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    Structured Review

    Millipore dimethyl sulfoxide dmso
    Effects of <t>BPA</t> on the mRNA expression level of caspase8 ( Casp8 ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with <t>DMSO</t> or BPA (0.1, 1, 5, and 10 μg/ml). Casp8 expression was measured using qPCR. Relative
    Dimethyl Sulfoxide Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries"

    Article Title: Bisphenol A exposure inhibits germ cell nest breakdown by reducing apoptosis in cultured neonatal mouse ovaries

    Journal: Reproductive toxicology (Elmsford, N.Y.)

    doi: 10.1016/j.reprotox.2015.05.012

    Effects of BPA on the mRNA expression level of caspase8 ( Casp8 ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Casp8 expression was measured using qPCR. Relative
    Figure Legend Snippet: Effects of BPA on the mRNA expression level of caspase8 ( Casp8 ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Casp8 expression was measured using qPCR. Relative

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Effects of BPA on the ratios of Bcl2 family members. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1 day with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2 , Bax , and Bad expression were measured using qPCR. Ratios of
    Figure Legend Snippet: Effects of BPA on the ratios of Bcl2 family members. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1 day with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2 , Bax , and Bad expression were measured using qPCR. Ratios of

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Effects of BPA on the mRNA expression level of Fas cell surface death receptor ( Fas ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Fas expression was measured
    Figure Legend Snippet: Effects of BPA on the mRNA expression level of Fas cell surface death receptor ( Fas ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Fas expression was measured

    Techniques Used: Expressing, Cell Culture

    Effects of BPA on ROS levels in cultured neonatal ovaries. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured with DMSO or BPA (0.1, 1, 5, and 10 μg/ml) for 2, 4, and 8 days and then subjected to in vitro ROS assays to measure
    Figure Legend Snippet: Effects of BPA on ROS levels in cultured neonatal ovaries. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured with DMSO or BPA (0.1, 1, 5, and 10 μg/ml) for 2, 4, and 8 days and then subjected to in vitro ROS assays to measure

    Techniques Used: Cell Culture, In Vitro

    Effects of BPA on percentage of germ cells and follicles in the ovary. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 2, 4, and 8 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml), and subjected to histological evaluation
    Figure Legend Snippet: Effects of BPA on percentage of germ cells and follicles in the ovary. Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 2, 4, and 8 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml), and subjected to histological evaluation

    Techniques Used: Cell Culture

    Effects of BPA on the mRNA expression level of catalase ( Cat ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Cat expression was measured in the cultured neonatal
    Figure Legend Snippet: Effects of BPA on the mRNA expression level of catalase ( Cat ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Cat expression was measured in the cultured neonatal

    Techniques Used: Expressing, Cell Culture

    Effects of BPA on the mRNA expression level of glutathione peroxidase ( Gpx ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gpx expression was measured in
    Figure Legend Snippet: Effects of BPA on the mRNA expression level of glutathione peroxidase ( Gpx ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gpx expression was measured in

    Techniques Used: Expressing, Cell Culture

    Effects of BPA on the mRNA expression level of B cell leukemia/lymphoma 2 ( Bcl2 ) and Bcl2-like 1 ( Bclxl ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2
    Figure Legend Snippet: Effects of BPA on the mRNA expression level of B cell leukemia/lymphoma 2 ( Bcl2 ) and Bcl2-like 1 ( Bclxl ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Bcl2

    Techniques Used: Expressing, Cell Culture

    Effects of BPA on the mRNA expression level of glutathione reductase ( Gsr ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gsr expression was measured in the
    Figure Legend Snippet: Effects of BPA on the mRNA expression level of glutathione reductase ( Gsr ). Neonatal ovaries were collected on postnatal day (PND) 0 and cultured for 1, 2, and 4 days with DMSO or BPA (0.1, 1, 5, and 10 μg/ml). Gsr expression was measured in the

    Techniques Used: Expressing, Cell Culture

    2) Product Images from "Differential Cytotoxic Potential of Acridocarpus orientalis Leaf and Stem Extracts with the Ability to Induce Multiple Cell Death Pathways"

    Article Title: Differential Cytotoxic Potential of Acridocarpus orientalis Leaf and Stem Extracts with the Ability to Induce Multiple Cell Death Pathways

    Journal: Molecules

    doi: 10.3390/molecules24213976

    Western blot analysis of MCF-7 cells treated with A. orientalis dichloromethane leaf fraction (AOD (L)) at 250 µg/mL and tested for the activation of caspases 7, 8 and 9, and poly ADB ribose polymerase (PARP). Actin antibody was used as a loading control. DMSO-treated, DMSO control having the same amount of DMSO as the test sample.
    Figure Legend Snippet: Western blot analysis of MCF-7 cells treated with A. orientalis dichloromethane leaf fraction (AOD (L)) at 250 µg/mL and tested for the activation of caspases 7, 8 and 9, and poly ADB ribose polymerase (PARP). Actin antibody was used as a loading control. DMSO-treated, DMSO control having the same amount of DMSO as the test sample.

    Techniques Used: Western Blot, Activation Assay

    Annexin V-FITC/propidium iodide (PI) analysis of HeLa cells treated with 250 µg/mL of the A. orientalis dichloromethane leaf (AOD (L)) fraction or 125 and 250 µg/mL n-butanol stem (AOB (S)) fraction for 24 h. Samples: ( A ) Untreated; ( B ) DMSO control; ( C ) Positive control-treated; ( D ) 250 µg/mL AOD (L)-treated; ( E ) 250 µg/mL AOD (L)-treated cells pretreated with ZVAD, or ( F ) NEC-1, or ( G ) both; ( H ) 125 µg/mL AOB (S)-treated; ( I ) 125 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( J ) NEC-1, or ( K ) both; ( L ) 250 µg/mL AOB (S)-treated; ( M ) 250 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( N ) NEC-1, or ( O ) both. ZVAD = Z-VAD-FMK; NEC-1, Necrostatin 1. The DMSO Control sample contains the same amount of DMSO as in the 250 µg/mL sample of the treatment.
    Figure Legend Snippet: Annexin V-FITC/propidium iodide (PI) analysis of HeLa cells treated with 250 µg/mL of the A. orientalis dichloromethane leaf (AOD (L)) fraction or 125 and 250 µg/mL n-butanol stem (AOB (S)) fraction for 24 h. Samples: ( A ) Untreated; ( B ) DMSO control; ( C ) Positive control-treated; ( D ) 250 µg/mL AOD (L)-treated; ( E ) 250 µg/mL AOD (L)-treated cells pretreated with ZVAD, or ( F ) NEC-1, or ( G ) both; ( H ) 125 µg/mL AOB (S)-treated; ( I ) 125 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( J ) NEC-1, or ( K ) both; ( L ) 250 µg/mL AOB (S)-treated; ( M ) 250 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( N ) NEC-1, or ( O ) both. ZVAD = Z-VAD-FMK; NEC-1, Necrostatin 1. The DMSO Control sample contains the same amount of DMSO as in the 250 µg/mL sample of the treatment.

    Techniques Used: Positive Control

    Photomicrographs of HeLa cells treated with 250 µg/mL of the A. orientalis dichloromethane leaf (AOD (L)) fraction or 125 and 250 µg/mL n-butanol stem (AOB (S)) fraction for 24 h. Samples: ( A ) Untreated; ( B ) DMSO control; ( C ) Positive control-treated; ( D ) 250 µg/mL AOD (L)-treated; ( E ) 250 µg/mL AOD (L)-treated cells pretreated with ZVAD, or ( F ) NEC-1, or ( G ) both; ( H ) 125 µg/mL AOB (S)-treated; ( I ) 125 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( J ) NEC-1, or ( K ) both; ( L ) 250 µg/mL AOB (S)-treated; ( M ) 250 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( N ) NEC-1, or ( O ) both. ZVAD = Z-VAD-FMK; NEC-1, Necrostatin 1. The DMSO Control sample contains the same amount of DMSO as in the 250 µg/mL sample of the treatment. Magnification: 400×.
    Figure Legend Snippet: Photomicrographs of HeLa cells treated with 250 µg/mL of the A. orientalis dichloromethane leaf (AOD (L)) fraction or 125 and 250 µg/mL n-butanol stem (AOB (S)) fraction for 24 h. Samples: ( A ) Untreated; ( B ) DMSO control; ( C ) Positive control-treated; ( D ) 250 µg/mL AOD (L)-treated; ( E ) 250 µg/mL AOD (L)-treated cells pretreated with ZVAD, or ( F ) NEC-1, or ( G ) both; ( H ) 125 µg/mL AOB (S)-treated; ( I ) 125 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( J ) NEC-1, or ( K ) both; ( L ) 250 µg/mL AOB (S)-treated; ( M ) 250 µg/mL AOB (S)-treated cells pretreated with ZVAD, or ( N ) NEC-1, or ( O ) both. ZVAD = Z-VAD-FMK; NEC-1, Necrostatin 1. The DMSO Control sample contains the same amount of DMSO as in the 250 µg/mL sample of the treatment. Magnification: 400×.

    Techniques Used: Positive Control

    Western blot analysis of HeLa cells treated with 250 µg/mL of A. orientalis dichloromethane leaf (AOD (L)) or n -butanol stem fractions (AOB (S)) for 24 and 48 h. Actin antibody was used as a loading control. Different oligomeric forms of SQSTM1 were observed on the gel, that have been indicated on the side. DMSO C, DMSO control having same amount of DMSO as the test treated sample.
    Figure Legend Snippet: Western blot analysis of HeLa cells treated with 250 µg/mL of A. orientalis dichloromethane leaf (AOD (L)) or n -butanol stem fractions (AOB (S)) for 24 and 48 h. Actin antibody was used as a loading control. Different oligomeric forms of SQSTM1 were observed on the gel, that have been indicated on the side. DMSO C, DMSO control having same amount of DMSO as the test treated sample.

    Techniques Used: Western Blot

    Cytotoxic effects of A. orientalis dichloromethane leaf fraction (AOD (L)) using the MTT assay on MCF-10A, MCF-7, MDA-MB-231, and HeLa cells in a dose- ( A – C ) and time- ( D – F ) dependent manner after normalizing to the effects of DMSO solvent used to dissolve the extract. *indicates statistically significant differences between the DMSO- and AOD (L)-treated samples (* p
    Figure Legend Snippet: Cytotoxic effects of A. orientalis dichloromethane leaf fraction (AOD (L)) using the MTT assay on MCF-10A, MCF-7, MDA-MB-231, and HeLa cells in a dose- ( A – C ) and time- ( D – F ) dependent manner after normalizing to the effects of DMSO solvent used to dissolve the extract. *indicates statistically significant differences between the DMSO- and AOD (L)-treated samples (* p

    Techniques Used: MTT Assay, Multiple Displacement Amplification

    Photomicrographs of MCF-7 cells treated with 250 µg/mL of A. orientalis dichloromethane leaf fraction (AOD (L)), 30 h post treatment. DMSO C, DMSO control having the same amount of DMSO as the test sample.
    Figure Legend Snippet: Photomicrographs of MCF-7 cells treated with 250 µg/mL of A. orientalis dichloromethane leaf fraction (AOD (L)), 30 h post treatment. DMSO C, DMSO control having the same amount of DMSO as the test sample.

    Techniques Used:

    Western blots of HeLa cells treated with 250 µg/mL of A. orientalis n -butanol stem (AOB (S)) or dichloromethane (AOD (L)) leaf fractions for the indicated time points and tested for the activation of caspase 7, 8, and 9, as well as poly ADB ribose polymerase (PARP). FL, full length. DMSO-treated, DMSO control having the same amount of DMSO as the test sample.
    Figure Legend Snippet: Western blots of HeLa cells treated with 250 µg/mL of A. orientalis n -butanol stem (AOB (S)) or dichloromethane (AOD (L)) leaf fractions for the indicated time points and tested for the activation of caspase 7, 8, and 9, as well as poly ADB ribose polymerase (PARP). FL, full length. DMSO-treated, DMSO control having the same amount of DMSO as the test sample.

    Techniques Used: Western Blot, Activation Assay

    Cytotoxic effects of A. orientalis dichloromethane leaf fraction (AOB (S)) using the MTT assay on MCF-10A, MCF-7, MDA-MB-231, and HeLa cells in a dose- ( A – C ) and time- ( D – F ) dependent manner after normalizing to the effects of DMSO solvent used to dissolve the extract. *indicates statistically significant differences between the DMSO- and AOD (L)-treated samples (* p
    Figure Legend Snippet: Cytotoxic effects of A. orientalis dichloromethane leaf fraction (AOB (S)) using the MTT assay on MCF-10A, MCF-7, MDA-MB-231, and HeLa cells in a dose- ( A – C ) and time- ( D – F ) dependent manner after normalizing to the effects of DMSO solvent used to dissolve the extract. *indicates statistically significant differences between the DMSO- and AOD (L)-treated samples (* p

    Techniques Used: MTT Assay, Multiple Displacement Amplification

    3) Product Images from "Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC"

    Article Title: Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011822

    The effects of calycosin treatment on blood vessel formation in SIVs of Tg(fli1:EGFP) zebrafish embryos. (A) Control: embryo treated with 0.1% DMSO at 96 hpf, SIVs appear as a smooth basket-like structure. (B–D) Calycosin: embryo treated with 10, 30, 100 µM calycosin at 72 hpf for 24 h, leads to enlarged SIV basket stretching into the posterior yolk extension. (a–d) Enlarged SIV region (×4.5) of A–D respectively. White arrows indicating the enlarged vessels, yellow and red arrows indicate sprouting and intersectioning branches respectively. (E) Calycosin increases SIV diameter in a dose-dependent manner. Data are plotted as mean±SEM, (n = 3), *P
    Figure Legend Snippet: The effects of calycosin treatment on blood vessel formation in SIVs of Tg(fli1:EGFP) zebrafish embryos. (A) Control: embryo treated with 0.1% DMSO at 96 hpf, SIVs appear as a smooth basket-like structure. (B–D) Calycosin: embryo treated with 10, 30, 100 µM calycosin at 72 hpf for 24 h, leads to enlarged SIV basket stretching into the posterior yolk extension. (a–d) Enlarged SIV region (×4.5) of A–D respectively. White arrows indicating the enlarged vessels, yellow and red arrows indicate sprouting and intersectioning branches respectively. (E) Calycosin increases SIV diameter in a dose-dependent manner. Data are plotted as mean±SEM, (n = 3), *P

    Techniques Used:

    The effects of calycosin, raloxifene and tamoxifen in SIVs of Tg(fli1:EGFP) . (A) Controls: were treated with 0.1% DMSO at 96 hpf, showing no effect on vessel formation (B–E) were treated with 10 µM raloxifene, 3 µM tamoxifen, 10 µM 17-β-Estradiol and 100 µM calycosin at 72 hpf for 24 h. (a–e) Enlarged SIV region (×4.5) of A–E respectively. Abnormal phenotype of blood vessel formation in SIVs was indicated by white arrow, showing slight increase in vessel diameter. Significant increase in vessel diameter was indicated by thick white arrow.
    Figure Legend Snippet: The effects of calycosin, raloxifene and tamoxifen in SIVs of Tg(fli1:EGFP) . (A) Controls: were treated with 0.1% DMSO at 96 hpf, showing no effect on vessel formation (B–E) were treated with 10 µM raloxifene, 3 µM tamoxifen, 10 µM 17-β-Estradiol and 100 µM calycosin at 72 hpf for 24 h. (a–e) Enlarged SIV region (×4.5) of A–E respectively. Abnormal phenotype of blood vessel formation in SIVs was indicated by white arrow, showing slight increase in vessel diameter. Significant increase in vessel diameter was indicated by thick white arrow.

