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Techniques Used: Mouse Assay, In Vivo, Labeling, Injection, Blocking Assay, Transmission Electron Microscopy, Inhibition

    4) Product Images from "Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis"

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.09.018

    TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p
    Figure Legend Snippet: TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p

    Techniques Used: Mouse Assay, In Vivo, Labeling, Blocking Assay, Injection, Transmission Electron Microscopy

    Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.
    Figure Legend Snippet: Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Techniques Used: Mouse Assay, In Vivo, Labeling, Injection, Blocking Assay, Transmission Electron Microscopy, Inhibition

    5) Product Images from "Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis"

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.09.018

    CXCL1 and CXCL2 Are Differentially Expressed in TNF-Stimulated Tissues (A–E) WT mice were treated i.s. with PBS or TNF and cremaster muscles were IF stained for CXCL1 or CXCL2 and CD31 (ECs), α-SMA (pericytes), and MRP14 (neutrophils). Representative confocal images of venules showing CXCL1 (A) or CXCL2 (C) staining within EC and pericyte isosurface masks and quantifications of CXCL1 (B) and CXCL2 (D) in ECs or pericytes, in terms of MFIs (n = 4 mice per group) from 4 independent experiments. (E) Confocal abluminal and cross-sectional images (acquired along the dashed line and presented at 90° rotation) of a venule showing overall CXCL2 staining representative of 4 independent experiments. (F) Cxcl1 and Cxcl2 mRNA levels relative to Gapdh in circulating neutrophils 2–3 hr after i.s. PBS or TNF injection as determined by real-time PCR (n = 4–5 mice per group, 2 independent experiments). (G–J) Purified mouse bone marrow neutrophils were treated with PBS or TNF (1 nM, 1 hr) on uncoated (G, H), BSA-coated, or CXCL1-coated wells (I, J). CXCL1 and/or CXCL2 levels in lysates (G and I) and supernatants (H and J) as quantified by ELISA (n = 4–12) from 2–4 independent experiments. Means ± SEM, ∗ p
    Figure Legend Snippet: CXCL1 and CXCL2 Are Differentially Expressed in TNF-Stimulated Tissues (A–E) WT mice were treated i.s. with PBS or TNF and cremaster muscles were IF stained for CXCL1 or CXCL2 and CD31 (ECs), α-SMA (pericytes), and MRP14 (neutrophils). Representative confocal images of venules showing CXCL1 (A) or CXCL2 (C) staining within EC and pericyte isosurface masks and quantifications of CXCL1 (B) and CXCL2 (D) in ECs or pericytes, in terms of MFIs (n = 4 mice per group) from 4 independent experiments. (E) Confocal abluminal and cross-sectional images (acquired along the dashed line and presented at 90° rotation) of a venule showing overall CXCL2 staining representative of 4 independent experiments. (F) Cxcl1 and Cxcl2 mRNA levels relative to Gapdh in circulating neutrophils 2–3 hr after i.s. PBS or TNF injection as determined by real-time PCR (n = 4–5 mice per group, 2 independent experiments). (G–J) Purified mouse bone marrow neutrophils were treated with PBS or TNF (1 nM, 1 hr) on uncoated (G, H), BSA-coated, or CXCL1-coated wells (I, J). CXCL1 and/or CXCL2 levels in lysates (G and I) and supernatants (H and J) as quantified by ELISA (n = 4–12) from 2–4 independent experiments. Means ± SEM, ∗ p

    Techniques Used: Mouse Assay, Staining, Injection, Real-time Polymerase Chain Reaction, Purification, Enzyme-linked Immunosorbent Assay

    Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. (C) Time-lapse IVM images ( Video S3 ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). (H) Time-lapse confocal IVM images ( Video S4 ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.
    Figure Legend Snippet: Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. (C) Time-lapse IVM images ( Video S3 ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). (H) Time-lapse confocal IVM images ( Video S4 ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Techniques Used: Mouse Assay, In Vivo, Labeling, Injection, Blocking Assay, Transmission Electron Microscopy, Inhibition

    TNF-Induced Neutrophil Migration Is Dependent on Both CXCL1 and CXCL2 WT mice pre-treated intrascrotally (i.s.) with control (ctr) or blocking mAbs or Cxcr2 −/− mice were subjected to i.s. injections of PBS or TNF. The cremaster muscles were immunostained for MRP14 (neutrophils) and α-SMA (pericytes) and analyzed for neutrophil infiltration by confocal microscopy. (A) and (C) are representative images and (B), (D), and (E) show quantifications (n = 4–10 mice per group) from 5–6 independent experiments. Means ± SEM, ∗∗∗ p
    Figure Legend Snippet: TNF-Induced Neutrophil Migration Is Dependent on Both CXCL1 and CXCL2 WT mice pre-treated intrascrotally (i.s.) with control (ctr) or blocking mAbs or Cxcr2 −/− mice were subjected to i.s. injections of PBS or TNF. The cremaster muscles were immunostained for MRP14 (neutrophils) and α-SMA (pericytes) and analyzed for neutrophil infiltration by confocal microscopy. (A) and (C) are representative images and (B), (D), and (E) show quantifications (n = 4–10 mice per group) from 5–6 independent experiments. Means ± SEM, ∗∗∗ p

    Techniques Used: Migration, Mouse Assay, Blocking Assay, Confocal Microscopy

    6) Product Images from "Multipotent luminal mammary cancer stem cells model tumor heterogeneity"

    Article Title: Multipotent luminal mammary cancer stem cells model tumor heterogeneity

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-015-0615-y

    MaCSCs have the capacity to differentiate into multiple cell types. ( a ) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). ( b ) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. ( c ) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. ( d ) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. ( e,f ) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. ( g ) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. ( h ) Confluent Py230 cells spontaneously form domes. ( i ) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. ( j ) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. ( k ) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm
    Figure Legend Snippet: MaCSCs have the capacity to differentiate into multiple cell types. ( a ) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). ( b ) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. ( c ) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. ( d ) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. ( e,f ) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. ( g ) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. ( h ) Confluent Py230 cells spontaneously form domes. ( i ) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. ( j ) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. ( k ) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm

    Techniques Used: Staining, Marker, Expressing

    7) Product Images from "Multipotent luminal mammary cancer stem cells model tumor heterogeneity"

    Article Title: Multipotent luminal mammary cancer stem cells model tumor heterogeneity

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-015-0615-y

    Py230 mammary cancer stem cells recapitulate spontaneous luminal tumorigenesis and metastasis. a-f , H E stained sections of Py230 MIN ( a ), tumor ( b ) and metastasis ( c ) compared with spontaneous PyVmT MIN, ( d ), tumor ( e ) and metastasis ( f ). Scale bar = 50 μm. g-j Whole mounts of normal mammary gland (NMG), pre-lactating mammary gland (PLMG), PyVmT MIN and a 10-day outgrowth of Py230 tumor cells in the cleared mammary fat pad. Scale bar = 100 μm. k-r , Representative dot plots of NMG ( k,o ), PLMG ( l,p ), PyVmT MIN ( m,q ) and Py230 tumor ( n,r ) labeled with CD24 and CD49f ( k-n ) or CD24 and CD29 ( o-r )
    Figure Legend Snippet: Py230 mammary cancer stem cells recapitulate spontaneous luminal tumorigenesis and metastasis. a-f , H E stained sections of Py230 MIN ( a ), tumor ( b ) and metastasis ( c ) compared with spontaneous PyVmT MIN, ( d ), tumor ( e ) and metastasis ( f ). Scale bar = 50 μm. g-j Whole mounts of normal mammary gland (NMG), pre-lactating mammary gland (PLMG), PyVmT MIN and a 10-day outgrowth of Py230 tumor cells in the cleared mammary fat pad. Scale bar = 100 μm. k-r , Representative dot plots of NMG ( k,o ), PLMG ( l,p ), PyVmT MIN ( m,q ) and Py230 tumor ( n,r ) labeled with CD24 and CD49f ( k-n ) or CD24 and CD29 ( o-r )

    Techniques Used: Staining, Labeling

    8) Product Images from "Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis"

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20200288

    tmTNF overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and nontg littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P
    Figure Legend Snippet: tmTNF overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and nontg littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P

    Techniques Used: Over Expression, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, MANN-WHITNEY

    Endochondral new bone formation is only present in tmTNF tg mouse. (A) Safranin-O/fast green stainings of ankle joints of a nontg, tmTNF tg , and TNF ΔARE mouse reveals that the tmTNF tg ankle has clear bone formation, while the TNF ΔARE and nontg do not. Scale bars, 500 µm. (B) Safranin-O/fast green stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE mouse. The tmTNF tg axial joint reveals inflammation adjacent to bone formation, whereas the TNF ΔARE and nontg do not have these features. Scale bars, 500 µm. (C) Magnified view of tmTNF tg axial and ankle joint of region with endochondral ossification. Scale bars, 200 µm. n = 3 mice per group.
    Figure Legend Snippet: Endochondral new bone formation is only present in tmTNF tg mouse. (A) Safranin-O/fast green stainings of ankle joints of a nontg, tmTNF tg , and TNF ΔARE mouse reveals that the tmTNF tg ankle has clear bone formation, while the TNF ΔARE and nontg do not. Scale bars, 500 µm. (B) Safranin-O/fast green stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE mouse. The tmTNF tg axial joint reveals inflammation adjacent to bone formation, whereas the TNF ΔARE and nontg do not have these features. Scale bars, 500 µm. (C) Magnified view of tmTNF tg axial and ankle joint of region with endochondral ossification. Scale bars, 200 µm. n = 3 mice per group.