    Techniques Used:

    The effects of calycosin on HUVECs in vitro . (A) Effects of calycosin on proliferation of HUVEC by XTT assay. HUVECs were seeded in 96-well plates and incubated with calycosin at different concentrations. Cell proliferation was assessed using XTT assay; (B) Tube formation of calycosin-treated HUVECs on Matrigel. HUVECs cultured on 3-dimensional Matrigel in treatment of calycosin (3 µM, 10 µM, 30 µM and 100 µM). Cells receiving 0.1% DMSO served as vehicle control. Number of branching points in different concentrations of calycosin-treated HUVECs was calculated by computer software (Metamorph). Results are expressed as percentage of control (100%) in mean±SEM (n≥3 independent experiments), *P
    Figure Legend Snippet: The effects of calycosin on HUVECs in vitro . (A) Effects of calycosin on proliferation of HUVEC by XTT assay. HUVECs were seeded in 96-well plates and incubated with calycosin at different concentrations. Cell proliferation was assessed using XTT assay; (B) Tube formation of calycosin-treated HUVECs on Matrigel. HUVECs cultured on 3-dimensional Matrigel in treatment of calycosin (3 µM, 10 µM, 30 µM and 100 µM). Cells receiving 0.1% DMSO served as vehicle control. Number of branching points in different concentrations of calycosin-treated HUVECs was calculated by computer software (Metamorph). Results are expressed as percentage of control (100%) in mean±SEM (n≥3 independent experiments), *P

    Techniques Used: In Vitro, XTT Assay, Incubation, Cell Culture, Software

    The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis in zebrafish embryos. (A) Control: embryo treated with 0.1% DMSO at 96 hpf. (B) Calycosin: embryo treated with calycosin (100 µM) at 72 hpf for 24 h. (C E) VRI: embryo treated with low concentration (100 ng/ml, C) and high concentration (1 µg/ml, E) of VRI at 72 hpf for 24 h. (D) VRI and calycosin: embryo treated with both VRI (100 ng/ml) and calycosin (100 µM) at 72 hpf for 24 h. (a–e) Enlarged SIV region (×4.5) of A–E respectively. (F) Effects of calycosin and/or VRI on the diameter of SIV compared with the control group. Data are plotted as mean±SEM, (n = 3), # P
    Figure Legend Snippet: The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis in zebrafish embryos. (A) Control: embryo treated with 0.1% DMSO at 96 hpf. (B) Calycosin: embryo treated with calycosin (100 µM) at 72 hpf for 24 h. (C E) VRI: embryo treated with low concentration (100 ng/ml, C) and high concentration (1 µg/ml, E) of VRI at 72 hpf for 24 h. (D) VRI and calycosin: embryo treated with both VRI (100 ng/ml) and calycosin (100 µM) at 72 hpf for 24 h. (a–e) Enlarged SIV region (×4.5) of A–E respectively. (F) Effects of calycosin and/or VRI on the diameter of SIV compared with the control group. Data are plotted as mean±SEM, (n = 3), # P

    Techniques Used: Concentration Assay

    The effects of calycosin on endothelial cells population in SIVs of Tg(fli1:nEGFP) zebrafish embryos. Each green light point represents one endothelial cell (GFP+). (A) Control: embryo treated with 0.1% DMSO at 96 hpf. (B–D) Calycosin: embryo treated with 10, 30, 100 µM calycosin at 72 hpf for 24 h, leads to an increase in endothelial cells. (a–d) Enlarged SIV region (×4.5) of A–D respectively. (E) Calycosin increases the number of endothelial cells in the SIV region in a dose-dependent manner. Data are plotted as mean±SEM, (n = 3), **P
    Figure Legend Snippet: The effects of calycosin on endothelial cells population in SIVs of Tg(fli1:nEGFP) zebrafish embryos. Each green light point represents one endothelial cell (GFP+). (A) Control: embryo treated with 0.1% DMSO at 96 hpf. (B–D) Calycosin: embryo treated with 10, 30, 100 µM calycosin at 72 hpf for 24 h, leads to an increase in endothelial cells. (a–d) Enlarged SIV region (×4.5) of A–D respectively. (E) Calycosin increases the number of endothelial cells in the SIV region in a dose-dependent manner. Data are plotted as mean±SEM, (n = 3), **P

    Techniques Used:

    4) Product Images from "Activation of aryl hydrocarbon receptor regulates the LPS/IFNγ-induced inflammatory response by inducing ubiquitin-proteosomal and lysosomal degradation of RelA/p65"

    Article Title: Activation of aryl hydrocarbon receptor regulates the LPS/IFNγ-induced inflammatory response by inducing ubiquitin-proteosomal and lysosomal degradation of RelA/p65

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2018.06.016

    Effect of βNF and TCDD on pro-inflammatory cytokine levels in Ahr -null mouse macrophages. Peritoneal macrophages (1 × 10 6 cells/mL) from Ahr -null mice were activated with LPS and IFNγ together (1 μg/mL and 20 ng/mL, respectively) and treated with1 μM βNF or 10 nM TCDD. After 12 h, TNF-α (A), IL-6 (B), and IL-12 (C) protein levels from supernatant cultures were determined by ELISA. DMSO was used as a control. The results are expressed as the mean ± S.D. of three independent experiments.
    Figure Legend Snippet: Effect of βNF and TCDD on pro-inflammatory cytokine levels in Ahr -null mouse macrophages. Peritoneal macrophages (1 × 10 6 cells/mL) from Ahr -null mice were activated with LPS and IFNγ together (1 μg/mL and 20 ng/mL, respectively) and treated with1 μM βNF or 10 nM TCDD. After 12 h, TNF-α (A), IL-6 (B), and IL-12 (C) protein levels from supernatant cultures were determined by ELISA. DMSO was used as a control. The results are expressed as the mean ± S.D. of three independent experiments.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    TCDD induces RelA/p65 lysosome-dependent degradation. Peritoneal macrophages (1 × 10 6 cells/mL) from WT mice were visualized by phase contrast microscopy after 10 nM TCDD treatment (A) and after cell cultures were washed and cultured for another 12 h in fresh control media (B). The arrows point to vacuoles. C) Peritoneal macrophages (1 × 10 6 cells/mL) from WT mice were treated with 10 nM TCDD or were co-treated with 10 nM TCDD and 10 μM chloroquine (CQ) at the indicated times, and RelA/p65 protein levels were determined by Western blot. Densitometric scanning of the blots are shown. DMSO was used as a control and β-actin as a loading control. The results are expressed as the mean ± S.D. of three independent experiments. * p
    Figure Legend Snippet: TCDD induces RelA/p65 lysosome-dependent degradation. Peritoneal macrophages (1 × 10 6 cells/mL) from WT mice were visualized by phase contrast microscopy after 10 nM TCDD treatment (A) and after cell cultures were washed and cultured for another 12 h in fresh control media (B). The arrows point to vacuoles. C) Peritoneal macrophages (1 × 10 6 cells/mL) from WT mice were treated with 10 nM TCDD or were co-treated with 10 nM TCDD and 10 μM chloroquine (CQ) at the indicated times, and RelA/p65 protein levels were determined by Western blot. Densitometric scanning of the blots are shown. DMSO was used as a control and β-actin as a loading control. The results are expressed as the mean ± S.D. of three independent experiments. * p

    Techniques Used: Mouse Assay, Microscopy, Cell Culture, Western Blot

    Effect of βNF and TCDD on pro-inflammatory cytokine levels in WT mouse macrophages. Peritoneal macrophages (1 × 10 6 cells/mL) from WT mice were activated with LPS and IFNγ together (1 μg/mL and 20 ng/mL, respectively) and treated with 1 μM βNF or 10 nM TCDD. After 12 h, TNF-α (A), IL-6 (B), and IL-12 (C) protein levels from supernatant cultures were determined by ELISA. DMSO was used as a control. The results are expressed as the mean ± S.D. of three independent experiments. * p
    Figure Legend Snippet: Effect of βNF and TCDD on pro-inflammatory cytokine levels in WT mouse macrophages. Peritoneal macrophages (1 × 10 6 cells/mL) from WT mice were activated with LPS and IFNγ together (1 μg/mL and 20 ng/mL, respectively) and treated with 1 μM βNF or 10 nM TCDD. After 12 h, TNF-α (A), IL-6 (B), and IL-12 (C) protein levels from supernatant cultures were determined by ELISA. DMSO was used as a control. The results are expressed as the mean ± S.D. of three independent experiments. * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    TCDD induces RelA/p65 26S proteasome-dependent degradation. Peritoneal macrophages (1 × 10 6 cells/mL) from WT (A) or Ahr -null (B) mice were treated with 10 nM TCDD. C) WT peritoneal macrophages were treated with cycloheximide (CHX, 10 μg/mL) for 6 h to inhibit the de novo protein synthesis and harvested at the indicated times. D) WT peritoneal macrophages were co-treated with 10 nM TCDD and 25 μM MG132 at the indicated times. RelA/p65 protein levels were determined by Western blot. Representative blots and densitometric scanning of the blots are shown. DMSO was used as a control and β-actin as a loading control. The data are presented as the mean ± S.D. of three independent experiments. * p
    Figure Legend Snippet: TCDD induces RelA/p65 26S proteasome-dependent degradation. Peritoneal macrophages (1 × 10 6 cells/mL) from WT (A) or Ahr -null (B) mice were treated with 10 nM TCDD. C) WT peritoneal macrophages were treated with cycloheximide (CHX, 10 μg/mL) for 6 h to inhibit the de novo protein synthesis and harvested at the indicated times. D) WT peritoneal macrophages were co-treated with 10 nM TCDD and 25 μM MG132 at the indicated times. RelA/p65 protein levels were determined by Western blot. Representative blots and densitometric scanning of the blots are shown. DMSO was used as a control and β-actin as a loading control. The data are presented as the mean ± S.D. of three independent experiments. * p

    Techniques Used: Mouse Assay, Western Blot

    TCDD failed to promote P-RelA/p65 degradation in Ahr -null mouse macrophages. Peritoneal macrophages (1 × 10 6 cells/mL) from Ahr -null mice were pretreated with 10 nM TCDD at the indicated times and activated with LPS/IFNγ (1 μg/mL and 20 ng/mL, respectively) for 30 min. Control cultures were activated with LPS/IFNγ. A) P-RelA/p65 and RelA/p65 were determined by Western blot. Representative blots and densitometric scanning of the blots are shown, β-actin was used as a loading control. The data are presented as the mean ± S.D. of three independent experiments. B) Representative confocal microscopy images of P-RelA/p65 are shown after macrophage activation only or after 12 h of TCDD pretreatment and LPS/IFNγ. P-RelA/p65, β-actin, and DAPI were visualized as green, red, and blue, respectively. DMSO was used as a control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: TCDD failed to promote P-RelA/p65 degradation in Ahr -null mouse macrophages. Peritoneal macrophages (1 × 10 6 cells/mL) from Ahr -null mice were pretreated with 10 nM TCDD at the indicated times and activated with LPS/IFNγ (1 μg/mL and 20 ng/mL, respectively) for 30 min. Control cultures were activated with LPS/IFNγ. A) P-RelA/p65 and RelA/p65 were determined by Western blot. Representative blots and densitometric scanning of the blots are shown, β-actin was used as a loading control. The data are presented as the mean ± S.D. of three independent experiments. B) Representative confocal microscopy images of P-RelA/p65 are shown after macrophage activation only or after 12 h of TCDD pretreatment and LPS/IFNγ. P-RelA/p65, β-actin, and DAPI were visualized as green, red, and blue, respectively. DMSO was used as a control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Mouse Assay, Western Blot, Confocal Microscopy, Activation Assay

    5) Product Images from "Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX2) Mediated TNF-α and IL-6 Production in Macrophages"

    Article Title: Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX2) Mediated TNF-α and IL-6 Production in Macrophages

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2013/325481

    Effect of propofol pretreatment on LPS-induced generation of ROS. After a 40 min incubation following 50 μ M propofol or dimethyl sulfoxide (DMSO) treatment, cells were stimulated with LPS for 40 min then stained with 2 μ M dye hydroethidine (DHE) and incubated for 30 min at 37°C in the dark. Nuclei were stained with DAPI.
    Figure Legend Snippet: Effect of propofol pretreatment on LPS-induced generation of ROS. After a 40 min incubation following 50 μ M propofol or dimethyl sulfoxide (DMSO) treatment, cells were stimulated with LPS for 40 min then stained with 2 μ M dye hydroethidine (DHE) and incubated for 30 min at 37°C in the dark. Nuclei were stained with DAPI.

    Techniques Used: Incubation, Staining

    Effect of propofol on LPS-induced TNF- α and IL-6 expression. (a) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 50 μ M propofol for 40 min and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (b) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P
    Figure Legend Snippet: Effect of propofol on LPS-induced TNF- α and IL-6 expression. (a) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 50 μ M propofol for 40 min and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (b) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    Effect of propofol pretreatment on LPS-induced NADPH oxidase expression. Cells were pretreated with dimethyl sulfoxide (DMSO) or propofol for 40 min and then stimulated with LPS for 8 h. (a) Protein expression of oxidase subunits was analyzed by Western blot analysis of whole cell lysates. (b, c, and d) The levels of subunit protein expression were quantified by measuring band intensities and displayed as fold increase relative to β -actin. Each value represents the means ± SD for n = 4. # and ∗ indicate that a value significantly ( P
    Figure Legend Snippet: Effect of propofol pretreatment on LPS-induced NADPH oxidase expression. Cells were pretreated with dimethyl sulfoxide (DMSO) or propofol for 40 min and then stimulated with LPS for 8 h. (a) Protein expression of oxidase subunits was analyzed by Western blot analysis of whole cell lysates. (b, c, and d) The levels of subunit protein expression were quantified by measuring band intensities and displayed as fold increase relative to β -actin. Each value represents the means ± SD for n = 4. # and ∗ indicate that a value significantly ( P

    Techniques Used: Expressing, Western Blot

    Effect of propofol pretreatment on LPS-induced phosphorylation of nuclear NF- κ B and Akt. (a), (c) The cells were pretreated with dimethyl sulfoxide (DMSO) or propofol for 40 min and then stimulated with LPS for 1 h. Nuclear NF- κ B and Akt activation, indicated by phosphorylation, was analyzed by Western blotting on whole cell lysates. (b), (d) The levels of phosphorylated NF- κ B (p-NF- κ B) and phosphorylated Akt (p-Akt) were quantified by measuring band intensities and represented as fold change over total NF- κ B and Akt. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P
    Figure Legend Snippet: Effect of propofol pretreatment on LPS-induced phosphorylation of nuclear NF- κ B and Akt. (a), (c) The cells were pretreated with dimethyl sulfoxide (DMSO) or propofol for 40 min and then stimulated with LPS for 1 h. Nuclear NF- κ B and Akt activation, indicated by phosphorylation, was analyzed by Western blotting on whole cell lysates. (b), (d) The levels of phosphorylated NF- κ B (p-NF- κ B) and phosphorylated Akt (p-Akt) were quantified by measuring band intensities and represented as fold change over total NF- κ B and Akt. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P

    Techniques Used: Activation Assay, Western Blot

    Effect of propofol on LPS-induced TNF- α and IL-6 secretion. Macrophages were pretreated with dimethyl sulfoxide (DMSO) or 1 μ M, 10 μ M, 50 μ M, and 100 μ M propofol for 40 min and stimulated with 100 ng/mL LPS for 8 h. The concentrations of TNF- α and IL-6 in culture supernatants were measured by ELISA. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P
    Figure Legend Snippet: Effect of propofol on LPS-induced TNF- α and IL-6 secretion. Macrophages were pretreated with dimethyl sulfoxide (DMSO) or 1 μ M, 10 μ M, 50 μ M, and 100 μ M propofol for 40 min and stimulated with 100 ng/mL LPS for 8 h. The concentrations of TNF- α and IL-6 in culture supernatants were measured by ELISA. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effect of propofol pretreatment on NADPH oxidase activity. Cells were preincubated with dimethyl sulfoxide (DMSO) or propofol for 40 min, followed by 6 h incubation with or without LPS. The activity of NADPH oxidase was measured with an oxidase activity assay kit. Each value represents the means ± SD for n = 4. # and ∗ indicate that a value significantly ( P
    Figure Legend Snippet: Effect of propofol pretreatment on NADPH oxidase activity. Cells were preincubated with dimethyl sulfoxide (DMSO) or propofol for 40 min, followed by 6 h incubation with or without LPS. The activity of NADPH oxidase was measured with an oxidase activity assay kit. Each value represents the means ± SD for n = 4. # and ∗ indicate that a value significantly ( P

    Techniques Used: Activity Assay, Incubation

    6) Product Images from "Bisphenol A inhibits cultured mouse ovarian follicle growth partially via the aryl hydrocarbon receptor signaling pathway"

    Article Title: Bisphenol A inhibits cultured mouse ovarian follicle growth partially via the aryl hydrocarbon receptor signaling pathway

    Journal: Reproductive toxicology (Elmsford, N.Y.)

    doi: 10.1016/j.reprotox.2013.07.022

    Effect of in vitro BPA exposure on Ahr KO mouse antral follicle growth. Antral follicles mechanically isolated from Ahr KO mice were individually cultured with vehicle control (DMSO) or BPA (0.004 - 438 μM) for 96 hours. During the culture, follicle diameters were measured and then were converted to percent change from baseline (time 0). Graph represents means ± SEM from 3-4 separate experiments (at least five follicles per treatment per experiment). Asterisks represent significant differences from DMSO per time point; p ≤0.05.
    Figure Legend Snippet: Effect of in vitro BPA exposure on Ahr KO mouse antral follicle growth. Antral follicles mechanically isolated from Ahr KO mice were individually cultured with vehicle control (DMSO) or BPA (0.004 - 438 μM) for 96 hours. During the culture, follicle diameters were measured and then were converted to percent change from baseline (time 0). Graph represents means ± SEM from 3-4 separate experiments (at least five follicles per treatment per experiment). Asterisks represent significant differences from DMSO per time point; p ≤0.05.

    Techniques Used: In Vitro, Isolation, Mouse Assay, Cell Culture

    Effect of in vitro BPA exposure on estradiol synthesis by wild-type mouse antral follicles. Antral follicles mechanically isolated from wild-type mice were individually cultured with vehicle control (DMSO) or BPA (0.004 - 438 μM). After 96 hours, the media were collected and analyzed for estradiol levels (at least three follicles per treatment, per experiment). Graph represents means ± SEM from three separate experiments. Asterisks represent significant differences from DMSO; p ≤0.05.
    Figure Legend Snippet: Effect of in vitro BPA exposure on estradiol synthesis by wild-type mouse antral follicles. Antral follicles mechanically isolated from wild-type mice were individually cultured with vehicle control (DMSO) or BPA (0.004 - 438 μM). After 96 hours, the media were collected and analyzed for estradiol levels (at least three follicles per treatment, per experiment). Graph represents means ± SEM from three separate experiments. Asterisks represent significant differences from DMSO; p ≤0.05.

    Techniques Used: In Vitro, Isolation, Mouse Assay, Cell Culture

    Effect of 96 hours in vitro BPA exposure on expression of Bcl2 and Bax . Antral follicles isolated from wild-type mice were treated with vehicle control (DMSO) or BPA (0.004 - 438 μM) and cultured for 96 hours. Expression levels of Bcl2 and Bax were measured using qPCR. Data represent means ± SEM from at least three separate experiments. Asterisks represent significant differences from DMSO; p≤0.05.
    Figure Legend Snippet: Effect of 96 hours in vitro BPA exposure on expression of Bcl2 and Bax . Antral follicles isolated from wild-type mice were treated with vehicle control (DMSO) or BPA (0.004 - 438 μM) and cultured for 96 hours. Expression levels of Bcl2 and Bax were measured using qPCR. Data represent means ± SEM from at least three separate experiments. Asterisks represent significant differences from DMSO; p≤0.05.

    Techniques Used: In Vitro, Expressing, Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Effect of in vitro BPA exposure on expression of major factors in the AHR signaling pathway, following 24 and 96 hours of culture. Antral follicles isolated from wild-type mice were treated with vehicle control (DMSO) or BPA (0.004 - 438 μM) and cultured for 24 or 96 hours. Expression levels of Cyp1b1 (a ,b), Arnt (c, d) , Ahr (e, f), and Ahrr (g, h) were measured using qPCR. Data represent means ± SEM from at least three separate experiments. Asterisks represent significant differences from DMSO; p ≤0.05.
    Figure Legend Snippet: Effect of in vitro BPA exposure on expression of major factors in the AHR signaling pathway, following 24 and 96 hours of culture. Antral follicles isolated from wild-type mice were treated with vehicle control (DMSO) or BPA (0.004 - 438 μM) and cultured for 24 or 96 hours. Expression levels of Cyp1b1 (a ,b), Arnt (c, d) , Ahr (e, f), and Ahrr (g, h) were measured using qPCR. Data represent means ± SEM from at least three separate experiments. Asterisks represent significant differences from DMSO; p ≤0.05.

    Techniques Used: In Vitro, Expressing, Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Effect of in vitro BPA exposure on wild-type mouse antral follicle growth. Antral follicles mechanically isolated from wild-type mice were individually cultured with vehicle control (DMSO) or BPA (0.004 - 438 μM) for 96 hours. During the culture, follicle diameters were measured and then were converted to percent change from baseline (time 0). Graph represents means ± SEM from 3-4 separate experiments (at least five follicles per treatment per replicate). Asterisks represent significant differences from DMSO per time point; p ≤0.05.
    Figure Legend Snippet: Effect of in vitro BPA exposure on wild-type mouse antral follicle growth. Antral follicles mechanically isolated from wild-type mice were individually cultured with vehicle control (DMSO) or BPA (0.004 - 438 μM) for 96 hours. During the culture, follicle diameters were measured and then were converted to percent change from baseline (time 0). Graph represents means ± SEM from 3-4 separate experiments (at least five follicles per treatment per replicate). Asterisks represent significant differences from DMSO per time point; p ≤0.05.

    Techniques Used: In Vitro, Isolation, Mouse Assay, Cell Culture

    7) Product Images from "Bornyl cis-4-Hydroxycinnamate Suppresses Cell Metastasis of Melanoma through FAK/PI3K/Akt/mTOR and MAPK Signaling Pathways and Inhibition of the Epithelial-to-Mesenchymal Transition"

    Article Title: Bornyl cis-4-Hydroxycinnamate Suppresses Cell Metastasis of Melanoma through FAK/PI3K/Akt/mTOR and MAPK Signaling Pathways and Inhibition of the Epithelial-to-Mesenchymal Transition

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19082152

    Suppression of epithelial to mesenchymal transition (EMT) by bornyl cis -4-hydroxycinnamate in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM), and ( A ) cytosol and ( B ) nucleus proteins were collected separately for western blot analysis. EMT-related proteins N -cadherin, E -cadherin, and Snail were validated. The protein levels of N -cadherin and Snail were decreased, while that of E -cadherin was increased in A2058 and A375 cells after treatment with bornyl cis -4-hydroxycinnamate. β-actin and Lamin A2 were used as the internal controls separately for cytosol and nucleus proteins. Mock: control, DMSO-treated cells.
    Figure Legend Snippet: Suppression of epithelial to mesenchymal transition (EMT) by bornyl cis -4-hydroxycinnamate in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM), and ( A ) cytosol and ( B ) nucleus proteins were collected separately for western blot analysis. EMT-related proteins N -cadherin, E -cadherin, and Snail were validated. The protein levels of N -cadherin and Snail were decreased, while that of E -cadherin was increased in A2058 and A375 cells after treatment with bornyl cis -4-hydroxycinnamate. β-actin and Lamin A2 were used as the internal controls separately for cytosol and nucleus proteins. Mock: control, DMSO-treated cells.

    Techniques Used: Western Blot

    Effects of bornyl cis -4-hydroxycinnamate on the activities of MMP-2/-9 and protein levels in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 1, 3, 6 µM) for 24 h, and conditioned media and cell lysates were collected for analysis. ( A ) Gelatin zymography showed that bornyl cis -4-hydroxycinnamate inhibited MMP-2/-9 activities in A2058 and A375 cells in a dose-dependent manner; ( B ) The expression levels of MMP-2/-9-related proteins, including MMP-2, MMP-9, uPA, TIMP-1, and TIMP-2, were validated by western blotting. In order to observe the effect of these proteins associated with bornyl cis -4-hydroxycinnamate at low concentrations, a 0.5 μM treated group was added. The results showed decreased MMP-2/-9 and uPA protein levels and increased TIMP-1 and TIMP-2 protein levels in A2058 and A375 cells after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: Effects of bornyl cis -4-hydroxycinnamate on the activities of MMP-2/-9 and protein levels in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 1, 3, 6 µM) for 24 h, and conditioned media and cell lysates were collected for analysis. ( A ) Gelatin zymography showed that bornyl cis -4-hydroxycinnamate inhibited MMP-2/-9 activities in A2058 and A375 cells in a dose-dependent manner; ( B ) The expression levels of MMP-2/-9-related proteins, including MMP-2, MMP-9, uPA, TIMP-1, and TIMP-2, were validated by western blotting. In order to observe the effect of these proteins associated with bornyl cis -4-hydroxycinnamate at low concentrations, a 0.5 μM treated group was added. The results showed decreased MMP-2/-9 and uPA protein levels and increased TIMP-1 and TIMP-2 protein levels in A2058 and A375 cells after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Zymography, Expressing, Western Blot

    The effects of bornyl cis -4-hydroxycinnamate on mitogen-activated protein kinase (MAPK) signaling pathways in A2058 and A375 cells. A2058 and A375 cells were incubated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK of MAPKs-related proteins were validated. Bornyl cis -4-hydroxycinnamate treatment inhibited p-JNK, p-Jun, p-p38, and p-ERK protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: The effects of bornyl cis -4-hydroxycinnamate on mitogen-activated protein kinase (MAPK) signaling pathways in A2058 and A375 cells. A2058 and A375 cells were incubated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK of MAPKs-related proteins were validated. Bornyl cis -4-hydroxycinnamate treatment inhibited p-JNK, p-Jun, p-p38, and p-ERK protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Incubation, Western Blot

    The effects of bornyl cis -4-hydroxycinnamate on the FAK/PI3K/Akt/mTOR signaling pathways in A2058 and A375 cells. A2058 and A375 cells were treated with bornyl cis -4-hydroxycinnamate at various concentrations (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. The FAK/PI3K/Akt/mTOR-related proteins were validated, including FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. Bornyl cis -4-hydroxycinnamate inhibited FAK, p-PI3K, p-Akt, and p-mTOR protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: The effects of bornyl cis -4-hydroxycinnamate on the FAK/PI3K/Akt/mTOR signaling pathways in A2058 and A375 cells. A2058 and A375 cells were treated with bornyl cis -4-hydroxycinnamate at various concentrations (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. The FAK/PI3K/Akt/mTOR-related proteins were validated, including FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. Bornyl cis -4-hydroxycinnamate inhibited FAK, p-PI3K, p-Akt, and p-mTOR protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Western Blot

    The effects of bornyl cis -4-hydroxycinnamate on the GRB2 signaling pathway in A2058 and A375 cells. A2058 and A375 cells were treated with various concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis to determine the expression levels of GRB2-related proteins, including GRB2, Rac, PKC, Ras, RhoA, MEKK3, and MEKK7. The protein expression levels were decreased after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: The effects of bornyl cis -4-hydroxycinnamate on the GRB2 signaling pathway in A2058 and A375 cells. A2058 and A375 cells were treated with various concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis to determine the expression levels of GRB2-related proteins, including GRB2, Rac, PKC, Ras, RhoA, MEKK3, and MEKK7. The protein expression levels were decreased after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Western Blot, Expressing

    8) Product Images from "Bornyl cis-4-Hydroxycinnamate Suppresses Cell Metastasis of Melanoma through FAK/PI3K/Akt/mTOR and MAPK Signaling Pathways and Inhibition of the Epithelial-to-Mesenchymal Transition"

    Article Title: Bornyl cis-4-Hydroxycinnamate Suppresses Cell Metastasis of Melanoma through FAK/PI3K/Akt/mTOR and MAPK Signaling Pathways and Inhibition of the Epithelial-to-Mesenchymal Transition

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19082152

    Suppression of epithelial to mesenchymal transition (EMT) by bornyl cis -4-hydroxycinnamate in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM), and ( A ) cytosol and ( B ) nucleus proteins were collected separately for western blot analysis. EMT-related proteins N -cadherin, E -cadherin, and Snail were validated. The protein levels of N -cadherin and Snail were decreased, while that of E -cadherin was increased in A2058 and A375 cells after treatment with bornyl cis -4-hydroxycinnamate. β-actin and Lamin A2 were used as the internal controls separately for cytosol and nucleus proteins. Mock: control, DMSO-treated cells.
    Figure Legend Snippet: Suppression of epithelial to mesenchymal transition (EMT) by bornyl cis -4-hydroxycinnamate in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM), and ( A ) cytosol and ( B ) nucleus proteins were collected separately for western blot analysis. EMT-related proteins N -cadherin, E -cadherin, and Snail were validated. The protein levels of N -cadherin and Snail were decreased, while that of E -cadherin was increased in A2058 and A375 cells after treatment with bornyl cis -4-hydroxycinnamate. β-actin and Lamin A2 were used as the internal controls separately for cytosol and nucleus proteins. Mock: control, DMSO-treated cells.

    Techniques Used: Western Blot

    Effects of bornyl cis -4-hydroxycinnamate on the activities of MMP-2/-9 and protein levels in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 1, 3, 6 µM) for 24 h, and conditioned media and cell lysates were collected for analysis. ( A ) Gelatin zymography showed that bornyl cis -4-hydroxycinnamate inhibited MMP-2/-9 activities in A2058 and A375 cells in a dose-dependent manner; ( B ) The expression levels of MMP-2/-9-related proteins, including MMP-2, MMP-9, uPA, TIMP-1, and TIMP-2, were validated by western blotting. In order to observe the effect of these proteins associated with bornyl cis -4-hydroxycinnamate at low concentrations, a 0.5 μM treated group was added. The results showed decreased MMP-2/-9 and uPA protein levels and increased TIMP-1 and TIMP-2 protein levels in A2058 and A375 cells after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: Effects of bornyl cis -4-hydroxycinnamate on the activities of MMP-2/-9 and protein levels in A2058 and A375 cells. A2058 and A375 cells were treated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 1, 3, 6 µM) for 24 h, and conditioned media and cell lysates were collected for analysis. ( A ) Gelatin zymography showed that bornyl cis -4-hydroxycinnamate inhibited MMP-2/-9 activities in A2058 and A375 cells in a dose-dependent manner; ( B ) The expression levels of MMP-2/-9-related proteins, including MMP-2, MMP-9, uPA, TIMP-1, and TIMP-2, were validated by western blotting. In order to observe the effect of these proteins associated with bornyl cis -4-hydroxycinnamate at low concentrations, a 0.5 μM treated group was added. The results showed decreased MMP-2/-9 and uPA protein levels and increased TIMP-1 and TIMP-2 protein levels in A2058 and A375 cells after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Zymography, Expressing, Western Blot

    The effects of bornyl cis -4-hydroxycinnamate on mitogen-activated protein kinase (MAPK) signaling pathways in A2058 and A375 cells. A2058 and A375 cells were incubated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK of MAPKs-related proteins were validated. Bornyl cis -4-hydroxycinnamate treatment inhibited p-JNK, p-Jun, p-p38, and p-ERK protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: The effects of bornyl cis -4-hydroxycinnamate on mitogen-activated protein kinase (MAPK) signaling pathways in A2058 and A375 cells. A2058 and A375 cells were incubated with different concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK of MAPKs-related proteins were validated. Bornyl cis -4-hydroxycinnamate treatment inhibited p-JNK, p-Jun, p-p38, and p-ERK protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Incubation, Western Blot

    The effects of bornyl cis -4-hydroxycinnamate on the FAK/PI3K/Akt/mTOR signaling pathways in A2058 and A375 cells. A2058 and A375 cells were treated with bornyl cis -4-hydroxycinnamate at various concentrations (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. The FAK/PI3K/Akt/mTOR-related proteins were validated, including FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. Bornyl cis -4-hydroxycinnamate inhibited FAK, p-PI3K, p-Akt, and p-mTOR protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: The effects of bornyl cis -4-hydroxycinnamate on the FAK/PI3K/Akt/mTOR signaling pathways in A2058 and A375 cells. A2058 and A375 cells were treated with bornyl cis -4-hydroxycinnamate at various concentrations (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis. The FAK/PI3K/Akt/mTOR-related proteins were validated, including FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. Bornyl cis -4-hydroxycinnamate inhibited FAK, p-PI3K, p-Akt, and p-mTOR protein levels in A2058 and A375 cells. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Western Blot

    The effects of bornyl cis -4-hydroxycinnamate on the GRB2 signaling pathway in A2058 and A375 cells. A2058 and A375 cells were treated with various concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis to determine the expression levels of GRB2-related proteins, including GRB2, Rac, PKC, Ras, RhoA, MEKK3, and MEKK7. The protein expression levels were decreased after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).
    Figure Legend Snippet: The effects of bornyl cis -4-hydroxycinnamate on the GRB2 signaling pathway in A2058 and A375 cells. A2058 and A375 cells were treated with various concentrations of bornyl cis -4-hydroxycinnamate (0, 0.5, 1, 3, 6 µM) for 24 h, and cell lysates were collected for Western blot analysis to determine the expression levels of GRB2-related proteins, including GRB2, Rac, PKC, Ras, RhoA, MEKK3, and MEKK7. The protein expression levels were decreased after bornyl cis -4-hydroxycinnamate treatment. β-actin was used as the protein loading control. Mock: cells treated with vehicle control (DMSO).