    Techniques Used: Mouse Assay

    tmTNF-overexpressing mice develop clinical symptoms of arthritis and spondylitis. (A) Percentage of weight gain compared with day 30 in tmTNF tg mice and nontg littermates. (B) Picture of 100-d-old nontg littermate and tmTNF tg mice, the latter displaying slight swelling of hind paws. (C) Arthritis incidence over time. (D) Grip strength in seconds over time is depicted (maximal 20 s of grip strength were measured per time point). (E) Spondylitis incidence over time, P
    Figure Legend Snippet: tmTNF-overexpressing mice develop clinical symptoms of arthritis and spondylitis. (A) Percentage of weight gain compared with day 30 in tmTNF tg mice and nontg littermates. (B) Picture of 100-d-old nontg littermate and tmTNF tg mice, the latter displaying slight swelling of hind paws. (C) Arthritis incidence over time. (D) Grip strength in seconds over time is depicted (maximal 20 s of grip strength were measured per time point). (E) Spondylitis incidence over time, P

    Techniques Used: Mouse Assay

    Inflammation and bone and cartilage destruction in peripheral and axial joints of tmTNF tg mice. (A) H E staining of ankle joint of a 100-d-old nontg littermate. (B) H E staining of a 100-d-old tmTNF tg mouse reveals synovitis (black arrow), osteitis (white arrow), enthesitis (white arrowhead), and lymphoid aggregates (black arrowhead). Scale bars, 200 µm. (C) Semi-quantitative score (0–3) of inflammation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D) H E staining of an axial joint of a 100-d-old nontg littermate. (E) H E staining of axial joint sections of a 100-d-old tmTNF tg mouse, displaying cellular infiltrate (black arrow) in the connective tissue next to the intervertebral disc. Scale bars, 200 µm. (F) Semi-quantitative score (0–3) of inflammation in axial joints of tmTNF tg mice compared with nontg littermates (nontg n = 30, tmTNF tg n = 44, in four independent experiments). (G) H E staining of an ankle of a 100-d-old tmTNF tg mouse with destruction (black arrow) and lymphoid aggregate (black arrowhead). Scale bars, 100 µm. (H) Semi-quantitative score (0–3) of destruction in peripheral joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (I) H E staining of an axial joint of a 100-d-old tmTNF tg mouse indicating destruction by disruption of the cartilage endplate (black arrows) and lymphoid aggregate (black arrowheads). Scale bars, 200 µm. (J) Semi-quantitative score (0–3) of destruction in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 29; tmTNF tg , n = 37, in four independent experiments). The P value was determined by a Mann–Whitney test (C, F, H, and J). Data are representative of two independent experiments with two replicates (A–F) per sample. Values are mean ± SEM; ***, P
    Figure Legend Snippet: Inflammation and bone and cartilage destruction in peripheral and axial joints of tmTNF tg mice. (A) H E staining of ankle joint of a 100-d-old nontg littermate. (B) H E staining of a 100-d-old tmTNF tg mouse reveals synovitis (black arrow), osteitis (white arrow), enthesitis (white arrowhead), and lymphoid aggregates (black arrowhead). Scale bars, 200 µm. (C) Semi-quantitative score (0–3) of inflammation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D) H E staining of an axial joint of a 100-d-old nontg littermate. (E) H E staining of axial joint sections of a 100-d-old tmTNF tg mouse, displaying cellular infiltrate (black arrow) in the connective tissue next to the intervertebral disc. Scale bars, 200 µm. (F) Semi-quantitative score (0–3) of inflammation in axial joints of tmTNF tg mice compared with nontg littermates (nontg n = 30, tmTNF tg n = 44, in four independent experiments). (G) H E staining of an ankle of a 100-d-old tmTNF tg mouse with destruction (black arrow) and lymphoid aggregate (black arrowhead). Scale bars, 100 µm. (H) Semi-quantitative score (0–3) of destruction in peripheral joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (I) H E staining of an axial joint of a 100-d-old tmTNF tg mouse indicating destruction by disruption of the cartilage endplate (black arrows) and lymphoid aggregate (black arrowheads). Scale bars, 200 µm. (J) Semi-quantitative score (0–3) of destruction in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 29; tmTNF tg , n = 37, in four independent experiments). The P value was determined by a Mann–Whitney test (C, F, H, and J). Data are representative of two independent experiments with two replicates (A–F) per sample. Values are mean ± SEM; ***, P

    Techniques Used: Mouse Assay, Staining, MANN-WHITNEY

    Joint fusion of vertebrae in tmTNF tg mice. (A) Picture of 100-d-old tmTNF tg mice and a nontg littermate revealing hunchback formation and a crinkled tail in the tmTNF tg mouse, which is absent in the nontg littermate control. (B) Radiographical images of the tail in 8-mo-old nontg and tmTNF tg mouse. Arrow indicates spinal fusion in the tmTNF tg tail, while there is intervertebral space in the nontg mouse. Scale bars, 1 mm. (C) H E staining of an 8-mo-old nontg littermate and tmTNF tg mouse showing ankylosis in the tmTNF tg tail and destruction of the intervertebral disc. Scale bars, 200 µm. (D) Photo of a tmTNF +/WT xTNF-RI −/− mouse without clinical symptoms and a photo of a tmTNF +/WT xTNF-RII −/− mouse with hunchback formation and a crinkled tail. n = 3 mice per group in two independent experiments.
    Figure Legend Snippet: Joint fusion of vertebrae in tmTNF tg mice. (A) Picture of 100-d-old tmTNF tg mice and a nontg littermate revealing hunchback formation and a crinkled tail in the tmTNF tg mouse, which is absent in the nontg littermate control. (B) Radiographical images of the tail in 8-mo-old nontg and tmTNF tg mouse. Arrow indicates spinal fusion in the tmTNF tg tail, while there is intervertebral space in the nontg mouse. Scale bars, 1 mm. (C) H E staining of an 8-mo-old nontg littermate and tmTNF tg mouse showing ankylosis in the tmTNF tg tail and destruction of the intervertebral disc. Scale bars, 200 µm. (D) Photo of a tmTNF +/WT xTNF-RI −/− mouse without clinical symptoms and a photo of a tmTNF +/WT xTNF-RII −/− mouse with hunchback formation and a crinkled tail. n = 3 mice per group in two independent experiments.

    Techniques Used: Mouse Assay, Staining

    Osteoproliferation and endochondral new bone formation in tmTNF tg mice. (A and B) Safranin-O/fast green staining to detect proteoglycans (square in A) in an ankle joint of a tmTNF tg mouse. Scale bars, 500 µm (A) and 50 µm (B). (C) Semi-quantitative score (0–3) of endochondral new bone formation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D and E) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). (F–J) Safranin-O/fast green staining to detect proteoglycans (square in F) in an axial joint of a tmTNF tg mouse. Scale bars, 500 µm (F) and 50 µm (G). (H) Semi-quantitative score (0–3) of endochondral new bone formation in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 28; tmTNF tg , n = 35, in four independent experiments). (I and J) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). The P value was determined by a Mann–Whitney test (C–E and H–J). Values are mean ± SEM; **, P
    Figure Legend Snippet: Osteoproliferation and endochondral new bone formation in tmTNF tg mice. (A and B) Safranin-O/fast green staining to detect proteoglycans (square in A) in an ankle joint of a tmTNF tg mouse. Scale bars, 500 µm (A) and 50 µm (B). (C) Semi-quantitative score (0–3) of endochondral new bone formation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D and E) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). (F–J) Safranin-O/fast green staining to detect proteoglycans (square in F) in an axial joint of a tmTNF tg mouse. Scale bars, 500 µm (F) and 50 µm (G). (H) Semi-quantitative score (0–3) of endochondral new bone formation in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 28; tmTNF tg , n = 35, in four independent experiments). (I and J) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). The P value was determined by a Mann–Whitney test (C–E and H–J). Values are mean ± SEM; **, P

    Techniques Used: Mouse Assay, Staining, MANN-WHITNEY

    Comparison of inflammation in tmTNF tg and TNF ΔARE mice axial and peripheral joints . (A) H E stainings of ankle joints of a nontg ( tmTNF tg littermate), tmTNF tg , and TNF ΔARE mouse reveals that both tmTNF tg and TNF ΔARE mice have inflammation and destruction (arrowhead). In the tmTNF tg mouse, the inflammation is along the Achilles tendon and in the synovium (arrow), whereas the inflammation in the TNF ΔARE mouse is mostly limited to the synovium (arrow). (B) H E stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE . The tmTNF tg axial joint reveals extensive inflammation along the ligaments (arrow) and in the bone marrow (arrowhead), while the TNF ΔARE mouse shows minimal inflammation along the ligaments without osteitis. n = 3 mice per group. Scale bars, 500 µm.
    Figure Legend Snippet: Comparison of inflammation in tmTNF tg and TNF ΔARE mice axial and peripheral joints . (A) H E stainings of ankle joints of a nontg ( tmTNF tg littermate), tmTNF tg , and TNF ΔARE mouse reveals that both tmTNF tg and TNF ΔARE mice have inflammation and destruction (arrowhead). In the tmTNF tg mouse, the inflammation is along the Achilles tendon and in the synovium (arrow), whereas the inflammation in the TNF ΔARE mouse is mostly limited to the synovium (arrow). (B) H E stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE . The tmTNF tg axial joint reveals extensive inflammation along the ligaments (arrow) and in the bone marrow (arrowhead), while the TNF ΔARE mouse shows minimal inflammation along the ligaments without osteitis. n = 3 mice per group. Scale bars, 500 µm.

    Techniques Used: Mouse Assay

    Joint fusion of vertebra and sacroiliitis in 8-mo-old tmTNF tg mice. (A and B) Radiographical images of an 8-mo-old nontg littermate and a tmTNF tg mouse. (A) Radiographical images of the lumbar spine. Arrow indicates site of joint margin blurring in the tmTNF tg mice, while blurring is absent in nontg littermates. Scale bars, 1 mm. (B) Radiographical images of the sacroiliac joints. Arrows indicate joint margin blurring in the tmTNF tg mouse, which is absent in the nontg littermate. Scale bars, 1 mm. n = 3 mice per group.
    Figure Legend Snippet: Joint fusion of vertebra and sacroiliitis in 8-mo-old tmTNF tg mice. (A and B) Radiographical images of an 8-mo-old nontg littermate and a tmTNF tg mouse. (A) Radiographical images of the lumbar spine. Arrow indicates site of joint margin blurring in the tmTNF tg mice, while blurring is absent in nontg littermates. Scale bars, 1 mm. (B) Radiographical images of the sacroiliac joints. Arrows indicate joint margin blurring in the tmTNF tg mouse, which is absent in the nontg littermate. Scale bars, 1 mm. n = 3 mice per group.