    Techniques Used: Western Blot, Expressing

    9) Product Images from "BOL-303242-X, a novel selective glucocorticoid receptor agonist, with full anti-inflammatory properties in human ocular cells"

    Article Title: BOL-303242-X, a novel selective glucocorticoid receptor agonist, with full anti-inflammatory properties in human ocular cells

    Journal: Molecular Vision

    doi:

    BOL-303242-X demonstrates similar activity as triamcinolone acetonide (TA) in reducing LPS-induced cytokine release in human optic nerve astrocytes. Cells were pretreated with BOL-303242-X or TA for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, TA, or combinations of them at the indicated doses for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus LPS. The dagger indicates a p≤0.05 versus TA at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data. A Student’s t-test was used to determine whether LPS was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar.
    Figure Legend Snippet: BOL-303242-X demonstrates similar activity as triamcinolone acetonide (TA) in reducing LPS-induced cytokine release in human optic nerve astrocytes. Cells were pretreated with BOL-303242-X or TA for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, TA, or combinations of them at the indicated doses for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus LPS. The dagger indicates a p≤0.05 versus TA at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data. A Student’s t-test was used to determine whether LPS was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar.

    Techniques Used: Activity Assay, Luminex, Concentration Assay

    BOL-303242-X demonstrates similar activity as dexamethasone (DEX) in reducing IL-1ß-induced cytokine release from human corneal epithelial cells. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated with vehicle (0.1% DMSO), IL-1ß, BOL-303242-X, DEX, or combinations of them for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus IL-1ß, and the dagger indicates a p≤0.05 versus BOL-303242-X at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data. A Student’s t-test was used to determine whether IL-1ß was effective. The solid bar is statistically significant versus the gray bar at increasing cytokine release.
    Figure Legend Snippet: BOL-303242-X demonstrates similar activity as dexamethasone (DEX) in reducing IL-1ß-induced cytokine release from human corneal epithelial cells. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated with vehicle (0.1% DMSO), IL-1ß, BOL-303242-X, DEX, or combinations of them for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus IL-1ß, and the dagger indicates a p≤0.05 versus BOL-303242-X at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data. A Student’s t-test was used to determine whether IL-1ß was effective. The solid bar is statistically significant versus the gray bar at increasing cytokine release.

    Techniques Used: Activity Assay, Luminex, Concentration Assay

    BOL-303242-X reduces LPS-induced cytokine release with similar activity as dexamethasone (DEX) from THP-1 cells. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, DEX, or their combinations at the indicated doses for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM or geometric means±SE estimated by the Taylor series expansion for the MCP-1 data; n=3. The asterisk indicates a p≤0.05 versus LPS. The dagger indicates a p≤0.05 versus DEX at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test, and data elevated to the power of 0.4 for IL-6, 0.8 for IL-12p40, and by taking the logarithm of MCP-1 data. A Student’s t-test was used to determine whether LPS was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar.
    Figure Legend Snippet: BOL-303242-X reduces LPS-induced cytokine release with similar activity as dexamethasone (DEX) from THP-1 cells. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, DEX, or their combinations at the indicated doses for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM or geometric means±SE estimated by the Taylor series expansion for the MCP-1 data; n=3. The asterisk indicates a p≤0.05 versus LPS. The dagger indicates a p≤0.05 versus DEX at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test, and data elevated to the power of 0.4 for IL-6, 0.8 for IL-12p40, and by taking the logarithm of MCP-1 data. A Student’s t-test was used to determine whether LPS was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar.

    Techniques Used: Activity Assay, Luminex, Concentration Assay

    BOL-303242-X demonstrates similar activity as triamcinolone acetonide (TA) in reducing LPS-induced IL-1ß and ICAM-1 levels in human retinal endothelial cell lysates. Cells were pretreated with BOL-303242-X or TA for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, TA, or combinations of them at the indicated doses for 18 h. Cytokine and ICAM-1 content in the cell lysates was determined by Luminex, and protein content in the lysates by the Micro BCA assay. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus the control, and the dagger indicates a p≤0.05 versus LPS. Statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test on raw data.
    Figure Legend Snippet: BOL-303242-X demonstrates similar activity as triamcinolone acetonide (TA) in reducing LPS-induced IL-1ß and ICAM-1 levels in human retinal endothelial cell lysates. Cells were pretreated with BOL-303242-X or TA for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, TA, or combinations of them at the indicated doses for 18 h. Cytokine and ICAM-1 content in the cell lysates was determined by Luminex, and protein content in the lysates by the Micro BCA assay. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus the control, and the dagger indicates a p≤0.05 versus LPS. Statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test on raw data.

    Techniques Used: Activity Assay, Luminex, BIA-KA

    BOL-303242-X demonstrates similar activity as dexamethasone (DEX) in reducing IL-1ß-induced cytokine release from human conjunctival fibroblasts. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated for 18 h with vehicle (0.1% DMSO), IL-1ß, BOL-303242-X, DEX, or combinations of them. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM or geometric means±SE estimated by the Taylor Series expansion for the MCP-1 data; n=3. The asterisk indicates a p≤0.05 versus that for IL-1ß. The dagger indicates a p≤0.05 versus BOL-303242-X at same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data for G-CSF and IL-8. Data were elevated to the power of 0.4 for IL-6 and by taking the logarithm of MCP-1 data. A Student’s t-test was used to determine whether IL-1ß was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar at increasing cytokine release.
    Figure Legend Snippet: BOL-303242-X demonstrates similar activity as dexamethasone (DEX) in reducing IL-1ß-induced cytokine release from human conjunctival fibroblasts. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated for 18 h with vehicle (0.1% DMSO), IL-1ß, BOL-303242-X, DEX, or combinations of them. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM or geometric means±SE estimated by the Taylor Series expansion for the MCP-1 data; n=3. The asterisk indicates a p≤0.05 versus that for IL-1ß. The dagger indicates a p≤0.05 versus BOL-303242-X at same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data for G-CSF and IL-8. Data were elevated to the power of 0.4 for IL-6 and by taking the logarithm of MCP-1 data. A Student’s t-test was used to determine whether IL-1ß was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar at increasing cytokine release.

    Techniques Used: Activity Assay, Luminex, Concentration Assay

    10) Product Images from "Dihydroaustrasulfone Alcohol (WA-25) Impedes Macrophage Foam Cell Formation by Regulating the Transforming Growth Factor-β1 Pathway"

    Article Title: Dihydroaustrasulfone Alcohol (WA-25) Impedes Macrophage Foam Cell Formation by Regulating the Transforming Growth Factor-β1 Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160510507

    SB-431542 (TGF-β1 inhibitor) blocks the lipolytic effect of WA-25 in RAW 264.7 macrophages stimulated by LPS. Cells were loaded with ( A ) DMSO; ( B ) LPS; ( C ) LPS + WA-25 (10 μM); or ( D ) LPS + WA-25 (10 μM) + SB-431542 (20 μM) in the presence of OA for 16 h. The arrows indicate lipid droplets; ( E ) The integrated intensity of immunofluorescent staining of lipid droplet. Scale bar, 50 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p
    Figure Legend Snippet: SB-431542 (TGF-β1 inhibitor) blocks the lipolytic effect of WA-25 in RAW 264.7 macrophages stimulated by LPS. Cells were loaded with ( A ) DMSO; ( B ) LPS; ( C ) LPS + WA-25 (10 μM); or ( D ) LPS + WA-25 (10 μM) + SB-431542 (20 μM) in the presence of OA for 16 h. The arrows indicate lipid droplets; ( E ) The integrated intensity of immunofluorescent staining of lipid droplet. Scale bar, 50 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p

    Techniques Used: Staining

    Effects of WA-25 on CD36 expression and LDL uptake in LPS-induced RAW 264.7 murine macrophages. ( A ) Western blots for CD36 and histogram showing the relative intensity; Cells were treated with LDL conjugated to DyLight™ 549 and incubated for 16 h with ( B ) DMSO (control); ( C ) LPS; ( D ) LPS + WA-25 (10 μM); or ( E ) WA-25 (10 μM). Scale bar, 30 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis.
    Figure Legend Snippet: Effects of WA-25 on CD36 expression and LDL uptake in LPS-induced RAW 264.7 murine macrophages. ( A ) Western blots for CD36 and histogram showing the relative intensity; Cells were treated with LDL conjugated to DyLight™ 549 and incubated for 16 h with ( B ) DMSO (control); ( C ) LPS; ( D ) LPS + WA-25 (10 μM); or ( E ) WA-25 (10 μM). Scale bar, 30 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis.

    Techniques Used: Expressing, Western Blot, Incubation

    Nile red staining of lipid droplets in RAW 264.7 macrophages. Cells were loaded with ( A ) Dimethyl sulfoxide (DMSO); ( B ) LPS; ( C ) LPS and WA-25 (10 μM); ( D ) LPS and simvastatin (10 μM) in the presence of oleic acid (OA) for 16 h. The arrows indicate lipid droplets; ( E ) The integrated intensity of immunofluorescent staining of lipid droplet. The number of lipid droplets was significantly decreased after WA-25 and simvastatin treatment. Scale bar, 50 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p
    Figure Legend Snippet: Nile red staining of lipid droplets in RAW 264.7 macrophages. Cells were loaded with ( A ) Dimethyl sulfoxide (DMSO); ( B ) LPS; ( C ) LPS and WA-25 (10 μM); ( D ) LPS and simvastatin (10 μM) in the presence of oleic acid (OA) for 16 h. The arrows indicate lipid droplets; ( E ) The integrated intensity of immunofluorescent staining of lipid droplet. The number of lipid droplets was significantly decreased after WA-25 and simvastatin treatment. Scale bar, 50 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p

    Techniques Used: Staining

    Effects of WA-25 or simvastatin on the expression of TGF-β1 in RAW 264.7 murine macrophages induced by LPS. Cells were immunostained with anti TGF-β1 proteins. RAW 264.7 were incubated for 16 h with ( A ) the DMSO control; ( B ) LPS; ( C ) LPS + WA-25 (10 μM); or ( D ) LPS and simvastatin (10 μM); ( E ) The integrated intensity of immunofluorescent staining of TGF-β1 protein. Scale bar, 50 μm. These experiments were repeated three times, and images of at least 6 different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p
    Figure Legend Snippet: Effects of WA-25 or simvastatin on the expression of TGF-β1 in RAW 264.7 murine macrophages induced by LPS. Cells were immunostained with anti TGF-β1 proteins. RAW 264.7 were incubated for 16 h with ( A ) the DMSO control; ( B ) LPS; ( C ) LPS + WA-25 (10 μM); or ( D ) LPS and simvastatin (10 μM); ( E ) The integrated intensity of immunofluorescent staining of TGF-β1 protein. Scale bar, 50 μm. These experiments were repeated three times, and images of at least 6 different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p

    Techniques Used: Expressing, Incubation, Staining

    Effects of WA-25 and simvastatin on the expression of lysosome and cAMP level in RAW 264.7 murine macrophages induced by LPS. Cells were immunostained with anti-LAMP1 proteins. RAW 264.7 were incubated for 16 h with ( A ) the DMSO control; ( B ) LPS; ( C ) LPS + WA-25 (10 μM); or ( D ) LPS + simvastatin (10 μM); ( E ) The integrated intensity of immunofluorescent staining of LAMP-1 protein; ( F ) The cAMP level. Scale bar, 50 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p
    Figure Legend Snippet: Effects of WA-25 and simvastatin on the expression of lysosome and cAMP level in RAW 264.7 murine macrophages induced by LPS. Cells were immunostained with anti-LAMP1 proteins. RAW 264.7 were incubated for 16 h with ( A ) the DMSO control; ( B ) LPS; ( C ) LPS + WA-25 (10 μM); or ( D ) LPS + simvastatin (10 μM); ( E ) The integrated intensity of immunofluorescent staining of LAMP-1 protein; ( F ) The cAMP level. Scale bar, 50 μm. These experiments were repeated three times, and images of at least six different fields were taken for analysis. *, significantly different from the LPS-stimulated group ( p

    Techniques Used: Expressing, Incubation, Staining

    11) Product Images from "Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR"

    Article Title: Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

    Journal: Toxicology letters

    doi: 10.1016/j.toxlet.2014.12.015

    DIM induces PXR transactivation of CYP3A4 promoter activity: (A, B C) CYP3A4 promoter activity was determined in HepG2 and LS174 T cells. The cells were transiently cotransfected with pGL3-CYP3A4-luc and either pcDNA3 (empty vector) or pcDNA3-hPXR or pcDNA3-mPXR plasmids. After 24 h of transfection, the cells were treated with the vehicle control DMSO, RIF, PCN or DIM as indicated for another 24 h. CYP3A4 promoter activity was determined by measuring the firefly luciferase activity 24 h after the treatments. The firefly luciferase activity was normalized to number of live cells measured using the CellTiter-Glo Luminescent Cell Viability Assay and presented as Relative Luminescence Units. DIM did not exert a noticeable cytotoxicity at the tested concentrations (data not shown). The values represent the means of eight independent experiments, and the bars denote the standard deviation. *, p
    Figure Legend Snippet: DIM induces PXR transactivation of CYP3A4 promoter activity: (A, B C) CYP3A4 promoter activity was determined in HepG2 and LS174 T cells. The cells were transiently cotransfected with pGL3-CYP3A4-luc and either pcDNA3 (empty vector) or pcDNA3-hPXR or pcDNA3-mPXR plasmids. After 24 h of transfection, the cells were treated with the vehicle control DMSO, RIF, PCN or DIM as indicated for another 24 h. CYP3A4 promoter activity was determined by measuring the firefly luciferase activity 24 h after the treatments. The firefly luciferase activity was normalized to number of live cells measured using the CellTiter-Glo Luminescent Cell Viability Assay and presented as Relative Luminescence Units. DIM did not exert a noticeable cytotoxicity at the tested concentrations (data not shown). The values represent the means of eight independent experiments, and the bars denote the standard deviation. *, p

    Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Luciferase, Cell Viability Assay, Standard Deviation

    DIM activation of rPXR WT and rPXR F305L: HepG2 cells were transiently cotransfected with pGL3-CYP3A4-luc, CMV-Renilla and pEF-rPXR or pEF-rPXR F305L plasmids. After 24 h transfection, the cells were treated with the vehicle control DMSO, PCN or DIM as indicated for another 24 h. The firefly and renilla luciferase activities were measured 24 h after the treatments using Dual-Glo luciferase assay system. The normalized CYP3A4 promoter activity is presented as fold activation over the DMSO control and represent the average from triplicate assays. P
    Figure Legend Snippet: DIM activation of rPXR WT and rPXR F305L: HepG2 cells were transiently cotransfected with pGL3-CYP3A4-luc, CMV-Renilla and pEF-rPXR or pEF-rPXR F305L plasmids. After 24 h transfection, the cells were treated with the vehicle control DMSO, PCN or DIM as indicated for another 24 h. The firefly and renilla luciferase activities were measured 24 h after the treatments using Dual-Glo luciferase assay system. The normalized CYP3A4 promoter activity is presented as fold activation over the DMSO control and represent the average from triplicate assays. P

    Techniques Used: Activation Assay, Transfection, Luciferase, Activity Assay

    DIM induces CYP3A4 and MDR1 gene expression in primary human hepatocytes and LS174 T cells: CYP3A4 and MDR1 mRNA expression was analyzed by quantitative RT-PCR in primary human hepatocytes (HU1488, Invitrogen) (A B) and LS174 T cells (C D) after treatment with vehicle DMSO, RIF, or increasing concentrations of DIM as indicated for 48 h. Results are presented as fold increase over the untreated control. Data represent mean ± SEM from three independent experiments: *, p
    Figure Legend Snippet: DIM induces CYP3A4 and MDR1 gene expression in primary human hepatocytes and LS174 T cells: CYP3A4 and MDR1 mRNA expression was analyzed by quantitative RT-PCR in primary human hepatocytes (HU1488, Invitrogen) (A B) and LS174 T cells (C D) after treatment with vehicle DMSO, RIF, or increasing concentrations of DIM as indicated for 48 h. Results are presented as fold increase over the untreated control. Data represent mean ± SEM from three independent experiments: *, p