    Techniques Used: Mouse Assay

    9) Product Images from "Phosphoinositide 3-kinase Akt signaling pathway interacts with protein kinase Cβ2 in the regulation of physiologic developmental hypertrophy and heart function"

    Article Title: Phosphoinositide 3-kinase Akt signaling pathway interacts with protein kinase Cβ2 in the regulation of physiologic developmental hypertrophy and heart function

    Journal:

    doi: 10.1152/ajpheart.00562.2008

    Comparison of organ weights and cardiomyocyte size in transgenic (Tg) mice. A : representation of heart size from wild-type (WT), protein kinase C (PKC) β2, dominant-negative (dn) phosphatidylinositol 3-kinase [PI3-kinase (PI3K)],
    Figure Legend Snippet: Comparison of organ weights and cardiomyocyte size in transgenic (Tg) mice. A : representation of heart size from wild-type (WT), protein kinase C (PKC) β2, dominant-negative (dn) phosphatidylinositol 3-kinase [PI3-kinase (PI3K)],

    Techniques Used: Transgenic Assay, Mouse Assay, Dominant Negative Mutation

    10) Product Images from "Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors 1"

    Article Title: Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors 1

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2016.05.002

    The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, TPA-treated skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the DMBA-TPA treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P
    Figure Legend Snippet: The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, TPA-treated skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the DMBA-TPA treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P

    Techniques Used: Over Expression, Papanicolaou Stain, FACS, Mouse Assay

    DMBA-TPA-induced skin carcinogenesis in VEGF-D knockout mice. Tumor incidence (A) and cumulative multiplicity (B) in the KO males (N = 26) and the WT males (N = 27) did not show statistically significant differences between the genotypes. ( C-D ) Expression of VEGF-D in mouse skin tumors. Representative immunohistochemical stainings showing VEGF-D expression in mouse cSCCs. (C) Consecutive sections of the WT cSCC stained with antibodies against VEGF-D and F4/80 demonstrate that VEGF-D is expressed by F4/80-positive macrophages (arrowheads) within the WT cSCC stroma. Asterisks indicate vessels that facilitate the identification of co-stained cells in the consecutive sections. (D) VEGF-D expression by invasive carcinoma cells (arrowheads) within the WT cSCC. The KO cSCC is used as a negative control for VEGF-D staining. (E) The quantification of lymphatic vessel density in the untreated (UT) skin, TPA-treated skin, and papillomas (PAP) and cSCCs collected at different time points of skin carcinogenesis. The KO tumors showed a trend for reduced lymphangiogenesis. In the UT skin, and in cSCCs at week 29, the difference between the genotypes was significant. (F) No significant differences in blood vessel densities between the KO and WT males in any type of sample. Scale bars: 50 μm (C) and 100 μm (D): Error bars: SD. *: P
    Figure Legend Snippet: DMBA-TPA-induced skin carcinogenesis in VEGF-D knockout mice. Tumor incidence (A) and cumulative multiplicity (B) in the KO males (N = 26) and the WT males (N = 27) did not show statistically significant differences between the genotypes. ( C-D ) Expression of VEGF-D in mouse skin tumors. Representative immunohistochemical stainings showing VEGF-D expression in mouse cSCCs. (C) Consecutive sections of the WT cSCC stained with antibodies against VEGF-D and F4/80 demonstrate that VEGF-D is expressed by F4/80-positive macrophages (arrowheads) within the WT cSCC stroma. Asterisks indicate vessels that facilitate the identification of co-stained cells in the consecutive sections. (D) VEGF-D expression by invasive carcinoma cells (arrowheads) within the WT cSCC. The KO cSCC is used as a negative control for VEGF-D staining. (E) The quantification of lymphatic vessel density in the untreated (UT) skin, TPA-treated skin, and papillomas (PAP) and cSCCs collected at different time points of skin carcinogenesis. The KO tumors showed a trend for reduced lymphangiogenesis. In the UT skin, and in cSCCs at week 29, the difference between the genotypes was significant. (F) No significant differences in blood vessel densities between the KO and WT males in any type of sample. Scale bars: 50 μm (C) and 100 μm (D): Error bars: SD. *: P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Immunohistochemistry, Staining, Negative Control

    Impaired skin tumor formation in TG K14-mVEGF-D mice in a chemical skin carcinogenesis model. Tumors were induced in the mouse skin using a multistage DMBA-TPA protocol and their growth was monitored for up to 30 weeks (Wk). Data from two separate experimental groups of males (WT N = 23, TG N = 25) are combined. (A) Representative photographs of the WT and K14-mVEGF-D TG male mice at different time points. Black arrowheads indicate exophytic papillomas and white arrowheads regressing papillomas. (B) Tumor incidence showed a significant delay in the TG mice. (C) The TG mice developed markedly less tumors than the WT mice. Cumulative tumor multiplicity is shown. (D) The percentage of regressing papillomas (the black portion of the column) was higher in the TG mice (red) than in the WT mice (green). (E) Smaller tumor size in the TG mice than in the WT mice. Tumor sizes are shown as a percentage of tumors in three different size categories. Statistical significance for each size category is shown in between the column pairs. (F) At weeks 20–30 the percentage of TG male mice with clinically apparent cSCC was lower than that of WT control males. (G) A trend towards less cSCCs per mouse was observed in TG males. N = number of mice. Error bars: SD.*: P
    Figure Legend Snippet: Impaired skin tumor formation in TG K14-mVEGF-D mice in a chemical skin carcinogenesis model. Tumors were induced in the mouse skin using a multistage DMBA-TPA protocol and their growth was monitored for up to 30 weeks (Wk). Data from two separate experimental groups of males (WT N = 23, TG N = 25) are combined. (A) Representative photographs of the WT and K14-mVEGF-D TG male mice at different time points. Black arrowheads indicate exophytic papillomas and white arrowheads regressing papillomas. (B) Tumor incidence showed a significant delay in the TG mice. (C) The TG mice developed markedly less tumors than the WT mice. Cumulative tumor multiplicity is shown. (D) The percentage of regressing papillomas (the black portion of the column) was higher in the TG mice (red) than in the WT mice (green). (E) Smaller tumor size in the TG mice than in the WT mice. Tumor sizes are shown as a percentage of tumors in three different size categories. Statistical significance for each size category is shown in between the column pairs. (F) At weeks 20–30 the percentage of TG male mice with clinically apparent cSCC was lower than that of WT control males. (G) A trend towards less cSCCs per mouse was observed in TG males. N = number of mice. Error bars: SD.*: P

    Techniques Used: Mouse Assay

    11) Product Images from "A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma"

    Article Title: A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-18-1319

    SSTC3 acts downstream of SMO to attenuate SHH activity. A, LIGHT2 cells were treated with 1 μg/mL recombinant SHH for 24 hours, followed by addition of the indicated concentrations of SSTC3 or SSTC111 for an additional 48 hours. Firefly luciferase activity was then determined and normalized to Renilla luciferase activity. B, NIH-3T3 cells were incubated with the SAG (100 nmol/L) and the indicated concentrations of SSTC3. The expression of GLI1 was determined at the indicated timepoints and normalized to that of the housekeeping gene GAPDH . Data were normalized to a vehicle control. C, LIGHT2 cells were transduced with lentivirus expressing the indicated shRNA to generate stable polyclonal cell lines. These cells were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 48 hours. The expression of GLI1 was determined and normalized to that of GAPDH . D, GPCs were incubated in the presence of DMSO, SAG (100 nmol/L), or SAG (100 nmol/L) and SSTC3 (200 nmol/L), for 24 hours prior to quantification of BrdU incorporation. E, Representative images from similarly treated GPC are shown. F, GPC were treated with vehicle, vismodegib (100 nmol/L), or SAG (100 nmol/L) and SSTC3 (200 nmol/L), and expression of the indicated genes determined 6 hours later. Data were normalized to a SAG-treated vehicle control. G, SUFU −/− MEFs were treated for 48 hours with vehicle, the indicated concentrations of SSTC3, or vismodegib (200 nmol/L). The expression of GLI1 was then determined and normalized to that of GAPDH . Data were normalized to a vehicle control. H, LIGHT2 cells expressing WT SMO , the vismodegib-resistant oncogenic SMO mutant M2-D473 , or GLI1 , were treated with vehicle, vismodegib (200 nmol/L), or SSTC3 (200 nmol/L). Firefly luciferase activity was then determined and normalized to Renilla luciferase activity. Data were normalized to a SMO WT vehicle control. I, The indicated MEFs were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 24 hours, and the levels of PTCH1 expression determined and normalized to that of housekeeping gene GAPDH by qRT-PCR. Data were normalized to a SAG-treated vehicle control. J, The indicated MEFs were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 24 hours, and the levels of GLI1 expression determined and normalized to that of housekeeping gene GAPDH by qRT-PCR. Data were normalized to a SAG-treated vehicle control.
    Figure Legend Snippet: SSTC3 acts downstream of SMO to attenuate SHH activity. A, LIGHT2 cells were treated with 1 μg/mL recombinant SHH for 24 hours, followed by addition of the indicated concentrations of SSTC3 or SSTC111 for an additional 48 hours. Firefly luciferase activity was then determined and normalized to Renilla luciferase activity. B, NIH-3T3 cells were incubated with the SAG (100 nmol/L) and the indicated concentrations of SSTC3. The expression of GLI1 was determined at the indicated timepoints and normalized to that of the housekeeping gene GAPDH . Data were normalized to a vehicle control. C, LIGHT2 cells were transduced with lentivirus expressing the indicated shRNA to generate stable polyclonal cell lines. These cells were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 48 hours. The expression of GLI1 was determined and normalized to that of GAPDH . D, GPCs were incubated in the presence of DMSO, SAG (100 nmol/L), or SAG (100 nmol/L) and SSTC3 (200 nmol/L), for 24 hours prior to quantification of BrdU incorporation. E, Representative images from similarly treated GPC are shown. F, GPC were treated with vehicle, vismodegib (100 nmol/L), or SAG (100 nmol/L) and SSTC3 (200 nmol/L), and expression of the indicated genes determined 6 hours later. Data were normalized to a SAG-treated vehicle control. G, SUFU −/− MEFs were treated for 48 hours with vehicle, the indicated concentrations of SSTC3, or vismodegib (200 nmol/L). The expression of GLI1 was then determined and normalized to that of GAPDH . Data were normalized to a vehicle control. H, LIGHT2 cells expressing WT SMO , the vismodegib-resistant oncogenic SMO mutant M2-D473 , or GLI1 , were treated with vehicle, vismodegib (200 nmol/L), or SSTC3 (200 nmol/L). Firefly luciferase activity was then determined and normalized to Renilla luciferase activity. Data were normalized to a SMO WT vehicle control. I, The indicated MEFs were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 24 hours, and the levels of PTCH1 expression determined and normalized to that of housekeeping gene GAPDH by qRT-PCR. Data were normalized to a SAG-treated vehicle control. J, The indicated MEFs were treated with SAG (100 nmol/L) and SSTC3 (200 nmol/L) for 24 hours, and the levels of GLI1 expression determined and normalized to that of housekeeping gene GAPDH by qRT-PCR. Data were normalized to a SAG-treated vehicle control.