    Techniques Used: Expressing, Quantitative RT-PCR

    DIM affects MDR1 substrate accumulation in LS174 T and LS180 cells: LS174 T (A) and LS180 (B) cells were treated with DMSO, RIF or DIM as indicated for 48 h. Rhodamine 123 accumulation was then determined in the absence or presence of the MDR1 specific inhibitor Valspodar (PSC-833). The ratio of intracellular rhodamine 123 fluorescence in the absence or presence of PSC-833 indicates the functionality of MDR1. The data are normalized to the DMSO controls and are presented as mean ± SD of three independent experiments (* P
    Figure Legend Snippet: DIM affects MDR1 substrate accumulation in LS174 T and LS180 cells: LS174 T (A) and LS180 (B) cells were treated with DMSO, RIF or DIM as indicated for 48 h. Rhodamine 123 accumulation was then determined in the absence or presence of the MDR1 specific inhibitor Valspodar (PSC-833). The ratio of intracellular rhodamine 123 fluorescence in the absence or presence of PSC-833 indicates the functionality of MDR1. The data are normalized to the DMSO controls and are presented as mean ± SD of three independent experiments (* P

    Techniques Used: Fluorescence

    Knockdown of hPXR expression attenuates DIM-induced hPXR target gene expression in HepG2 and LS174 T cells. (A) HepG2 cells stably expressing FLAG-hPXR were transfected with control non-silencing siRNA or hPXR siRNA. Whole-cell lysates were collected 72 h after siRNA transfections and subjected to western blot analysis using anti-FLAG and anti-actin antibodies (as a loading control). COS7 cells transiently transfected with FLAG-hPXR or FLAG-pcDNA served as positive or negative controls, respectively, for hPXR expression. Data shown are from a representative experiment. (B) 48 h after siRNA transfections, the cells were transiently cotransfected with CYP3A4-luc and CMV-Renilla (transfection control) plasmids, and 24 h after transfection, the cells were treated for another 24 h with DMSO, RIF, or DIM, as indicated. The firefly and renilla luciferase activities were measured 24 h after the treatments using Dual-Glo luciferase assay system. The normalized CYP3A4 promoter activity is expressed as relative luciferase units and presented as fold change over the DMSO control. Data represent mean ± SD from three independent experiments. Statistical significance (*, p
    Figure Legend Snippet: Knockdown of hPXR expression attenuates DIM-induced hPXR target gene expression in HepG2 and LS174 T cells. (A) HepG2 cells stably expressing FLAG-hPXR were transfected with control non-silencing siRNA or hPXR siRNA. Whole-cell lysates were collected 72 h after siRNA transfections and subjected to western blot analysis using anti-FLAG and anti-actin antibodies (as a loading control). COS7 cells transiently transfected with FLAG-hPXR or FLAG-pcDNA served as positive or negative controls, respectively, for hPXR expression. Data shown are from a representative experiment. (B) 48 h after siRNA transfections, the cells were transiently cotransfected with CYP3A4-luc and CMV-Renilla (transfection control) plasmids, and 24 h after transfection, the cells were treated for another 24 h with DMSO, RIF, or DIM, as indicated. The firefly and renilla luciferase activities were measured 24 h after the treatments using Dual-Glo luciferase assay system. The normalized CYP3A4 promoter activity is expressed as relative luciferase units and presented as fold change over the DMSO control. Data represent mean ± SD from three independent experiments. Statistical significance (*, p

    Techniques Used: Expressing, Stable Transfection, Transfection, Western Blot, Luciferase, Activity Assay

    12) Product Images from "Unlike for Human Monocytes after LPS Activation, Release of TNF-? by THP-1 Cells Is Produced by a TACE Catalytically Different from Constitutive TACE"

    Article Title: Unlike for Human Monocytes after LPS Activation, Release of TNF-? by THP-1 Cells Is Produced by a TACE Catalytically Different from Constitutive TACE

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034184

    Effects of Ro 32-7315, Celastrol and TQ on MOCA and ABZ substrates hydrolysis by THP-1 cells. The viable intact THP-1 cells (0.5×10 6 cells/ml) were incubated with 5 µM of the MOCA or ABZ substrate in Ca 2+ and Mg 2+ free HBSS in the presence of various concentrations of Ro 32-7315, Celastrol or TQ. The final DMSO concentration in the reaction mixture was 0.1%. The fluorescence intensity was monitored for 600 s and the rates of the substrates hydrolysis were calculated from the linear section of the fluorescence curve. To estimate the accurate inhibitory potency of the tested compounds, the reaction rates of MOCA hydrolysis obtained for different compounds concentrations (V(MOCA) [inhibitor] ) were divided by MOCA hydrolysis reaction rate in the presence of 0.1%DMSO ( V(MOCA) [0.1% DMSO] ). Inhibitory potency (%) = 100%−(V(MOCA) [inhibitor] /V(MOCA) [0.1% DMSO] )×100%.
    Figure Legend Snippet: Effects of Ro 32-7315, Celastrol and TQ on MOCA and ABZ substrates hydrolysis by THP-1 cells. The viable intact THP-1 cells (0.5×10 6 cells/ml) were incubated with 5 µM of the MOCA or ABZ substrate in Ca 2+ and Mg 2+ free HBSS in the presence of various concentrations of Ro 32-7315, Celastrol or TQ. The final DMSO concentration in the reaction mixture was 0.1%. The fluorescence intensity was monitored for 600 s and the rates of the substrates hydrolysis were calculated from the linear section of the fluorescence curve. To estimate the accurate inhibitory potency of the tested compounds, the reaction rates of MOCA hydrolysis obtained for different compounds concentrations (V(MOCA) [inhibitor] ) were divided by MOCA hydrolysis reaction rate in the presence of 0.1%DMSO ( V(MOCA) [0.1% DMSO] ). Inhibitory potency (%) = 100%−(V(MOCA) [inhibitor] /V(MOCA) [0.1% DMSO] )×100%.

    Techniques Used: Incubation, Concentration Assay, Fluorescence

    THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment. Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p
    Figure Legend Snippet: THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment. Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    PBMC surface accumulation of the membrane-bound TNF-α after Ro 32-7315, Celastrol and TQ treatment. Cells were stimulated for 24 hours with 5 µg/ml of LPS in the presence of 10 µM of Ro 32-7315, TQ and Celastrol. 0.1% (v/v) DMSO was used as a control (silver histogram). The non-permeabilized cells were then stained with FITC-anti-m-TNF-α MAb. Membrane TNF-α expression was evaluated by flow cytometry. The representative histograms are shown. The fluorescence intensity (AU) is plotted against the number of cells (y-axis). For all 3 compounds, membrane–bound TNF-α expression increased after THP-1 cells were treated (p
    Figure Legend Snippet: PBMC surface accumulation of the membrane-bound TNF-α after Ro 32-7315, Celastrol and TQ treatment. Cells were stimulated for 24 hours with 5 µg/ml of LPS in the presence of 10 µM of Ro 32-7315, TQ and Celastrol. 0.1% (v/v) DMSO was used as a control (silver histogram). The non-permeabilized cells were then stained with FITC-anti-m-TNF-α MAb. Membrane TNF-α expression was evaluated by flow cytometry. The representative histograms are shown. The fluorescence intensity (AU) is plotted against the number of cells (y-axis). For all 3 compounds, membrane–bound TNF-α expression increased after THP-1 cells were treated (p

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Fluorescence

    Inhibition of TNF-α secretion by different human cell types in presence of Ro 32-7315, TQ and Celastrol. Cells (5×10 5 /ml) were incubated for 4 hours (for human monocytes and THP-1 cells) or 24 hours (for PBMC) with 1or 5 µg/ml LPS respectively, in the presence of ranging compounds concentrations or 0.1% (v/v) DMSO as a control. The TNF-α levels were then assessed in culture supernatant by ELISA technique. Dose-response curves were fitted on GraphPad PRISM (v.4.0, GraphPad Software, Inc. La Jolla, CA,USA) sigmoidal dose-response curve-fit model (n = 3 independent experiments); s-TNF-α correspond to the secreted form of TNF-α. Statistical difference between IC50 of Ro 32-7315, TQ and Celastrol are indicated in Table 1 .
    Figure Legend Snippet: Inhibition of TNF-α secretion by different human cell types in presence of Ro 32-7315, TQ and Celastrol. Cells (5×10 5 /ml) were incubated for 4 hours (for human monocytes and THP-1 cells) or 24 hours (for PBMC) with 1or 5 µg/ml LPS respectively, in the presence of ranging compounds concentrations or 0.1% (v/v) DMSO as a control. The TNF-α levels were then assessed in culture supernatant by ELISA technique. Dose-response curves were fitted on GraphPad PRISM (v.4.0, GraphPad Software, Inc. La Jolla, CA,USA) sigmoidal dose-response curve-fit model (n = 3 independent experiments); s-TNF-α correspond to the secreted form of TNF-α. Statistical difference between IC50 of Ro 32-7315, TQ and Celastrol are indicated in Table 1 .

    Techniques Used: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay, Software

    Effects of Ro 32-7315, Celastrol and TQ on MOCA and ABZ substrates hydrolysis by human monocytes as compared to the THP-1 cell line (inset). The enzymatic activity was determined for viable intact cells using MOCA and ABZ substrates (n = 3). Thus, 0.5×10 6 /ml of viable cells were incubated with 5 µM of the substrate in Ca 2+ and Mg 2+ free HBSS in the presence of compounds (10 µM). Fluorescence intensity was monitored for 600 s, and the rates of the peptide hydrolysis were then calculated from the linear section of the fluorescence curve. The inhibitory potency of the compounds was calculated by divining the reaction rates of substrate hydrolysis obtained for different compounds concentrations (V(substrate) [inhibitor] ) by substrate hydrolysis reaction rate with DMSO addition ( V(substrate) [0.1% DMSO] ). Inhibitory potency (%) = 100%−(V(substrate)[inhibitor]/V(substrate)[0.1% DMSO])×100%.
    Figure Legend Snippet: Effects of Ro 32-7315, Celastrol and TQ on MOCA and ABZ substrates hydrolysis by human monocytes as compared to the THP-1 cell line (inset). The enzymatic activity was determined for viable intact cells using MOCA and ABZ substrates (n = 3). Thus, 0.5×10 6 /ml of viable cells were incubated with 5 µM of the substrate in Ca 2+ and Mg 2+ free HBSS in the presence of compounds (10 µM). Fluorescence intensity was monitored for 600 s, and the rates of the peptide hydrolysis were then calculated from the linear section of the fluorescence curve. The inhibitory potency of the compounds was calculated by divining the reaction rates of substrate hydrolysis obtained for different compounds concentrations (V(substrate) [inhibitor] ) by substrate hydrolysis reaction rate with DMSO addition ( V(substrate) [0.1% DMSO] ). Inhibitory potency (%) = 100%−(V(substrate)[inhibitor]/V(substrate)[0.1% DMSO])×100%.

    Techniques Used: Activity Assay, Incubation, Fluorescence

    13) Product Images from "Lysine suppresses myofibrillar protein degradation by regulating the autophagic-lysosomal system through phosphorylation of Akt in C2C12 cells"

    Article Title: Lysine suppresses myofibrillar protein degradation by regulating the autophagic-lysosomal system through phosphorylation of Akt in C2C12 cells

    Journal: SpringerPlus

    doi: 10.1186/2193-1801-3-584

    Lys regulates the mTOR and AMPK pathways by activating Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of 4E-BP1 (a) and AMPK (b) in the lysates was analyzed by Western blotting. The level of 4E-BP1 phosphorylation was estimated from the ratio of the γ-form to that of total 4E-BP1. Results are expressed as the level relative to the level in the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p
    Figure Legend Snippet: Lys regulates the mTOR and AMPK pathways by activating Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of 4E-BP1 (a) and AMPK (b) in the lysates was analyzed by Western blotting. The level of 4E-BP1 phosphorylation was estimated from the ratio of the γ-form to that of total 4E-BP1. Results are expressed as the level relative to the level in the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p

    Techniques Used: Western Blot

    Lys suppresses myofibrillar protein degradation through activation of Akt in C2C12 myotubes. Cells were incubated in medium containing DMSO (C), 10 μM Akti (Ai), 10 mM Lys (K), or 10 mM Lys with 10 μM Akti (AiK) for 4 h. The amount of MeHis released from the cells was measured by HPLC. Values are means with SE (n = 3). Different letters indicate significant differences among the groups (p
    Figure Legend Snippet: Lys suppresses myofibrillar protein degradation through activation of Akt in C2C12 myotubes. Cells were incubated in medium containing DMSO (C), 10 μM Akti (Ai), 10 mM Lys (K), or 10 mM Lys with 10 μM Akti (AiK) for 4 h. The amount of MeHis released from the cells was measured by HPLC. Values are means with SE (n = 3). Different letters indicate significant differences among the groups (p

    Techniques Used: Activation Assay, Incubation, High Performance Liquid Chromatography

    Lys suppresses the autophagic-lysosomal system through activation of Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The ratio of LC3-II to total LC3 (LC3-I + LC3-II) in the lysates was determined by Western blotting. Results are expressed as the level relative to the control group. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p
    Figure Legend Snippet: Lys suppresses the autophagic-lysosomal system through activation of Akt in C2C12 myotubes. Cells were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The ratio of LC3-II to total LC3 (LC3-I + LC3-II) in the lysates was determined by Western blotting. Results are expressed as the level relative to the control group. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p

    Techniques Used: Activation Assay, Western Blot

    Lys induces phosphorylation of Akt in C2C12 myotubes. C2C12 myotubes were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of Akt Ser473 (a) and Akt Thr308 (b) in the lysates was analyzed by Western blotting. Results are expressed as the level relative to the expression level of the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p
    Figure Legend Snippet: Lys induces phosphorylation of Akt in C2C12 myotubes. C2C12 myotubes were treated for 30 min with DMSO (C), 10 μM Akti (Ai), 10 mM Leu (L), 10 mM Leu and 10 μM Akti (AiL), 10 mM Lys (K), or 10 mM Lys and 10 μM Akti (AiK). The phosphorylation level of Akt Ser473 (a) and Akt Thr308 (b) in the lysates was analyzed by Western blotting. Results are expressed as the level relative to the expression level of the control group. Representative immunoblots are shown. Values are means with SE (n = 3–4). Different letters indicate significant differences among the groups (p

    Techniques Used: Western Blot, Expressing

    Effect of Lys and/or AICAR treatment on myofibrillar protein degradation in C2C12 myotubes. Cells were incubated in medium containing DMSO (C), 1 mM AICAR (Ac), 10 mM Lys (K), or 10 mM Lys and 1 mM AICAR (AcK) for 4 h. MeHis released from the cells was measured by HPLC. Values are means with SE (n = 3). Different letters indicate significant differences among the groups (p
    Figure Legend Snippet: Effect of Lys and/or AICAR treatment on myofibrillar protein degradation in C2C12 myotubes. Cells were incubated in medium containing DMSO (C), 1 mM AICAR (Ac), 10 mM Lys (K), or 10 mM Lys and 1 mM AICAR (AcK) for 4 h. MeHis released from the cells was measured by HPLC. Values are means with SE (n = 3). Different letters indicate significant differences among the groups (p

    Techniques Used: Incubation, High Performance Liquid Chromatography

    14) Product Images from "Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells"

    Article Title: Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22862-0

    Cytotoxicity of scoulerine ( 1 ), ( 2 ), ( 3 ) and ( 4 ) following a single-dose exposure at a concentration of 10 µM. Cell proliferation and viability of Jurkat, MOLT-4, Raji, HL-60, U-937 and HEL 92.1.7 cells measured by using XTT assay 48 h after treatment. Viability is referred to cells treated with 0.1% DMSO (Control DMSO). Data are shown as mean values ± SD of at least three independent experiments. *Significantly different to control (P ≤ 0.05) ( A ). Dynamic real-time xCELLigence screen of proliferation and cytotoxicity over 62 h. The human A2780 ovarian carcinoma cells in the logarithmic growth phase were treated. The negative control cells were exposed to 0.1% DMSO (vehicle) and 5% DMSO was used as a positive control. The plot is representative of at least three experiments performed ( B ).
    Figure Legend Snippet: Cytotoxicity of scoulerine ( 1 ), ( 2 ), ( 3 ) and ( 4 ) following a single-dose exposure at a concentration of 10 µM. Cell proliferation and viability of Jurkat, MOLT-4, Raji, HL-60, U-937 and HEL 92.1.7 cells measured by using XTT assay 48 h after treatment. Viability is referred to cells treated with 0.1% DMSO (Control DMSO). Data are shown as mean values ± SD of at least three independent experiments. *Significantly different to control (P ≤ 0.05) ( A ). Dynamic real-time xCELLigence screen of proliferation and cytotoxicity over 62 h. The human A2780 ovarian carcinoma cells in the logarithmic growth phase were treated. The negative control cells were exposed to 0.1% DMSO (vehicle) and 5% DMSO was used as a positive control. The plot is representative of at least three experiments performed ( B ).

    Techniques Used: Concentration Assay, XTT Assay, Negative Control, Positive Control

    Western blot analysis of proteins that regulate cell cycle progression or cell death in Jurkat and MOLT-4 cells after scoulerine treatment for 24 h. Control cells were mock treated with 0.1% DMSO (DMSO). Cells treated with cisplatin at 5 µM were used for positive control in Western blot analysis. These experiments were performed at least three times with similar results and a cropped blot is shown from one representative experiment.
    Figure Legend Snippet: Western blot analysis of proteins that regulate cell cycle progression or cell death in Jurkat and MOLT-4 cells after scoulerine treatment for 24 h. Control cells were mock treated with 0.1% DMSO (DMSO). Cells treated with cisplatin at 5 µM were used for positive control in Western blot analysis. These experiments were performed at least three times with similar results and a cropped blot is shown from one representative experiment.

    Techniques Used: Western Blot, Positive Control

    Microscopic images of A549 cells stained with an anti-β-tubulin antibody (red) and counterstained with DAPI (blue). The cells were treated for 24 h with scoulerine or a solvent (0.1% DMSO) as vehicle control. Nocodazole, an antineoplastic agent that disrupts microtubule function by binding to tubulin was used as a reference compound in this assay. Scale bar: 10 µm. Experiments were performed in triplicate using epi-fluorescence microscopy. Photographs from representative chambers are shown. Compared with controls, thicker and denser microtubule bundles were evident in scoulerine-treated cells.
    Figure Legend Snippet: Microscopic images of A549 cells stained with an anti-β-tubulin antibody (red) and counterstained with DAPI (blue). The cells were treated for 24 h with scoulerine or a solvent (0.1% DMSO) as vehicle control. Nocodazole, an antineoplastic agent that disrupts microtubule function by binding to tubulin was used as a reference compound in this assay. Scale bar: 10 µm. Experiments were performed in triplicate using epi-fluorescence microscopy. Photographs from representative chambers are shown. Compared with controls, thicker and denser microtubule bundles were evident in scoulerine-treated cells.

    Techniques Used: Staining, Binding Assay, Fluorescence, Microscopy

    Dynamic real-time monitoring of proliferation and cytotoxicity using the xCELLigence system dedicated to adherent cell lines. Growth kinetics of human A549 lung carcinoma ( A ), A2780 ovarian carcinoma ( B ), SK-BR-3 breast adenocarcinoma ( C ) and MCF-7 breast adenocarcinoma ( D ) cells treated with scoulerine. Cells treated with 0.1% DMSO were used as vehicle control and 5% DMSO treated cells were used as positive control. The normalized cell index was measured over 72 h. Plots shown are representative of at least three replicate experiments in each case.
    Figure Legend Snippet: Dynamic real-time monitoring of proliferation and cytotoxicity using the xCELLigence system dedicated to adherent cell lines. Growth kinetics of human A549 lung carcinoma ( A ), A2780 ovarian carcinoma ( B ), SK-BR-3 breast adenocarcinoma ( C ) and MCF-7 breast adenocarcinoma ( D ) cells treated with scoulerine. Cells treated with 0.1% DMSO were used as vehicle control and 5% DMSO treated cells were used as positive control. The normalized cell index was measured over 72 h. Plots shown are representative of at least three replicate experiments in each case.

    Techniques Used: Positive Control

    15) Product Images from "Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells"

    Article Title: Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22862-0

    Cytotoxicity of scoulerine ( 1 ), ( 2 ), ( 3 ) and ( 4 ) following a single-dose exposure at a concentration of 10 µM. Cell proliferation and viability of Jurkat, MOLT-4, Raji, HL-60, U-937 and HEL 92.1.7 cells measured by using XTT assay 48 h after treatment. Viability is referred to cells treated with 0.1% DMSO (Control DMSO). Data are shown as mean values ± SD of at least three independent experiments. *Significantly different to control (P ≤ 0.05) ( A ). Dynamic real-time xCELLigence screen of proliferation and cytotoxicity over 62 h. The human A2780 ovarian carcinoma cells in the logarithmic growth phase were treated. The negative control cells were exposed to 0.1% DMSO (vehicle) and 5% DMSO was used as a positive control. The plot is representative of at least three experiments performed ( B ).
    Figure Legend Snippet: Cytotoxicity of scoulerine ( 1 ), ( 2 ), ( 3 ) and ( 4 ) following a single-dose exposure at a concentration of 10 µM. Cell proliferation and viability of Jurkat, MOLT-4, Raji, HL-60, U-937 and HEL 92.1.7 cells measured by using XTT assay 48 h after treatment. Viability is referred to cells treated with 0.1% DMSO (Control DMSO). Data are shown as mean values ± SD of at least three independent experiments. *Significantly different to control (P ≤ 0.05) ( A ). Dynamic real-time xCELLigence screen of proliferation and cytotoxicity over 62 h. The human A2780 ovarian carcinoma cells in the logarithmic growth phase were treated. The negative control cells were exposed to 0.1% DMSO (vehicle) and 5% DMSO was used as a positive control. The plot is representative of at least three experiments performed ( B ).

    Techniques Used: Concentration Assay, XTT Assay, Negative Control, Positive Control

    Western blot analysis of proteins that regulate cell cycle progression or cell death in Jurkat and MOLT-4 cells after scoulerine treatment for 24 h. Control cells were mock treated with 0.1% DMSO (DMSO). Cells treated with cisplatin at 5 µM were used for positive control in Western blot analysis. These experiments were performed at least three times with similar results and a cropped blot is shown from one representative experiment.
    Figure Legend Snippet: Western blot analysis of proteins that regulate cell cycle progression or cell death in Jurkat and MOLT-4 cells after scoulerine treatment for 24 h. Control cells were mock treated with 0.1% DMSO (DMSO). Cells treated with cisplatin at 5 µM were used for positive control in Western blot analysis. These experiments were performed at least three times with similar results and a cropped blot is shown from one representative experiment.

    Techniques Used: Western Blot, Positive Control

    Microscopic images of A549 cells stained with an anti-β-tubulin antibody (red) and counterstained with DAPI (blue). The cells were treated for 24 h with scoulerine or a solvent (0.1% DMSO) as vehicle control. Nocodazole, an antineoplastic agent that disrupts microtubule function by binding to tubulin was used as a reference compound in this assay. Scale bar: 10 µm. Experiments were performed in triplicate using epi-fluorescence microscopy. Photographs from representative chambers are shown. Compared with controls, thicker and denser microtubule bundles were evident in scoulerine-treated cells.
    Figure Legend Snippet: Microscopic images of A549 cells stained with an anti-β-tubulin antibody (red) and counterstained with DAPI (blue). The cells were treated for 24 h with scoulerine or a solvent (0.1% DMSO) as vehicle control. Nocodazole, an antineoplastic agent that disrupts microtubule function by binding to tubulin was used as a reference compound in this assay. Scale bar: 10 µm. Experiments were performed in triplicate using epi-fluorescence microscopy. Photographs from representative chambers are shown. Compared with controls, thicker and denser microtubule bundles were evident in scoulerine-treated cells.

    Techniques Used: Staining, Binding Assay, Fluorescence, Microscopy

    Dynamic real-time monitoring of proliferation and cytotoxicity using the xCELLigence system dedicated to adherent cell lines. Growth kinetics of human A549 lung carcinoma ( A ), A2780 ovarian carcinoma ( B ), SK-BR-3 breast adenocarcinoma ( C ) and MCF-7 breast adenocarcinoma ( D ) cells treated with scoulerine. Cells treated with 0.1% DMSO were used as vehicle control and 5% DMSO treated cells were used as positive control. The normalized cell index was measured over 72 h. Plots shown are representative of at least three replicate experiments in each case.
    Figure Legend Snippet: Dynamic real-time monitoring of proliferation and cytotoxicity using the xCELLigence system dedicated to adherent cell lines. Growth kinetics of human A549 lung carcinoma ( A ), A2780 ovarian carcinoma ( B ), SK-BR-3 breast adenocarcinoma ( C ) and MCF-7 breast adenocarcinoma ( D ) cells treated with scoulerine. Cells treated with 0.1% DMSO were used as vehicle control and 5% DMSO treated cells were used as positive control. The normalized cell index was measured over 72 h. Plots shown are representative of at least three replicate experiments in each case.

    Techniques Used: Positive Control

    16) Product Images from "Itraconazole, A Commonly Used Antifungal, Inhibits Fcγ Receptor-Mediated Phagocytosis: Alteration of Fcγ Receptor Glycosylation And Gene Expression *"

    Article Title: Itraconazole, A Commonly Used Antifungal, Inhibits Fcγ Receptor-Mediated Phagocytosis: Alteration of Fcγ Receptor Glycosylation And Gene Expression *

    Journal: Shock (Augusta, Ga.)

    doi: 10.1097/SHK.0000000000000169

    Itraconazole decreased expression and altered the electrophoretic mobility of Fcγ receptors J774.1 Cells (∼1 × 10 6 ) were treated for 16 h at 37°C with ICZ (1 μM) or vehicle control (DMSO). A and B , cell surface and C and D , total cellular expression of FcγI and FcγIII/II receptors was determined by flow cytometry using APC-labeled anti-mouse CD64 and FITC-labeled rat anti-mouse CD16/CD32 antibodies. E , ICZ altered the electrophoretic mobility of FcγIII/II receptors. At the end of the incubation period with either ICZ or DMSO, cells were lysed, equal amounts of protein for each sample (50 μg) were resolved by SDS-PAGE, and the presence of FcγRIII/II was determined by Western blotting. F , mRNA levels of FcγRI: Fcgr1 (CD64), FcγRII: Fcgr2b (CD32), FcγRIII: Fcgr3 (CD16) were determined using qRT-PCR. Values were determined using a standard curve and expressed as copy number. All values were normalized to GAPDH mRNA levels. Results from three independent experiments performed in triplicate for each sample were used to calculate the mean and standard error depicted. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to Control (DMSO).
    Figure Legend Snippet: Itraconazole decreased expression and altered the electrophoretic mobility of Fcγ receptors J774.1 Cells (∼1 × 10 6 ) were treated for 16 h at 37°C with ICZ (1 μM) or vehicle control (DMSO). A and B , cell surface and C and D , total cellular expression of FcγI and FcγIII/II receptors was determined by flow cytometry using APC-labeled anti-mouse CD64 and FITC-labeled rat anti-mouse CD16/CD32 antibodies. E , ICZ altered the electrophoretic mobility of FcγIII/II receptors. At the end of the incubation period with either ICZ or DMSO, cells were lysed, equal amounts of protein for each sample (50 μg) were resolved by SDS-PAGE, and the presence of FcγRIII/II was determined by Western blotting. F , mRNA levels of FcγRI: Fcgr1 (CD64), FcγRII: Fcgr2b (CD32), FcγRIII: Fcgr3 (CD16) were determined using qRT-PCR. Values were determined using a standard curve and expressed as copy number. All values were normalized to GAPDH mRNA levels. Results from three independent experiments performed in triplicate for each sample were used to calculate the mean and standard error depicted. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to Control (DMSO).

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Labeling, Incubation, SDS Page, Western Blot, Quantitative RT-PCR

    ICZ decreased phagocytosis of opsonized bacterial particles A , Phagocytosis dose response curve. J774.1 cells (∼1 × 10 6 ) were treated with ICZ at various doses (0.5, 1, 2 or 10 μM) for 16 h at 37°C or equivalent concentrations of vehicle (DMSO) then challenged with opsonized fluorescent bacterial particles at a ratio of 10:1 (bioparticles:Møs) for 60 min. Phagocytosis was determined by flow cytometry. The phagocytic rate was calculated for each treatment dose from the mean fluorescence intensity (MFI) after quenching cell surface fluorescence using trypan blue. Results from three independent experiments performed in triplicate for each sample are presented. B , Phagocytosis time response curve. The phagocytic rate of opsonized fluorescent bioparticles (ratio of 10:1 bioparticles:Møs, 60 min challenge) was determined (as described above) after incubation of J774.1 cells (∼1 × 10 6 ) with ICZ (1 μM) or vehicle (DMSO) for 0, 3, 6, 9, 12 or 16 h. C , Phagocytic rate after ICZ treatment. J774.1 cells (∼1 × 10 6 ) were treated for 16 h at 37°C with ICZ (1 μM) or vehicle (DMSO), then challenged with opsonized fluorescent bacterial particles (ratio of 10:1 bioparticles:Møs) for 15, 30 and 60 min. Results from three independent experiments performed in triplicate for each sample are presented. D , Representative flow cytometry plots at 15, 30 and 60 min of bacterial challenge (as described in C ) are depicted: ICZ, dotted lines, DMSO, solid lines, non-stained control, grey lines. E , Phagocytosis of opsonized bacterial particles is Fcγ-receptor-mediated. The specificity of Fcγ-receptor-mediated phagocytosis of opsonized bacterial particles was determined by competition with purified mouse IgG applied concomitantly to bacterial particles. J774.1 Møs (∼1 × 10 6 ) were treated with ICZ (1 μM) or vehicle (DMSO) for 16 h and then challenged with opsonized bacterial particles as described above for 60 min in the presence or absence of purified mouse IgG (10 μg/mL). F , Non FcγR-mediated phagocytosis is unaffected by ICZ. A global effect on phagocytosis was evaluated using fluorescent latex beads. After incubation with ICZ (1 μM) or vehicle (DMSO) for 16 h, J774.1 cells (∼1 × 10 6 ) were exposed to fluorescent latex beads at a ratio of 10:1 (beads:Møs) for 15, 30 and 60 min at 37°C. Representative flow cytometry plots are depicted: ICZ, dotted lines, DMSO, solid lines, non-stained control, grey lines. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to Control (DMSO).
    Figure Legend Snippet: ICZ decreased phagocytosis of opsonized bacterial particles A , Phagocytosis dose response curve. J774.1 cells (∼1 × 10 6 ) were treated with ICZ at various doses (0.5, 1, 2 or 10 μM) for 16 h at 37°C or equivalent concentrations of vehicle (DMSO) then challenged with opsonized fluorescent bacterial particles at a ratio of 10:1 (bioparticles:Møs) for 60 min. Phagocytosis was determined by flow cytometry. The phagocytic rate was calculated for each treatment dose from the mean fluorescence intensity (MFI) after quenching cell surface fluorescence using trypan blue. Results from three independent experiments performed in triplicate for each sample are presented. B , Phagocytosis time response curve. The phagocytic rate of opsonized fluorescent bioparticles (ratio of 10:1 bioparticles:Møs, 60 min challenge) was determined (as described above) after incubation of J774.1 cells (∼1 × 10 6 ) with ICZ (1 μM) or vehicle (DMSO) for 0, 3, 6, 9, 12 or 16 h. C , Phagocytic rate after ICZ treatment. J774.1 cells (∼1 × 10 6 ) were treated for 16 h at 37°C with ICZ (1 μM) or vehicle (DMSO), then challenged with opsonized fluorescent bacterial particles (ratio of 10:1 bioparticles:Møs) for 15, 30 and 60 min. Results from three independent experiments performed in triplicate for each sample are presented. D , Representative flow cytometry plots at 15, 30 and 60 min of bacterial challenge (as described in C ) are depicted: ICZ, dotted lines, DMSO, solid lines, non-stained control, grey lines. E , Phagocytosis of opsonized bacterial particles is Fcγ-receptor-mediated. The specificity of Fcγ-receptor-mediated phagocytosis of opsonized bacterial particles was determined by competition with purified mouse IgG applied concomitantly to bacterial particles. J774.1 Møs (∼1 × 10 6 ) were treated with ICZ (1 μM) or vehicle (DMSO) for 16 h and then challenged with opsonized bacterial particles as described above for 60 min in the presence or absence of purified mouse IgG (10 μg/mL). F , Non FcγR-mediated phagocytosis is unaffected by ICZ. A global effect on phagocytosis was evaluated using fluorescent latex beads. After incubation with ICZ (1 μM) or vehicle (DMSO) for 16 h, J774.1 cells (∼1 × 10 6 ) were exposed to fluorescent latex beads at a ratio of 10:1 (beads:Møs) for 15, 30 and 60 min at 37°C. Representative flow cytometry plots are depicted: ICZ, dotted lines, DMSO, solid lines, non-stained control, grey lines. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to Control (DMSO).