    Techniques Used: Activity Assay, Recombinant, Luciferase, Incubation, Expressing, Transduction, shRNA, BrdU Incorporation Assay, Gel Permeation Chromatography, Mutagenesis, Quantitative RT-PCR

    SSTC3 increases the symptom-free survival of a GEMM of SHH subgroup medulloblastoma. A, SAG (100 nmol/L)-induced WT- or ND2:SMOA1 -derived GPC were treated with the indicated concentrations of vismodegib or SSTC3. GPC proliferation was subsequently determined by BrdU incorporation 24 hours later. Data were normalized to a vehicle control. B, Two-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (20 mg/kg i.p. in DMSO) daily for 1 month. Representative IVIS images from vehicle or SSTC3-treated mice are shown. C, Similar mice were treated every other day with SSTC3 (10 mg/kg i.p. in DMSO) for a month. Representative images of brains and H E staining from WT vehicle, ND2:SMOA1 vehicle, or SSTC3-treated mice are shown. D, The area of tumor from the vehicle and SSTC3-treated mice was determined ( N = 6). E, Metastatic lesions were quantified from similarly treated mice ( N = 6). F, Tumors from these mice were immunostained for the proliferation biomarker PCNA and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. G, Representative images of PCNA immunostaining are shown. H, Orthotopic tumors were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. I, Representative images of Cleaved CASPASE-3 immunostaining are shown. J, Four-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) for 2 consecutive days. The mice were then sacrificed and their cerebella harvested 6 hours after the last injection. The expression of the indicated genes was then determined and normalized to the expression of GAPDH . Data was normalized to a vehicle control ( N = 5). K, The levels of indicated proteins in similarly treated mice were determined by immunoblotting from a cohort of vehicle (1–5) or SSTC3 (6–10)-treated mice. L, Two-month-old ND2:SMOA1 mice were treated with SSTC3 (10 mg/kg i.p. in DMSO) every other day for 1 month and medulloblastoma symptom-free survival monitored for 9 additional months ( N = 10).
    Figure Legend Snippet: SSTC3 increases the symptom-free survival of a GEMM of SHH subgroup medulloblastoma. A, SAG (100 nmol/L)-induced WT- or ND2:SMOA1 -derived GPC were treated with the indicated concentrations of vismodegib or SSTC3. GPC proliferation was subsequently determined by BrdU incorporation 24 hours later. Data were normalized to a vehicle control. B, Two-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (20 mg/kg i.p. in DMSO) daily for 1 month. Representative IVIS images from vehicle or SSTC3-treated mice are shown. C, Similar mice were treated every other day with SSTC3 (10 mg/kg i.p. in DMSO) for a month. Representative images of brains and H E staining from WT vehicle, ND2:SMOA1 vehicle, or SSTC3-treated mice are shown. D, The area of tumor from the vehicle and SSTC3-treated mice was determined ( N = 6). E, Metastatic lesions were quantified from similarly treated mice ( N = 6). F, Tumors from these mice were immunostained for the proliferation biomarker PCNA and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. G, Representative images of PCNA immunostaining are shown. H, Orthotopic tumors were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. I, Representative images of Cleaved CASPASE-3 immunostaining are shown. J, Four-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) for 2 consecutive days. The mice were then sacrificed and their cerebella harvested 6 hours after the last injection. The expression of the indicated genes was then determined and normalized to the expression of GAPDH . Data was normalized to a vehicle control ( N = 5). K, The levels of indicated proteins in similarly treated mice were determined by immunoblotting from a cohort of vehicle (1–5) or SSTC3 (6–10)-treated mice. L, Two-month-old ND2:SMOA1 mice were treated with SSTC3 (10 mg/kg i.p. in DMSO) every other day for 1 month and medulloblastoma symptom-free survival monitored for 9 additional months ( N = 10).

    Techniques Used: Derivative Assay, Gel Permeation Chromatography, BrdU Incorporation Assay, Mouse Assay, Staining, Biomarker Assay, Immunostaining, Injection, Expressing

    12) Product Images from "Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis"

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20200288

    tmTNF overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and nontg littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P
    Figure Legend Snippet: tmTNF overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and nontg littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P

    Techniques Used: Over Expression, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, MANN-WHITNEY

    Endochondral new bone formation is only present in tmTNF tg mouse. (A) Safranin-O/fast green stainings of ankle joints of a nontg, tmTNF tg , and TNF ΔARE mouse reveals that the tmTNF tg ankle has clear bone formation, while the TNF ΔARE and nontg do not. Scale bars, 500 µm. (B) Safranin-O/fast green stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE mouse. The tmTNF tg axial joint reveals inflammation adjacent to bone formation, whereas the TNF ΔARE and nontg do not have these features. Scale bars, 500 µm. (C) Magnified view of tmTNF tg axial and ankle joint of region with endochondral ossification. Scale bars, 200 µm. n = 3 mice per group.
    Figure Legend Snippet: Endochondral new bone formation is only present in tmTNF tg mouse. (A) Safranin-O/fast green stainings of ankle joints of a nontg, tmTNF tg , and TNF ΔARE mouse reveals that the tmTNF tg ankle has clear bone formation, while the TNF ΔARE and nontg do not. Scale bars, 500 µm. (B) Safranin-O/fast green stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE mouse. The tmTNF tg axial joint reveals inflammation adjacent to bone formation, whereas the TNF ΔARE and nontg do not have these features. Scale bars, 500 µm. (C) Magnified view of tmTNF tg axial and ankle joint of region with endochondral ossification. Scale bars, 200 µm. n = 3 mice per group.

    Techniques Used: Mouse Assay

    tmTNF-overexpressing mice develop clinical symptoms of arthritis and spondylitis. (A) Percentage of weight gain compared with day 30 in tmTNF tg mice and nontg littermates. (B) Picture of 100-d-old nontg littermate and tmTNF tg mice, the latter displaying slight swelling of hind paws. (C) Arthritis incidence over time. (D) Grip strength in seconds over time is depicted (maximal 20 s of grip strength were measured per time point). (E) Spondylitis incidence over time, P
    Figure Legend Snippet: tmTNF-overexpressing mice develop clinical symptoms of arthritis and spondylitis. (A) Percentage of weight gain compared with day 30 in tmTNF tg mice and nontg littermates. (B) Picture of 100-d-old nontg littermate and tmTNF tg mice, the latter displaying slight swelling of hind paws. (C) Arthritis incidence over time. (D) Grip strength in seconds over time is depicted (maximal 20 s of grip strength were measured per time point). (E) Spondylitis incidence over time, P

    Techniques Used: Mouse Assay

    Inflammation and bone and cartilage destruction in peripheral and axial joints of tmTNF tg mice. (A) H E staining of ankle joint of a 100-d-old nontg littermate. (B) H E staining of a 100-d-old tmTNF tg mouse reveals synovitis (black arrow), osteitis (white arrow), enthesitis (white arrowhead), and lymphoid aggregates (black arrowhead). Scale bars, 200 µm. (C) Semi-quantitative score (0–3) of inflammation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D) H E staining of an axial joint of a 100-d-old nontg littermate. (E) H E staining of axial joint sections of a 100-d-old tmTNF tg mouse, displaying cellular infiltrate (black arrow) in the connective tissue next to the intervertebral disc. Scale bars, 200 µm. (F) Semi-quantitative score (0–3) of inflammation in axial joints of tmTNF tg mice compared with nontg littermates (nontg n = 30, tmTNF tg n = 44, in four independent experiments). (G) H E staining of an ankle of a 100-d-old tmTNF tg mouse with destruction (black arrow) and lymphoid aggregate (black arrowhead). Scale bars, 100 µm. (H) Semi-quantitative score (0–3) of destruction in peripheral joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (I) H E staining of an axial joint of a 100-d-old tmTNF tg mouse indicating destruction by disruption of the cartilage endplate (black arrows) and lymphoid aggregate (black arrowheads). Scale bars, 200 µm. (J) Semi-quantitative score (0–3) of destruction in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 29; tmTNF tg , n = 37, in four independent experiments). The P value was determined by a Mann–Whitney test (C, F, H, and J). Data are representative of two independent experiments with two replicates (A–F) per sample. Values are mean ± SEM; ***, P
    Figure Legend Snippet: Inflammation and bone and cartilage destruction in peripheral and axial joints of tmTNF tg mice. (A) H E staining of ankle joint of a 100-d-old nontg littermate. (B) H E staining of a 100-d-old tmTNF tg mouse reveals synovitis (black arrow), osteitis (white arrow), enthesitis (white arrowhead), and lymphoid aggregates (black arrowhead). Scale bars, 200 µm. (C) Semi-quantitative score (0–3) of inflammation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D) H E staining of an axial joint of a 100-d-old nontg littermate. (E) H E staining of axial joint sections of a 100-d-old tmTNF tg mouse, displaying cellular infiltrate (black arrow) in the connective tissue next to the intervertebral disc. Scale bars, 200 µm. (F) Semi-quantitative score (0–3) of inflammation in axial joints of tmTNF tg mice compared with nontg littermates (nontg n = 30, tmTNF tg n = 44, in four independent experiments). (G) H E staining of an ankle of a 100-d-old tmTNF tg mouse with destruction (black arrow) and lymphoid aggregate (black arrowhead). Scale bars, 100 µm. (H) Semi-quantitative score (0–3) of destruction in peripheral joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (I) H E staining of an axial joint of a 100-d-old tmTNF tg mouse indicating destruction by disruption of the cartilage endplate (black arrows) and lymphoid aggregate (black arrowheads). Scale bars, 200 µm. (J) Semi-quantitative score (0–3) of destruction in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 29; tmTNF tg , n = 37, in four independent experiments). The P value was determined by a Mann–Whitney test (C, F, H, and J). Data are representative of two independent experiments with two replicates (A–F) per sample. Values are mean ± SEM; ***, P

    Techniques Used: Mouse Assay, Staining, MANN-WHITNEY

    Joint fusion of vertebrae in tmTNF tg mice. (A) Picture of 100-d-old tmTNF tg mice and a nontg littermate revealing hunchback formation and a crinkled tail in the tmTNF tg mouse, which is absent in the nontg littermate control. (B) Radiographical images of the tail in 8-mo-old nontg and tmTNF tg mouse. Arrow indicates spinal fusion in the tmTNF tg tail, while there is intervertebral space in the nontg mouse. Scale bars, 1 mm. (C) H E staining of an 8-mo-old nontg littermate and tmTNF tg mouse showing ankylosis in the tmTNF tg tail and destruction of the intervertebral disc. Scale bars, 200 µm. (D) Photo of a tmTNF +/WT xTNF-RI −/− mouse without clinical symptoms and a photo of a tmTNF +/WT xTNF-RII −/− mouse with hunchback formation and a crinkled tail. n = 3 mice per group in two independent experiments.
    Figure Legend Snippet: Joint fusion of vertebrae in tmTNF tg mice. (A) Picture of 100-d-old tmTNF tg mice and a nontg littermate revealing hunchback formation and a crinkled tail in the tmTNF tg mouse, which is absent in the nontg littermate control. (B) Radiographical images of the tail in 8-mo-old nontg and tmTNF tg mouse. Arrow indicates spinal fusion in the tmTNF tg tail, while there is intervertebral space in the nontg mouse. Scale bars, 1 mm. (C) H E staining of an 8-mo-old nontg littermate and tmTNF tg mouse showing ankylosis in the tmTNF tg tail and destruction of the intervertebral disc. Scale bars, 200 µm. (D) Photo of a tmTNF +/WT xTNF-RI −/− mouse without clinical symptoms and a photo of a tmTNF +/WT xTNF-RII −/− mouse with hunchback formation and a crinkled tail. n = 3 mice per group in two independent experiments.

    Techniques Used: Mouse Assay, Staining

    Osteoproliferation and endochondral new bone formation in tmTNF tg mice. (A and B) Safranin-O/fast green staining to detect proteoglycans (square in A) in an ankle joint of a tmTNF tg mouse. Scale bars, 500 µm (A) and 50 µm (B). (C) Semi-quantitative score (0–3) of endochondral new bone formation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D and E) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). (F–J) Safranin-O/fast green staining to detect proteoglycans (square in F) in an axial joint of a tmTNF tg mouse. Scale bars, 500 µm (F) and 50 µm (G). (H) Semi-quantitative score (0–3) of endochondral new bone formation in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 28; tmTNF tg , n = 35, in four independent experiments). (I and J) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). The P value was determined by a Mann–Whitney test (C–E and H–J). Values are mean ± SEM; **, P
    Figure Legend Snippet: Osteoproliferation and endochondral new bone formation in tmTNF tg mice. (A and B) Safranin-O/fast green staining to detect proteoglycans (square in A) in an ankle joint of a tmTNF tg mouse. Scale bars, 500 µm (A) and 50 µm (B). (C) Semi-quantitative score (0–3) of endochondral new bone formation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D and E) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). (F–J) Safranin-O/fast green staining to detect proteoglycans (square in F) in an axial joint of a tmTNF tg mouse. Scale bars, 500 µm (F) and 50 µm (G). (H) Semi-quantitative score (0–3) of endochondral new bone formation in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 28; tmTNF tg , n = 35, in four independent experiments). (I and J) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). The P value was determined by a Mann–Whitney test (C–E and H–J). Values are mean ± SEM; **, P

    Techniques Used: Mouse Assay, Staining, MANN-WHITNEY

    Comparison of inflammation in tmTNF tg and TNF ΔARE mice axial and peripheral joints . (A) H E stainings of ankle joints of a nontg ( tmTNF tg littermate), tmTNF tg , and TNF ΔARE mouse reveals that both tmTNF tg and TNF ΔARE mice have inflammation and destruction (arrowhead). In the tmTNF tg mouse, the inflammation is along the Achilles tendon and in the synovium (arrow), whereas the inflammation in the TNF ΔARE mouse is mostly limited to the synovium (arrow). (B) H E stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE . The tmTNF tg axial joint reveals extensive inflammation along the ligaments (arrow) and in the bone marrow (arrowhead), while the TNF ΔARE mouse shows minimal inflammation along the ligaments without osteitis. n = 3 mice per group. Scale bars, 500 µm.
    Figure Legend Snippet: Comparison of inflammation in tmTNF tg and TNF ΔARE mice axial and peripheral joints . (A) H E stainings of ankle joints of a nontg ( tmTNF tg littermate), tmTNF tg , and TNF ΔARE mouse reveals that both tmTNF tg and TNF ΔARE mice have inflammation and destruction (arrowhead). In the tmTNF tg mouse, the inflammation is along the Achilles tendon and in the synovium (arrow), whereas the inflammation in the TNF ΔARE mouse is mostly limited to the synovium (arrow). (B) H E stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE . The tmTNF tg axial joint reveals extensive inflammation along the ligaments (arrow) and in the bone marrow (arrowhead), while the TNF ΔARE mouse shows minimal inflammation along the ligaments without osteitis. n = 3 mice per group. Scale bars, 500 µm.

    Techniques Used: Mouse Assay

    Joint fusion of vertebra and sacroiliitis in 8-mo-old tmTNF tg mice. (A and B) Radiographical images of an 8-mo-old nontg littermate and a tmTNF tg mouse. (A) Radiographical images of the lumbar spine. Arrow indicates site of joint margin blurring in the tmTNF tg mice, while blurring is absent in nontg littermates. Scale bars, 1 mm. (B) Radiographical images of the sacroiliac joints. Arrows indicate joint margin blurring in the tmTNF tg mouse, which is absent in the nontg littermate. Scale bars, 1 mm. n = 3 mice per group.
    Figure Legend Snippet: Joint fusion of vertebra and sacroiliitis in 8-mo-old tmTNF tg mice. (A and B) Radiographical images of an 8-mo-old nontg littermate and a tmTNF tg mouse. (A) Radiographical images of the lumbar spine. Arrow indicates site of joint margin blurring in the tmTNF tg mice, while blurring is absent in nontg littermates. Scale bars, 1 mm. (B) Radiographical images of the sacroiliac joints. Arrows indicate joint margin blurring in the tmTNF tg mouse, which is absent in the nontg littermate. Scale bars, 1 mm. n = 3 mice per group.

    Techniques Used: Mouse Assay

    13) Product Images from "A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma"