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Incubation, Staining, Purification

    Itraconazole exerted similar effects on isolated mouse naïve peritoneal macrophages Resident naïve peritoneal Møs (PMøs ∼1 × 10 6 ) were isolated by peritoneal cavity lavage from CD1 mice (8–10 weeks old) and treated for 16 h at 37°C with ICZ (1 μM) or vehicle control (DMSO). At the end of the ICZ incubation period, the phagocytic rate ( A ) and FcγRI and FcγRIII/II cell surface expression levels ( B ) were determined. A, Phagocytosis of opsonized bioparticles (ratio of 10:1, 60 min exposure) was evaluated by flow cytometry. B , Cell surface expression of Fcγ I and Fcγ III/II receptors was determined by flow cytometry using APC-labeled anti-mouse CD64 and FITC-labeled rat anti-mouse CD16/CD32 antibodies. C , ICZ treatment resulted in increased Alexa Fluor 488-ConA binding to PMø cell surface glycoproteins as quantified by flow cytometry. Results from three independent experiments performed in triplicate for each sample were used to calculate the mean and standard error depicted. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to Control (DMSO).
    Figure Legend Snippet: Itraconazole exerted similar effects on isolated mouse naïve peritoneal macrophages Resident naïve peritoneal Møs (PMøs ∼1 × 10 6 ) were isolated by peritoneal cavity lavage from CD1 mice (8–10 weeks old) and treated for 16 h at 37°C with ICZ (1 μM) or vehicle control (DMSO). At the end of the ICZ incubation period, the phagocytic rate ( A ) and FcγRI and FcγRIII/II cell surface expression levels ( B ) were determined. A, Phagocytosis of opsonized bioparticles (ratio of 10:1, 60 min exposure) was evaluated by flow cytometry. B , Cell surface expression of Fcγ I and Fcγ III/II receptors was determined by flow cytometry using APC-labeled anti-mouse CD64 and FITC-labeled rat anti-mouse CD16/CD32 antibodies. C , ICZ treatment resulted in increased Alexa Fluor 488-ConA binding to PMø cell surface glycoproteins as quantified by flow cytometry. Results from three independent experiments performed in triplicate for each sample were used to calculate the mean and standard error depicted. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to Control (DMSO).

    Techniques Used: Isolation, Mouse Assay, Incubation, Expressing, Flow Cytometry, Cytometry, Labeling, Binding Assay

    LDL prevented the effect of ICZ J774.1 Cells (∼1 × 10 6 ) were treated for 16 h at 37°C with ICZ (1μM) or DMSO in the presence or absence of LDL (100 μg/mL). A , LDL inhibits the ICZ effect on phagocytosis. At the end of the incubation period, cells were challenged with opsonized fluorescent bacterial particles at a ratio of 10:1 (bioparticles:Møs) for 30 min. Cells were then washed and fixed. Cell surface fluorescence was quenched with trypan blue in the fixing solution and phagocytosis determined by flow cytometry. Representative flow cytometry plots are depicted. B , LDL inhibits the ICZ effect on FcγR levels. Cell surface and total cellular expression of Fcγ III/II receptors was determined by flow cytometry using FITC-labeled rat anti-mouse CD16/CD32 antibodies: ICZ, dotted lines, DMSO, solid lines, non-stained control, grey lines C , LDL reverses ICZ effect on cell surface glycoproteins, as evidenced by decreased fluorescent ConA binding. Representative flow cytometry plots. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to vehicle control (DMSO).
    Figure Legend Snippet: LDL prevented the effect of ICZ J774.1 Cells (∼1 × 10 6 ) were treated for 16 h at 37°C with ICZ (1μM) or DMSO in the presence or absence of LDL (100 μg/mL). A , LDL inhibits the ICZ effect on phagocytosis. At the end of the incubation period, cells were challenged with opsonized fluorescent bacterial particles at a ratio of 10:1 (bioparticles:Møs) for 30 min. Cells were then washed and fixed. Cell surface fluorescence was quenched with trypan blue in the fixing solution and phagocytosis determined by flow cytometry. Representative flow cytometry plots are depicted. B , LDL inhibits the ICZ effect on FcγR levels. Cell surface and total cellular expression of Fcγ III/II receptors was determined by flow cytometry using FITC-labeled rat anti-mouse CD16/CD32 antibodies: ICZ, dotted lines, DMSO, solid lines, non-stained control, grey lines C , LDL reverses ICZ effect on cell surface glycoproteins, as evidenced by decreased fluorescent ConA binding. Representative flow cytometry plots. Statistical analysis was performed using ANOVA. * P ≤ 0.05 compared to vehicle control (DMSO).

    Techniques Used: Incubation, Fluorescence, Flow Cytometry, Cytometry, Expressing, Labeling, Staining, Binding Assay

    17) Product Images from "Eugenol as a Promising Molecule for the Treatment of Dermatitis: Antioxidant and Anti-inflammatory Activities and Its Nanoformulation"

    Article Title: Eugenol as a Promising Molecule for the Treatment of Dermatitis: Antioxidant and Anti-inflammatory Activities and Its Nanoformulation

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/8194849

    Evaluation of antioxidant activity of EUG in human neutrophils by chemiluminescence (CL). The inhibitory effect of EUG in human neutrophil oxidative metabolism was assessed by luminol (LumCL) (a) or lucigenin (LucCL) (b). Data from three to eight samples. ∗ vs. control (DMSO), p
    Figure Legend Snippet: Evaluation of antioxidant activity of EUG in human neutrophils by chemiluminescence (CL). The inhibitory effect of EUG in human neutrophil oxidative metabolism was assessed by luminol (LumCL) (a) or lucigenin (LucCL) (b). Data from three to eight samples. ∗ vs. control (DMSO), p

    Techniques Used: Antioxidant Activity Assay

    Effect of EUG on the percentage of measurement of membrane integrity of human neutrophils as monitored by flow cytometry using the sensitive fluorochrome propidium iodide. Neutrophils were cultured for 30 min in the presence of EUG at various concentrations (10–50 μ g/mL). Values are presented as means ± SEM of 3 separated experiments in triplicate and by analysis of 10,000 events. The control group consists of cells treated with vehicle (DMSO 1%). ∗ vs. HBSS: nontreated cells. p
    Figure Legend Snippet: Effect of EUG on the percentage of measurement of membrane integrity of human neutrophils as monitored by flow cytometry using the sensitive fluorochrome propidium iodide. Neutrophils were cultured for 30 min in the presence of EUG at various concentrations (10–50 μ g/mL). Values are presented as means ± SEM of 3 separated experiments in triplicate and by analysis of 10,000 events. The control group consists of cells treated with vehicle (DMSO 1%). ∗ vs. HBSS: nontreated cells. p

    Techniques Used: Flow Cytometry, Cytometry, Cell Culture

    Evaluation of EUG toxicity on MTT test in human neutrophils. Data from three to eight samples. The control group consists of cells treated with vehicle (DMSO 1%). Triton X-100 (Tx, 0.02%) was used as cytotoxic standard. ∗ vs. HBSS: nontreated cells. p
    Figure Legend Snippet: Evaluation of EUG toxicity on MTT test in human neutrophils. Data from three to eight samples. The control group consists of cells treated with vehicle (DMSO 1%). Triton X-100 (Tx, 0.02%) was used as cytotoxic standard. ∗ vs. HBSS: nontreated cells. p

    Techniques Used: MTT Assay

    Evaluation of EUG and NCEUG toxicity on MTT test for 24 hours, 48 hours, and 72 hours in human neutrophils. Data from two to eight samples. The control group consists of cells treated with vehicle (DMSO 1%). ∗ vs. DMEM: untreated cells. p
    Figure Legend Snippet: Evaluation of EUG and NCEUG toxicity on MTT test for 24 hours, 48 hours, and 72 hours in human neutrophils. Data from two to eight samples. The control group consists of cells treated with vehicle (DMSO 1%). ∗ vs. DMEM: untreated cells. p

    Techniques Used: MTT Assay

    18) Product Images from "Andrographolide Inhibits Nuclear Factor- κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells"

    Article Title: Andrographolide Inhibits Nuclear Factor- κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    Journal: The Scientific World Journal

    doi: 10.1155/2014/130381

    Effects of andrographolide on I κ B α degradation and p65 activation in TNF- α -stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μ M) or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 30 min. (a) I κ B α degradation and (b) p65 phosphorylation were evaluated as described in Section 2 .* P
    Figure Legend Snippet: Effects of andrographolide on I κ B α degradation and p65 activation in TNF- α -stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μ M) or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 30 min. (a) I κ B α degradation and (b) p65 phosphorylation were evaluated as described in Section 2 .* P

    Techniques Used: Activation Assay

    Regulatory effects of various signal inhibitors on p65 activation and Akt and JNK phosphorylation in TNF- α -stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with LY294002 (10 μ M), SP600125 (10 μ M), or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 10 min ((b) and (c)) or 30 min (a). (a) p65 phosphorylation, (b) Akt phosphorylation, and (c) JNK phosphorylation were evaluated as described in Section 2 .* P
    Figure Legend Snippet: Regulatory effects of various signal inhibitors on p65 activation and Akt and JNK phosphorylation in TNF- α -stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with LY294002 (10 μ M), SP600125 (10 μ M), or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 10 min ((b) and (c)) or 30 min (a). (a) p65 phosphorylation, (b) Akt phosphorylation, and (c) JNK phosphorylation were evaluated as described in Section 2 .* P

    Techniques Used: Activation Assay

    Effects of andrographolide on iNOS expression in TNF- α -stimulated VSMCs. (a) Photomicrograph showing the primary cultured rat aortic VSMCs (magnification ×100). (b) The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μ M) or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 24 h. The iNOS protein level was evaluated as described in Section 2 .** P
    Figure Legend Snippet: Effects of andrographolide on iNOS expression in TNF- α -stimulated VSMCs. (a) Photomicrograph showing the primary cultured rat aortic VSMCs (magnification ×100). (b) The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μ M) or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 24 h. The iNOS protein level was evaluated as described in Section 2 .** P

    Techniques Used: Expressing, Cell Culture

    Effects of andrographolide on p38MAPK, ERK1/2, JNK, and Akt signaling pathways in TNF- α -stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μ M) or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 10 min. (a) p38MAPK phosphorylation, (b) ERK1/2 phosphorylation, (c) JNK phosphorylation, and (d) Akt phosphorylation were evaluated as described in Section 2 .* P
    Figure Legend Snippet: Effects of andrographolide on p38MAPK, ERK1/2, JNK, and Akt signaling pathways in TNF- α -stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μ M) or an equal volume of DMSO (solvent control) for 20 min, and TNF- α (10 ng/mL) was subsequently added for 10 min. (a) p38MAPK phosphorylation, (b) ERK1/2 phosphorylation, (c) JNK phosphorylation, and (d) Akt phosphorylation were evaluated as described in Section 2 .* P

    Techniques Used:

    19) Product Images from "Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells"

    Article Title: Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170233

    DNA fragmentation analysed in 1% agarose gel after 72 h incubation with different concentration of either FKB or APN. MW: DNA marker; DC: DMSO treated control; all concentrations are in μM.
    Figure Legend Snippet: DNA fragmentation analysed in 1% agarose gel after 72 h incubation with different concentration of either FKB or APN. MW: DNA marker; DC: DMSO treated control; all concentrations are in μM.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Concentration Assay, Marker

    (A) Expression of UCK2 mRNA in HT-29 cells analysed in 1% agarose gel. (a)Levels of UCK2 mRNA expression in cells treated with increasing concentration of crude hexane (IC 25 : 10.52, IC 50 : 21.05, and IC 75 :42.1 μg/mL) and chloroform (IC 25 : 9.5, IC 50 : 19.09, and IC 75 :38.18 μg/mL) extracts; (b) Levels of UCK2 mRNA expressed in cells treated with FKB at 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL); (c) Levels of UCK2 mRNA expressed in cells treated with APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). The housekeeping gene, GAPDH was used as loading control. C, Untreated control; D, DMSO used as negative control at a final concentration of 0.1%. (B) Western blot analysis of UCK2 protein expressed in HT-29 cells. (a) Levels of UCK2 protein expression in cells treated with increasing concentration of crude hexane (IC 25 : 10.52, IC 50 : 21.05, and IC 75 :42.1 μg/mL) and chloroform (IC 25 : 9.5, IC 50 : 19.09, and IC 75 :38.18 μg/mL) extracts. (b) Levels of UCK2 protein expressed in cells treated with FKB at a concentration of 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL). (c) Levels of UCK2 protein expressed in cells treated with APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). (C) Levels of UCK2 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT-29 cells treated with (a) Crude hexane and chloroform extract, and (b) Bioactive compounds of FKB and APN;. DC: DMSO treated control at a final concentration of 0.1%. Data are expressed as Mean±SD; ns: non-significant; *p
    Figure Legend Snippet: (A) Expression of UCK2 mRNA in HT-29 cells analysed in 1% agarose gel. (a)Levels of UCK2 mRNA expression in cells treated with increasing concentration of crude hexane (IC 25 : 10.52, IC 50 : 21.05, and IC 75 :42.1 μg/mL) and chloroform (IC 25 : 9.5, IC 50 : 19.09, and IC 75 :38.18 μg/mL) extracts; (b) Levels of UCK2 mRNA expressed in cells treated with FKB at 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL); (c) Levels of UCK2 mRNA expressed in cells treated with APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). The housekeeping gene, GAPDH was used as loading control. C, Untreated control; D, DMSO used as negative control at a final concentration of 0.1%. (B) Western blot analysis of UCK2 protein expressed in HT-29 cells. (a) Levels of UCK2 protein expression in cells treated with increasing concentration of crude hexane (IC 25 : 10.52, IC 50 : 21.05, and IC 75 :42.1 μg/mL) and chloroform (IC 25 : 9.5, IC 50 : 19.09, and IC 75 :38.18 μg/mL) extracts. (b) Levels of UCK2 protein expressed in cells treated with FKB at a concentration of 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL). (c) Levels of UCK2 protein expressed in cells treated with APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). (C) Levels of UCK2 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT-29 cells treated with (a) Crude hexane and chloroform extract, and (b) Bioactive compounds of FKB and APN;. DC: DMSO treated control at a final concentration of 0.1%. Data are expressed as Mean±SD; ns: non-significant; *p

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Concentration Assay, Negative Control, Western Blot, Software

    (A) Live cell imaging of the controls of HT-29 cells with SmartFlare Cy5-Uptake, Cy5-Scramble or Cy5-18S probes, imaged using fluorescence microscope in exposure settings at 20× magnification. (a) Uptake probe used as positive control that quenched on inside the cell. (b) Scramble probe used as negative control where the probe is not able to recognize any RNA sequence inside the cell nucleus. (c) Cells with 18S probe after 48 h of 0.1% ( v / v ) DMSO treatment as negative control. (B) Live cell imaging of HT-29 cells with SmartFlare Cy5-18S probes, and imaged using fluorescence microscope in exposure settings at 20× magnification. Cells with 18S probe after 48h treatment with crude extract of (d) hexane extract (21.05 μg/mL), (e) chloroform extract (19.09 μg/mL), (f) FKB (8.47 μg/mL), and (g) APN (13.12 μg/mL).
    Figure Legend Snippet: (A) Live cell imaging of the controls of HT-29 cells with SmartFlare Cy5-Uptake, Cy5-Scramble or Cy5-18S probes, imaged using fluorescence microscope in exposure settings at 20× magnification. (a) Uptake probe used as positive control that quenched on inside the cell. (b) Scramble probe used as negative control where the probe is not able to recognize any RNA sequence inside the cell nucleus. (c) Cells with 18S probe after 48 h of 0.1% ( v / v ) DMSO treatment as negative control. (B) Live cell imaging of HT-29 cells with SmartFlare Cy5-18S probes, and imaged using fluorescence microscope in exposure settings at 20× magnification. Cells with 18S probe after 48h treatment with crude extract of (d) hexane extract (21.05 μg/mL), (e) chloroform extract (19.09 μg/mL), (f) FKB (8.47 μg/mL), and (g) APN (13.12 μg/mL).