    Article Title: A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-18-1319

    SSTC3 increases the symptom-free survival of a GEMM of SHH subgroup medulloblastoma. A, SAG (100 nmol/L)-induced WT- or ND2:SMOA1 -derived GPC were treated with the indicated concentrations of vismodegib or SSTC3. GPC proliferation was subsequently determined by BrdU incorporation 24 hours later. Data were normalized to a vehicle control. B, Two-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (20 mg/kg i.p. in DMSO) daily for 1 month. Representative IVIS images from vehicle or SSTC3-treated mice are shown. C, Similar mice were treated every other day with SSTC3 (10 mg/kg i.p. in DMSO) for a month. Representative images of brains and H E staining from WT vehicle, ND2:SMOA1 vehicle, or SSTC3-treated mice are shown. D, The area of tumor from the vehicle and SSTC3-treated mice was determined ( N = 6). E, Metastatic lesions were quantified from similarly treated mice ( N = 6). F, Tumors from these mice were immunostained for the proliferation biomarker PCNA and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. G, Representative images of PCNA immunostaining are shown. H, Orthotopic tumors were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. I, Representative images of Cleaved CASPASE-3 immunostaining are shown. J, Four-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) for 2 consecutive days. The mice were then sacrificed and their cerebella harvested 6 hours after the last injection. The expression of the indicated genes was then determined and normalized to the expression of GAPDH . Data was normalized to a vehicle control ( N = 5). K, The levels of indicated proteins in similarly treated mice were determined by immunoblotting from a cohort of vehicle (1–5) or SSTC3 (6–10)-treated mice. L, Two-month-old ND2:SMOA1 mice were treated with SSTC3 (10 mg/kg i.p. in DMSO) every other day for 1 month and medulloblastoma symptom-free survival monitored for 9 additional months ( N = 10).
    Figure Legend Snippet: SSTC3 increases the symptom-free survival of a GEMM of SHH subgroup medulloblastoma. A, SAG (100 nmol/L)-induced WT- or ND2:SMOA1 -derived GPC were treated with the indicated concentrations of vismodegib or SSTC3. GPC proliferation was subsequently determined by BrdU incorporation 24 hours later. Data were normalized to a vehicle control. B, Two-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (20 mg/kg i.p. in DMSO) daily for 1 month. Representative IVIS images from vehicle or SSTC3-treated mice are shown. C, Similar mice were treated every other day with SSTC3 (10 mg/kg i.p. in DMSO) for a month. Representative images of brains and H E staining from WT vehicle, ND2:SMOA1 vehicle, or SSTC3-treated mice are shown. D, The area of tumor from the vehicle and SSTC3-treated mice was determined ( N = 6). E, Metastatic lesions were quantified from similarly treated mice ( N = 6). F, Tumors from these mice were immunostained for the proliferation biomarker PCNA and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. G, Representative images of PCNA immunostaining are shown. H, Orthotopic tumors were immunostained for the apoptosis biomarker Cleaved CASPASE-3 and the numbers of positive cells per field in 4 fields from 3 independent mice quantified. I, Representative images of Cleaved CASPASE-3 immunostaining are shown. J, Four-month-old ND2:SMOA1 mice were treated with vehicle or SSTC3 (10 mg/kg i.p. in DMSO) for 2 consecutive days. The mice were then sacrificed and their cerebella harvested 6 hours after the last injection. The expression of the indicated genes was then determined and normalized to the expression of GAPDH . Data was normalized to a vehicle control ( N = 5). K, The levels of indicated proteins in similarly treated mice were determined by immunoblotting from a cohort of vehicle (1–5) or SSTC3 (6–10)-treated mice. L, Two-month-old ND2:SMOA1 mice were treated with SSTC3 (10 mg/kg i.p. in DMSO) every other day for 1 month and medulloblastoma symptom-free survival monitored for 9 additional months ( N = 10).

    Techniques Used: Derivative Assay, Gel Permeation Chromatography, BrdU Incorporation Assay, Mouse Assay, Staining, Biomarker Assay, Immunostaining, Injection, Expressing

    14) Product Images from "Multipotent luminal mammary cancer stem cells model tumor heterogeneity"

    Article Title: Multipotent luminal mammary cancer stem cells model tumor heterogeneity

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-015-0615-y

    MaCSCs have the capacity to differentiate into multiple cell types. ( a ) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). ( b ) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. ( c ) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. ( d ) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. ( e,f ) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. ( g ) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. ( h ) Confluent Py230 cells spontaneously form domes. ( i ) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. ( j ) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. ( k ) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm
    Figure Legend Snippet: MaCSCs have the capacity to differentiate into multiple cell types. ( a ) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). ( b ) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. ( c ) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. ( d ) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. ( e,f ) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. ( g ) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. ( h ) Confluent Py230 cells spontaneously form domes. ( i ) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. ( j ) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. ( k ) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm

    Techniques Used: Staining, Marker, Expressing

    15) Product Images from "Functionally Distinct Subgroups of Oligodendrocyte Precursor Cells Integrate Neural Activity and Execute Myelin Formation"

    Article Title: Functionally Distinct Subgroups of Oligodendrocyte Precursor Cells Integrate Neural Activity and Execute Myelin Formation

    Journal: Nature neuroscience

    doi: 10.1038/s41593-019-0581-2

    Effects of chronic 4-AP incubation on zebrafish. a) Minimum intensity projections of a two-minute time-lapse of fish freely swimming in a 3 cm petri dish in different treatment conditions (n=6/7/3/3 animals in control/4-AP/TTX/4-AP+TTX, 3 independent experiments). b) Traces of GCaMP transients Tg(elavl3:h2b-GCaMP6s) zebrafish at 4 dpf and after overnight incubation in 0.1 mM 4-AP, and before / after 10 μM TTX (7 animals per conditions in 2 experiments). c) Confocal images of Tg(mfap4:memCerulean), Tg(olig1:nls-mApple) zebrafish at 4 dpf after treatment with 0.1 mM 4-AP, 0.5 mM 4-AP, or Danieau’s solution as control. Transmitted light images to show spinal cord morphology and tissue integrity following drug treatment. Scale bars: 100 μm. The graph shows that number of macrophages which accumulate in 400 μM length of spinal cord of Tg(mfap4:memCerulean) zebrafish after 1 day of control (2±0.25/2 cells), 0.1 mM (2±1/2 cells), and 0.5 mM (3±0.25/2 cells) 4-AP treatment (median (25%/75% percentiles); p=0.43 (control vs . 0.1mM 4-AP), p=0.03 (control vs. 0.5 mM 4-AP) (Kruskal-Wallis test, test statistic=3.003), n=16/19/8 animals in 3 experiments. d) Representative images of Tg(mbp:nls-EGFP), Tg(olig1:nls-mApple) zebrafish in control and after 2 days of 0.1 mM 4-AP treatment (see for n numbers). Scale bar: 20 μm. Fig 7g
    Figure Legend Snippet: Effects of chronic 4-AP incubation on zebrafish. a) Minimum intensity projections of a two-minute time-lapse of fish freely swimming in a 3 cm petri dish in different treatment conditions (n=6/7/3/3 animals in control/4-AP/TTX/4-AP+TTX, 3 independent experiments). b) Traces of GCaMP transients Tg(elavl3:h2b-GCaMP6s) zebrafish at 4 dpf and after overnight incubation in 0.1 mM 4-AP, and before / after 10 μM TTX (7 animals per conditions in 2 experiments). c) Confocal images of Tg(mfap4:memCerulean), Tg(olig1:nls-mApple) zebrafish at 4 dpf after treatment with 0.1 mM 4-AP, 0.5 mM 4-AP, or Danieau’s solution as control. Transmitted light images to show spinal cord morphology and tissue integrity following drug treatment. Scale bars: 100 μm. The graph shows that number of macrophages which accumulate in 400 μM length of spinal cord of Tg(mfap4:memCerulean) zebrafish after 1 day of control (2±0.25/2 cells), 0.1 mM (2±1/2 cells), and 0.5 mM (3±0.25/2 cells) 4-AP treatment (median (25%/75% percentiles); p=0.43 (control vs . 0.1mM 4-AP), p=0.03 (control vs. 0.5 mM 4-AP) (Kruskal-Wallis test, test statistic=3.003), n=16/19/8 animals in 3 experiments. d) Representative images of Tg(mbp:nls-EGFP), Tg(olig1:nls-mApple) zebrafish in control and after 2 days of 0.1 mM 4-AP treatment (see for n numbers). Scale bar: 20 μm. Fig 7g

    Techniques Used: Incubation, Fluorescence In Situ Hybridization

    Related Articles

    Isolation:

    Article Title: c-Maf-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont
    Article Snippet: .. Isolation of lymphocytes Intestinal tissues were sequentially treated with PBS containing 1 mM DTT at room temperature for 10 min, and 5 mM EDTA at 37°C for 20 min to remove epithelial cells, and then minced and dissociated in RPMI containing collagenase (1 mg/ml collagenase II; Roche), DNase I (100 μg/ml; Sigma), dispase (0.05 U/ml; Worthington) and 10% FBS with constant stirring at 37°C for 45 min (SI) or 60 min (LI). .. Leukocytes were collected at the interface of a 40%/80% Percoll gradient (GE Healthcare).

    Incubation:

    Article Title: High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor
    Article Snippet: .. Single-cell RNA-seq Cochleae were dissected out and incubated in solution (50% accutase (Innovative Cell Technologies, San Diego), 0.02% Trypsin(Thermofisher), 125 μg/ml Thermolysin (SIGMA-ALDRICH)) at 37°C for 3 min. 0.02 mg/ml collagenase IV (SIGMA-ALDRICH) and dispase (Worthington Biochemicals Corp) were further added to the enzyme solution and incubated for 4 min at 37°C. ..

    Article Title: Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment
    Article Snippet: .. For controlled proteolysis, hexon or AdV capsids were incubated with a 6-fold molar excess of dispase (neutral protease; Worthington Biochemicals, Freehold, NJ) over hexon in dispase buffer (100 mM Trizma-maleate, 120 mM NaCl, 2.5 mM CaCl2 ), and the pH was adjusted to either pH 7.4 (neutral) or pH 4.4 (acidic) for 5 min at 37°C until the reaction was stopped by the addition of either protein sample buffer (PSB) or 5 mM EDTA to the mixture. .. Sedimentation analysis of hexon was performed on linear 5 to 20% sucrose gradients of a volume of 1.4 ml in Tris-maleate buffer (50 mM Trizma-maleate, 10 mM NaCl, 1 mM EDTA, and 0.1% Tween 20) at the indicated pH values.

    RNA Sequencing Assay:

    Article Title: High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor
    Article Snippet: .. Single-cell RNA-seq Cochleae were dissected out and incubated in solution (50% accutase (Innovative Cell Technologies, San Diego), 0.02% Trypsin(Thermofisher), 125 μg/ml Thermolysin (SIGMA-ALDRICH)) at 37°C for 3 min. 0.02 mg/ml collagenase IV (SIGMA-ALDRICH) and dispase (Worthington Biochemicals Corp) were further added to the enzyme solution and incubated for 4 min at 37°C. ..

    Microscopy:

    Article Title: Delivery of Proteases in Aqueous Two-Phase Systems Enables Direct Purification of Stem Cell Colonies from Feeder Cell Co-Cultures for Differentiation into Functional Cardiomyocytes
    Article Snippet: .. Cells were immediately transferred to the microscope with the pneumatic ejection system and a capillary loaded with a solution of DMEM/F12 containing 16% DEX 10,000 kDa, 0.2 mg/mL collagenase (Worthington, 280 U/mg) and 0.2 mg/mL dispase (Worthington, 1.46 U/mg). ..

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    Worthington Biochemical acta2 rfpcherry tg mice
    TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions <t>Lyz2-EGFP-ki;Acta2-RFPcherry-Tg</t> mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). (E) Time-lapse IVM images ( Video S1 ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). (G) IVM images ( Video S2 ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p
    Acta2 Rfpcherry Tg Mice, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    <t>tmTNF</t> overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and <t>nontg</t> littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P
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    <t>tmTNF</t> overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and <t>nontg</t> littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P
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    TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). (E) Time-lapse IVM images ( Video S1 ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). (G) IVM images ( Video S2 ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p

    Journal: Immunity

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    doi: 10.1016/j.immuni.2018.09.018

    Figure Lengend Snippet: TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). (E) Time-lapse IVM images ( Video S1 ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). (G) IVM images ( Video S2 ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p

    Article Snippet: Pericyte isolation and culture Cremaster muscles from Acta2-RFPcherry-Tg mice were digested with 500 U/mL Collagenase II (Worthington) in PBS for 45 min at 37°C and 50 U/mL DNase I (Sigma-Aldrich) was added during the last 20 min.

    Techniques: Mouse Assay, In Vivo, Labeling, Blocking Assay, Injection, Transmission Electron Microscopy

    Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. (C) Time-lapse IVM images ( Video S3 ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). (H) Time-lapse confocal IVM images ( Video S4 ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Journal: Immunity

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    doi: 10.1016/j.immuni.2018.09.018

    Figure Lengend Snippet: Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. (C) Time-lapse IVM images ( Video S3 ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). (H) Time-lapse confocal IVM images ( Video S4 ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Article Snippet: Pericyte isolation and culture Cremaster muscles from Acta2-RFPcherry-Tg mice were digested with 500 U/mL Collagenase II (Worthington) in PBS for 45 min at 37°C and 50 U/mL DNase I (Sigma-Aldrich) was added during the last 20 min.

    Techniques: Mouse Assay, In Vivo, Labeling, Injection, Blocking Assay, Transmission Electron Microscopy, Inhibition

    TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p

    Journal: Immunity

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    doi: 10.1016/j.immuni.2018.09.018

    Figure Lengend Snippet: TNF-Elicited CXCL1 and CXCL2 Support Distinct Phases of Neutrophil-EC Interactions Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were treated with ctr or blocking mAbs (i.v. 10 min prior to TNF in C and D and i.s. 100 min post TNF in E–H) and neutrophil responses in cremaster muscles injected locally with PBS or TNF quantified by confocal IVM. (A) Illustrative images of the employed IVM model (scale bar, 20 μm). (B) Scheme depicting neutrophil responses quantified in (C)–(H). (C and D) Quantification of neutrophil adhesion and intraluminal crawling (n = 5–6 mice per group, 23 independent experiments). ) of a neutrophil TEM response in a TNF-stimulated tissue showing a neutrophil migrating from the lumen (0 min) through an EC junction (2–4 min) into the sub-EC space (6 min). Representative of 11 independent experiments; cross sections, top; luminal views, bottom; scale bars, 5 μm. (F) Quantifications of neutrophil TEM (n = 4–11 mice per group, 27 independent experiments). ) of an aborted TEM response in a mouse treated with local TNF+anti-CXCL2 mAb. The images show a luminal neutrophil extending a protrusion through an EC junction (1 min), retracting the protrusion, and re-entering the circulation (5–8 min). Representative of 6 independent experiments; cross sections and luminal views; scale bars, 5 μm. (H) Quantification of aborted neutrophil TEM (n = 4–11 mice per group, 21 independent experiments). Means ± SEM, ∗ p

    Article Snippet: Cremaster muscles from Acta2-RFPcherry-Tg mice were digested with 500 U/mL Collagenase II (Worthington) in PBS for 45 min at 37°C and 50 U/mL DNase I (Sigma-Aldrich) was added during the last 20 min.

    Techniques: Mouse Assay, In Vivo, Labeling, Blocking Assay, Injection, Transmission Electron Microscopy

    Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Journal: Immunity

    Article Title: Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

    doi: 10.1016/j.immuni.2018.09.018

    Figure Lengend Snippet: Neutrophil-Pericyte Interactions Are Selectively Mediated by Endogenous CXCL1 Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice, subjected to in vivo CD31 labeling, were stimulated locally with TNF and 100 min later i.s. injected with ctr or blocking mAbs, as indicated. (A) Neutrophil responses quantified in cremasteric venules by confocal IVM in (C)–(G). (B) Representative confocal IVM luminal and cross-sectional views depicting a neutrophil localized between TNF-stimulated ECs and pericytes 1 min post TEM. ) showing a neutrophil crawling on pericytes (tracks, dashed lines) in a TNF-stimulated tissue (top) and the inhibition of this response in tissues treated with anti-CXCL1 mAb (bottom). Scale bars in (B) and (C), 10 μm. (D–G) Crawling profiles of neutrophils on pericytes (20 cells per group for clarity) (D) as normalized for their origin and associated quantifications of displacement (E), straightness index (displacement/track length) (F), and breaching of the pericyte layer (G). ) illustrating neutrophil reverse TEM in a tissue treated with TNF+anti-CXCL1 mAb (luminal and cross-sectional views; scale bars, 5 μm). (I) Quantifications of neutrophil reverse TEM. Images are representative of 5–10 independent experiments and quantifications (n = 5–10 mice per group) involve 20 independent experiments.

    Article Snippet: Cremaster muscles from Acta2-RFPcherry-Tg mice were digested with 500 U/mL Collagenase II (Worthington) in PBS for 45 min at 37°C and 50 U/mL DNase I (Sigma-Aldrich) was added during the last 20 min.

    Techniques: Mouse Assay, In Vivo, Labeling, Injection, Blocking Assay, Transmission Electron Microscopy, Inhibition

    tmTNF overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and nontg littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: tmTNF overexpression on calvarial fibroblasts enhances osteoblast differentiation in an inflammatory environment. Calvarial fibroblasts from tmTNF tg mice and nontg littermates were differentiated (diff.) toward osteoblasts in osteogenic differentiation medium with or without rIL-17A. (A) Relative alkaline phosphatase mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in respectively nontg littermates and tmTNF tg mice. (B) Relative Col1a1 mRNA expression, measured by quantitative real-time PCR at day 7 after differentiation in, respectively, nontg littermates and tmTNF tg mice. (C) Quantification of Alizarin red staining for mineralization at day 28 after differentiation in, respectively, nontg littermates and tmTNF tg mice. The P value was determined by an unpaired t test (A, B, and C) or a Mann–Whitney test (A and C). Values depicted are means ± SEM; n = 9 or 10 mice per group, representative of two independent experiments; *, P

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Over Expression, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, MANN-WHITNEY

    Endochondral new bone formation is only present in tmTNF tg mouse. (A) Safranin-O/fast green stainings of ankle joints of a nontg, tmTNF tg , and TNF ΔARE mouse reveals that the tmTNF tg ankle has clear bone formation, while the TNF ΔARE and nontg do not. Scale bars, 500 µm. (B) Safranin-O/fast green stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE mouse. The tmTNF tg axial joint reveals inflammation adjacent to bone formation, whereas the TNF ΔARE and nontg do not have these features. Scale bars, 500 µm. (C) Magnified view of tmTNF tg axial and ankle joint of region with endochondral ossification. Scale bars, 200 µm. n = 3 mice per group.

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: Endochondral new bone formation is only present in tmTNF tg mouse. (A) Safranin-O/fast green stainings of ankle joints of a nontg, tmTNF tg , and TNF ΔARE mouse reveals that the tmTNF tg ankle has clear bone formation, while the TNF ΔARE and nontg do not. Scale bars, 500 µm. (B) Safranin-O/fast green stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE mouse. The tmTNF tg axial joint reveals inflammation adjacent to bone formation, whereas the TNF ΔARE and nontg do not have these features. Scale bars, 500 µm. (C) Magnified view of tmTNF tg axial and ankle joint of region with endochondral ossification. Scale bars, 200 µm. n = 3 mice per group.

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay

    tmTNF-overexpressing mice develop clinical symptoms of arthritis and spondylitis. (A) Percentage of weight gain compared with day 30 in tmTNF tg mice and nontg littermates. (B) Picture of 100-d-old nontg littermate and tmTNF tg mice, the latter displaying slight swelling of hind paws. (C) Arthritis incidence over time. (D) Grip strength in seconds over time is depicted (maximal 20 s of grip strength were measured per time point). (E) Spondylitis incidence over time, P

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: tmTNF-overexpressing mice develop clinical symptoms of arthritis and spondylitis. (A) Percentage of weight gain compared with day 30 in tmTNF tg mice and nontg littermates. (B) Picture of 100-d-old nontg littermate and tmTNF tg mice, the latter displaying slight swelling of hind paws. (C) Arthritis incidence over time. (D) Grip strength in seconds over time is depicted (maximal 20 s of grip strength were measured per time point). (E) Spondylitis incidence over time, P

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay

    Inflammation and bone and cartilage destruction in peripheral and axial joints of tmTNF tg mice. (A) H E staining of ankle joint of a 100-d-old nontg littermate. (B) H E staining of a 100-d-old tmTNF tg mouse reveals synovitis (black arrow), osteitis (white arrow), enthesitis (white arrowhead), and lymphoid aggregates (black arrowhead). Scale bars, 200 µm. (C) Semi-quantitative score (0–3) of inflammation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D) H E staining of an axial joint of a 100-d-old nontg littermate. (E) H E staining of axial joint sections of a 100-d-old tmTNF tg mouse, displaying cellular infiltrate (black arrow) in the connective tissue next to the intervertebral disc. Scale bars, 200 µm. (F) Semi-quantitative score (0–3) of inflammation in axial joints of tmTNF tg mice compared with nontg littermates (nontg n = 30, tmTNF tg n = 44, in four independent experiments). (G) H E staining of an ankle of a 100-d-old tmTNF tg mouse with destruction (black arrow) and lymphoid aggregate (black arrowhead). Scale bars, 100 µm. (H) Semi-quantitative score (0–3) of destruction in peripheral joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (I) H E staining of an axial joint of a 100-d-old tmTNF tg mouse indicating destruction by disruption of the cartilage endplate (black arrows) and lymphoid aggregate (black arrowheads). Scale bars, 200 µm. (J) Semi-quantitative score (0–3) of destruction in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 29; tmTNF tg , n = 37, in four independent experiments). The P value was determined by a Mann–Whitney test (C, F, H, and J). Data are representative of two independent experiments with two replicates (A–F) per sample. Values are mean ± SEM; ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: Inflammation and bone and cartilage destruction in peripheral and axial joints of tmTNF tg mice. (A) H E staining of ankle joint of a 100-d-old nontg littermate. (B) H E staining of a 100-d-old tmTNF tg mouse reveals synovitis (black arrow), osteitis (white arrow), enthesitis (white arrowhead), and lymphoid aggregates (black arrowhead). Scale bars, 200 µm. (C) Semi-quantitative score (0–3) of inflammation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D) H E staining of an axial joint of a 100-d-old nontg littermate. (E) H E staining of axial joint sections of a 100-d-old tmTNF tg mouse, displaying cellular infiltrate (black arrow) in the connective tissue next to the intervertebral disc. Scale bars, 200 µm. (F) Semi-quantitative score (0–3) of inflammation in axial joints of tmTNF tg mice compared with nontg littermates (nontg n = 30, tmTNF tg n = 44, in four independent experiments). (G) H E staining of an ankle of a 100-d-old tmTNF tg mouse with destruction (black arrow) and lymphoid aggregate (black arrowhead). Scale bars, 100 µm. (H) Semi-quantitative score (0–3) of destruction in peripheral joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (I) H E staining of an axial joint of a 100-d-old tmTNF tg mouse indicating destruction by disruption of the cartilage endplate (black arrows) and lymphoid aggregate (black arrowheads). Scale bars, 200 µm. (J) Semi-quantitative score (0–3) of destruction in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 29; tmTNF tg , n = 37, in four independent experiments). The P value was determined by a Mann–Whitney test (C, F, H, and J). Data are representative of two independent experiments with two replicates (A–F) per sample. Values are mean ± SEM; ***, P

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay, Staining, MANN-WHITNEY

    Joint fusion of vertebrae in tmTNF tg mice. (A) Picture of 100-d-old tmTNF tg mice and a nontg littermate revealing hunchback formation and a crinkled tail in the tmTNF tg mouse, which is absent in the nontg littermate control. (B) Radiographical images of the tail in 8-mo-old nontg and tmTNF tg mouse. Arrow indicates spinal fusion in the tmTNF tg tail, while there is intervertebral space in the nontg mouse. Scale bars, 1 mm. (C) H E staining of an 8-mo-old nontg littermate and tmTNF tg mouse showing ankylosis in the tmTNF tg tail and destruction of the intervertebral disc. Scale bars, 200 µm. (D) Photo of a tmTNF +/WT xTNF-RI −/− mouse without clinical symptoms and a photo of a tmTNF +/WT xTNF-RII −/− mouse with hunchback formation and a crinkled tail. n = 3 mice per group in two independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: Joint fusion of vertebrae in tmTNF tg mice. (A) Picture of 100-d-old tmTNF tg mice and a nontg littermate revealing hunchback formation and a crinkled tail in the tmTNF tg mouse, which is absent in the nontg littermate control. (B) Radiographical images of the tail in 8-mo-old nontg and tmTNF tg mouse. Arrow indicates spinal fusion in the tmTNF tg tail, while there is intervertebral space in the nontg mouse. Scale bars, 1 mm. (C) H E staining of an 8-mo-old nontg littermate and tmTNF tg mouse showing ankylosis in the tmTNF tg tail and destruction of the intervertebral disc. Scale bars, 200 µm. (D) Photo of a tmTNF +/WT xTNF-RI −/− mouse without clinical symptoms and a photo of a tmTNF +/WT xTNF-RII −/− mouse with hunchback formation and a crinkled tail. n = 3 mice per group in two independent experiments.

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay, Staining

    Osteoproliferation and endochondral new bone formation in tmTNF tg mice. (A and B) Safranin-O/fast green staining to detect proteoglycans (square in A) in an ankle joint of a tmTNF tg mouse. Scale bars, 500 µm (A) and 50 µm (B). (C) Semi-quantitative score (0–3) of endochondral new bone formation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D and E) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). (F–J) Safranin-O/fast green staining to detect proteoglycans (square in F) in an axial joint of a tmTNF tg mouse. Scale bars, 500 µm (F) and 50 µm (G). (H) Semi-quantitative score (0–3) of endochondral new bone formation in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 28; tmTNF tg , n = 35, in four independent experiments). (I and J) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). The P value was determined by a Mann–Whitney test (C–E and H–J). Values are mean ± SEM; **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: Osteoproliferation and endochondral new bone formation in tmTNF tg mice. (A and B) Safranin-O/fast green staining to detect proteoglycans (square in A) in an ankle joint of a tmTNF tg mouse. Scale bars, 500 µm (A) and 50 µm (B). (C) Semi-quantitative score (0–3) of endochondral new bone formation in ankle joints of tmTNF tg mice compared with nontg littermates (nontg, n = 7; tmTNF tg , n = 7, both ankles per mouse, in two independent experiments). (D and E) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). (F–J) Safranin-O/fast green staining to detect proteoglycans (square in F) in an axial joint of a tmTNF tg mouse. Scale bars, 500 µm (F) and 50 µm (G). (H) Semi-quantitative score (0–3) of endochondral new bone formation in axial joints of tmTNF tg mice compared with nontg littermates (nontg, n = 28; tmTNF tg , n = 35, in four independent experiments). (I and J) Histomorphometric analyses: proliferation area and the hypertrophic chondrocyte area within this proliferation area (nontg, n = 7; tmTNF tg , n = 7, in two independent experiments). The P value was determined by a Mann–Whitney test (C–E and H–J). Values are mean ± SEM; **, P

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay, Staining, MANN-WHITNEY

    Comparison of inflammation in tmTNF tg and TNF ΔARE mice axial and peripheral joints . (A) H E stainings of ankle joints of a nontg ( tmTNF tg littermate), tmTNF tg , and TNF ΔARE mouse reveals that both tmTNF tg and TNF ΔARE mice have inflammation and destruction (arrowhead). In the tmTNF tg mouse, the inflammation is along the Achilles tendon and in the synovium (arrow), whereas the inflammation in the TNF ΔARE mouse is mostly limited to the synovium (arrow). (B) H E stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE . The tmTNF tg axial joint reveals extensive inflammation along the ligaments (arrow) and in the bone marrow (arrowhead), while the TNF ΔARE mouse shows minimal inflammation along the ligaments without osteitis. n = 3 mice per group. Scale bars, 500 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: Comparison of inflammation in tmTNF tg and TNF ΔARE mice axial and peripheral joints . (A) H E stainings of ankle joints of a nontg ( tmTNF tg littermate), tmTNF tg , and TNF ΔARE mouse reveals that both tmTNF tg and TNF ΔARE mice have inflammation and destruction (arrowhead). In the tmTNF tg mouse, the inflammation is along the Achilles tendon and in the synovium (arrow), whereas the inflammation in the TNF ΔARE mouse is mostly limited to the synovium (arrow). (B) H E stainings of axial joints of a nontg, tmTNF tg , and TNF ΔARE . The tmTNF tg axial joint reveals extensive inflammation along the ligaments (arrow) and in the bone marrow (arrowhead), while the TNF ΔARE mouse shows minimal inflammation along the ligaments without osteitis. n = 3 mice per group. Scale bars, 500 µm.

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay

    Joint fusion of vertebra and sacroiliitis in 8-mo-old tmTNF tg mice. (A and B) Radiographical images of an 8-mo-old nontg littermate and a tmTNF tg mouse. (A) Radiographical images of the lumbar spine. Arrow indicates site of joint margin blurring in the tmTNF tg mice, while blurring is absent in nontg littermates. Scale bars, 1 mm. (B) Radiographical images of the sacroiliac joints. Arrows indicate joint margin blurring in the tmTNF tg mouse, which is absent in the nontg littermate. Scale bars, 1 mm. n = 3 mice per group.

    Journal: The Journal of Experimental Medicine

    Article Title: Transmembrane TNF drives osteoproliferative joint inflammation reminiscent of human spondyloarthritis

    doi: 10.1084/jem.20200288

    Figure Lengend Snippet: Joint fusion of vertebra and sacroiliitis in 8-mo-old tmTNF tg mice. (A and B) Radiographical images of an 8-mo-old nontg littermate and a tmTNF tg mouse. (A) Radiographical images of the lumbar spine. Arrow indicates site of joint margin blurring in the tmTNF tg mice, while blurring is absent in nontg littermates. Scale bars, 1 mm. (B) Radiographical images of the sacroiliac joints. Arrows indicate joint margin blurring in the tmTNF tg mouse, which is absent in the nontg littermate. Scale bars, 1 mm. n = 3 mice per group.

    Article Snippet: Calvarial fibroblast cultures Primary osteoblasts were isolated from calvariae from tmTNF tg mice or nontg littermates (n = 9 or 10 per group) after aseptic dissection and treatment with Collagenase II (100 U/ml, LS004174; Worthington).

    Techniques: Mouse Assay