    Techniques Used: Live Cell Imaging, Fluorescence, Microscopy, Positive Control, Negative Control, Sequencing

    Cell cycle analysis examined using flow cytometry on HT-29 cells after 72 h treatment. (A) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) FKB at a concentration of 12.5 (3.55 μg/mL), (c) 25 (7.1 μg/mL), (d) 50 μM (14.2 μg/mL), and (e) Percentage of cell cycle distribution in different phases. (B) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) APN at 12.5 (3.37 μg/mL), (c) 25 (6.75 μg/mL), (d) 50 μM (13.5 μg/mL) concentrations, and (e) Percentage of cell cycle distribution in different phases. G0/G1, G2+M, and S are cell phases, respectively; subG0/G1 refers to cell death due to DNA fragmentation. Data are expressed as Mean±SD of three independent experiments, *p
    Figure Legend Snippet: Cell cycle analysis examined using flow cytometry on HT-29 cells after 72 h treatment. (A) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) FKB at a concentration of 12.5 (3.55 μg/mL), (c) 25 (7.1 μg/mL), (d) 50 μM (14.2 μg/mL), and (e) Percentage of cell cycle distribution in different phases. (B) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) APN at 12.5 (3.37 μg/mL), (c) 25 (6.75 μg/mL), (d) 50 μM (13.5 μg/mL) concentrations, and (e) Percentage of cell cycle distribution in different phases. G0/G1, G2+M, and S are cell phases, respectively; subG0/G1 refers to cell death due to DNA fragmentation. Data are expressed as Mean±SD of three independent experiments, *p

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Cytometry, Concentration Assay

    20) Product Images from "Hyperoxia Disrupts Extracellular Signal-Regulated Kinases 1/2-Induced Angiogenesis in the Developing Lungs"

    Article Title: Hyperoxia Disrupts Extracellular Signal-Regulated Kinases 1/2-Induced Angiogenesis in the Developing Lungs

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051525

    Suppression of ERK1/2 activity decreases HPAEC tubule and mesh formation. HPAECs were pre-treated with dimethylsulfoxide (DMSO) or 30 µM PD98059 (PD 30) for 30 min before being loaded on growth factor-reduced Matrigel (BD Bioscience) in 96-well plates. Following an incubation period of 18 h, tubule formation was quantified. ( A , B ) Representative photographs showing tubule formation in growth factor-reduced Matrigel. ( C , D ) Quantitative analysis of tubule ( C ) and mesh ( D ) formation. The values are presented as mean ± SD ( n = 9/group). Significant differences between DMSO- and PD-treated cells are indicated by * p
    Figure Legend Snippet: Suppression of ERK1/2 activity decreases HPAEC tubule and mesh formation. HPAECs were pre-treated with dimethylsulfoxide (DMSO) or 30 µM PD98059 (PD 30) for 30 min before being loaded on growth factor-reduced Matrigel (BD Bioscience) in 96-well plates. Following an incubation period of 18 h, tubule formation was quantified. ( A , B ) Representative photographs showing tubule formation in growth factor-reduced Matrigel. ( C , D ) Quantitative analysis of tubule ( C ) and mesh ( D ) formation. The values are presented as mean ± SD ( n = 9/group). Significant differences between DMSO- and PD-treated cells are indicated by * p

    Techniques Used: Activity Assay, Incubation

    Decreased phosphorylated ERK1/2 protein levels in HPAECs treated with PD98059. HPAECs were treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10) or 30 (PD 30) µM for 30 min, after which whole-cell proteins were extracted, and immunoblotting was performed using antibodies against total ERK1/2, phosphorylated ERK1/2, or β-actin. Representative immunoblot showing total ERK1/2 and phosphorylated ERK1/2 protein expression ( A ). Densitometric analyses wherein the phosphorylated ERK1/2 band intensities were quantified and normalized to those of total ERK1/2 ( B ). The values are presented as mean ± SD ( n = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by * p
    Figure Legend Snippet: Decreased phosphorylated ERK1/2 protein levels in HPAECs treated with PD98059. HPAECs were treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10) or 30 (PD 30) µM for 30 min, after which whole-cell proteins were extracted, and immunoblotting was performed using antibodies against total ERK1/2, phosphorylated ERK1/2, or β-actin. Representative immunoblot showing total ERK1/2 and phosphorylated ERK1/2 protein expression ( A ). Densitometric analyses wherein the phosphorylated ERK1/2 band intensities were quantified and normalized to those of total ERK1/2 ( B ). The values are presented as mean ± SD ( n = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by * p

    Techniques Used: Expressing

    Suppression of ERK1/2 activity decreases HPAEC proliferation. HPAECs were treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10), 20 (PD 20), or 30 (PD 30) µM for 24 or 48 h, following which cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. The values are presented as mean ± SD ( n = 10/group). Two-way ANOVA analysis showed an effect of PD and duration of exposure and an interaction between them for the dependent variable, absorbance at 570 nm, in this figure. Significant differences between DMSO- and PD-treated cells are indicated by * p
    Figure Legend Snippet: Suppression of ERK1/2 activity decreases HPAEC proliferation. HPAECs were treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10), 20 (PD 20), or 30 (PD 30) µM for 24 or 48 h, following which cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. The values are presented as mean ± SD ( n = 10/group). Two-way ANOVA analysis showed an effect of PD and duration of exposure and an interaction between them for the dependent variable, absorbance at 570 nm, in this figure. Significant differences between DMSO- and PD-treated cells are indicated by * p

    Techniques Used: Activity Assay, MTT Assay

    Suppression of ERK1/2 activity decreases HPAEC migration. HPAECs grown as monolayers in six-well plates were treated with 10 µg/mL of mitomycin for 2 h and scratched with a 200 µL pipette tip. The cells were then treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10), 20 (PD 20), or 30 (PD 30) µM. The wound closure area was analyzed using Image J software after 16 h of treatment. ( A – H ) Representative photographs showing cell migration. ( I ) Quantitative analysis of cell migration. The values are presented as mean ± SD ( n = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by *, (DMSO vs. PD 10 [ p
    Figure Legend Snippet: Suppression of ERK1/2 activity decreases HPAEC migration. HPAECs grown as monolayers in six-well plates were treated with 10 µg/mL of mitomycin for 2 h and scratched with a 200 µL pipette tip. The cells were then treated with dimethylsulfoxide (DMSO) or PD98059 at concentrations of 10 (PD 10), 20 (PD 20), or 30 (PD 30) µM. The wound closure area was analyzed using Image J software after 16 h of treatment. ( A – H ) Representative photographs showing cell migration. ( I ) Quantitative analysis of cell migration. The values are presented as mean ± SD ( n = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by *, (DMSO vs. PD 10 [ p

    Techniques Used: Activity Assay, Migration, Transferring, Software

    ERK1/2 inhibition affects the expression of cell cycle regulatory proteins. HPAECs were treated with dimethylsulfoxide (DMSO) or 30 µM PD98059 (PD 30) for 24 h, after which whole-cell protein were extracted, and immunoblotting was performed using antibodies against the following proteins: cyclin A, cyclin D, Cdk4, p27, and β-actin. Representative immunoblots showing the expression of the above proteins ( A ). Densitometric analyses wherein cyclin A ( B ), cyclin D ( C ), Cdk4 ( D ), and p27 Kip 1 ( E ) band intensities were quantified and normalized to those of total β-actin. The values are presented as mean ± SD ( n = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by * p
    Figure Legend Snippet: ERK1/2 inhibition affects the expression of cell cycle regulatory proteins. HPAECs were treated with dimethylsulfoxide (DMSO) or 30 µM PD98059 (PD 30) for 24 h, after which whole-cell protein were extracted, and immunoblotting was performed using antibodies against the following proteins: cyclin A, cyclin D, Cdk4, p27, and β-actin. Representative immunoblots showing the expression of the above proteins ( A ). Densitometric analyses wherein cyclin A ( B ), cyclin D ( C ), Cdk4 ( D ), and p27 Kip 1 ( E ) band intensities were quantified and normalized to those of total β-actin. The values are presented as mean ± SD ( n = 6/group). Significant differences between DMSO- and PD-treated cells are indicated by * p

    Techniques Used: Inhibition, Expressing, Western Blot

    21) Product Images from "Clotrimazole induces a late G1 cell cycle arrest and sensitizes glioblastoma cells to radiation in vitro"

    Article Title: Clotrimazole induces a late G1 cell cycle arrest and sensitizes glioblastoma cells to radiation in vitro

    Journal: Anti-cancer drugs

    doi: 10.1097/CAD.0b013e32833e8022

    Cell survival curves for the detection of radiosensitization by clotrimzole (CLT). U-87 MG cells were treated with 5 μmol/l CLT (◇), 10 μmol/l CLT (△), 20 μmol/l CLT (○) or vehicle [dimethyl sulfoxide (DMSO),
    Figure Legend Snippet: Cell survival curves for the detection of radiosensitization by clotrimzole (CLT). U-87 MG cells were treated with 5 μmol/l CLT (◇), 10 μmol/l CLT (△), 20 μmol/l CLT (○) or vehicle [dimethyl sulfoxide (DMSO),

    Techniques Used:

    The modulation of the expression of cell cycle regulatory proteins in U-87 MG cells after exposure to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO) treated control cells. Total cell lysates were collected and equal concentrations of
    Figure Legend Snippet: The modulation of the expression of cell cycle regulatory proteins in U-87 MG cells after exposure to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO) treated control cells. Total cell lysates were collected and equal concentrations of

    Techniques Used: Expressing

    The modulation of the retinoblastoma protein (pRb) and the phosphorylation of pRb in U-87 MG cells after exposure to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO)-treated control cells. Total cell lysates were collected and equal concentrations
    Figure Legend Snippet: The modulation of the retinoblastoma protein (pRb) and the phosphorylation of pRb in U-87 MG cells after exposure to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO)-treated control cells. Total cell lysates were collected and equal concentrations

    Techniques Used:

    Immunoblot analysis for the translocation of hexokinase II (HKII) and the release of cytochrome c (Cyto C) from the mitochondria to the cytoplasm after U-87 MG cells were exposed to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO) for
    Figure Legend Snippet: Immunoblot analysis for the translocation of hexokinase II (HKII) and the release of cytochrome c (Cyto C) from the mitochondria to the cytoplasm after U-87 MG cells were exposed to 40 μmol/l clotrimzole (CLT) or dimethyl sulfoxide (DMSO) for

    Techniques Used: Translocation Assay

    22) Product Images from "Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells"

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002723

    Inhibition of Kgp activity alters P. gingivalis -mediated RIPK1 and RIPK2 cleavage in HAEC. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
    Figure Legend Snippet: Inhibition of Kgp activity alters P. gingivalis -mediated RIPK1 and RIPK2 cleavage in HAEC. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Techniques Used: Inhibition, Activity Assay, Cell Culture

    General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis -induced modification of RIPK1 and RIPK2 in HUVEC. HUVEC were pretreated (Pre-Tx) with medium ( M ), 0.25% DMSO vehicle control ( C ), 25 µM z-VAD-FMK ( VAD ), or 100 µM Boc-D-FMK ( Boc ) with for 1.5 h. HUVEC were then treated with medium ( M ) or P. gingivalis strain 381 (MOI 100, 381 ) for 2 h. Whole cell lysates were analyzed for ( A ) RIPK1 or ( B ) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.
    Figure Legend Snippet: General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis -induced modification of RIPK1 and RIPK2 in HUVEC. HUVEC were pretreated (Pre-Tx) with medium ( M ), 0.25% DMSO vehicle control ( C ), 25 µM z-VAD-FMK ( VAD ), or 100 µM Boc-D-FMK ( Boc ) with for 1.5 h. HUVEC were then treated with medium ( M ) or P. gingivalis strain 381 (MOI 100, 381 ) for 2 h. Whole cell lysates were analyzed for ( A ) RIPK1 or ( B ) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.

    Techniques Used: Modification

    KYT inhibitors specifically inhibit P. gingivalis gingipain activity and do not alter host 3 caspase activity. Effect of gingipain inhibitors on P. gingivalis A ) Rgp or B ) Kgp protease activity. P. gingivalis was untreated (none) or pretreated with 10 µM Rgp-specific inhibitor KYT-1, 10 µM Kgp-specific inhibitor KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 10 min and monitored for arginine-X-specific or lysine-X-specific protease activity. Effect of inhibitors on P. gingivalis is presented as percent Rgp-X activity or Kgp-X activity relative to untreated P. gingivalis . C ) HUVEC were untreated or treated with 2 µM staurosporine (STS) for 5 h. Whole cell lysates were analyzed for caspase-3 activity in the presence of KYT-36 gingipain inhibitor (3 µM). Activity is represented as fold change relative to untreated. A reversible caspase inhibitor was included to demonstrate observed fluorescence is specific to caspase-3 like proteases. Statistical analysis was performed using unpaired T-test (α = 0.05), **p
    Figure Legend Snippet: KYT inhibitors specifically inhibit P. gingivalis gingipain activity and do not alter host 3 caspase activity. Effect of gingipain inhibitors on P. gingivalis A ) Rgp or B ) Kgp protease activity. P. gingivalis was untreated (none) or pretreated with 10 µM Rgp-specific inhibitor KYT-1, 10 µM Kgp-specific inhibitor KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 10 min and monitored for arginine-X-specific or lysine-X-specific protease activity. Effect of inhibitors on P. gingivalis is presented as percent Rgp-X activity or Kgp-X activity relative to untreated P. gingivalis . C ) HUVEC were untreated or treated with 2 µM staurosporine (STS) for 5 h. Whole cell lysates were analyzed for caspase-3 activity in the presence of KYT-36 gingipain inhibitor (3 µM). Activity is represented as fold change relative to untreated. A reversible caspase inhibitor was included to demonstrate observed fluorescence is specific to caspase-3 like proteases. Statistical analysis was performed using unpaired T-test (α = 0.05), **p

    Techniques Used: Activity Assay, Fluorescence

    Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis ; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).
    Figure Legend Snippet: Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis ; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).

    Techniques Used: Recombinant, Cell Culture, SDS Page, Western Blot, Incubation

    23) Product Images from "Propofol enhances BCR-ABL TKIs’ inhibitory effects in chronic myeloid leukemia through Akt/mTOR suppression"

    Article Title: Propofol enhances BCR-ABL TKIs’ inhibitory effects in chronic myeloid leukemia through Akt/mTOR suppression

    Journal: BMC Anesthesiology

    doi: 10.1186/s12871-017-0423-2

    Propofol is selectively active against CML CD34 progenitor cells and enhances dasatinib’s inhibitory effects. Propofol is more effective in inducing apoptosis ( a ) and inhibiting colony formation ( b ) in CML than NBM CD34 progenitor cells. Combination of propofol and dasatinib results in significant more apoptosis induction and colony formation inhibition than propofol or dasatinib alone in CML cells. Propofol at 5 μM and dasatinib at 200 nM were used for combination studies. DMSO (final concentration 0.5%) was used as control. * p
    Figure Legend Snippet: Propofol is selectively active against CML CD34 progenitor cells and enhances dasatinib’s inhibitory effects. Propofol is more effective in inducing apoptosis ( a ) and inhibiting colony formation ( b ) in CML than NBM CD34 progenitor cells. Combination of propofol and dasatinib results in significant more apoptosis induction and colony formation inhibition than propofol or dasatinib alone in CML cells. Propofol at 5 μM and dasatinib at 200 nM were used for combination studies. DMSO (final concentration 0.5%) was used as control. * p

    Techniques Used: Inhibition, Concentration Assay

    Propofol significantly inhibits CML growth in vivo and augments dasatinib’s inhibitor effect. Propofol dose-dependently decreases tumor size ( a ) and weight ( b ) in a CML xenograft mouse model. Combination of propofol and dasatinib is more effective in decreasing tumor size ( c ) and weight ( d ) than propofol or dasatinib alone. In combination studies, 20 mg/kg propofol and 5 mg/kg dasatinib was given to mice by intraperitoneal and oral administration, respectively. DMSO/Saline (20%/80%) was used as vehicle control. Tumour volume was calculated using the formula: length × width 2 /2. * p
    Figure Legend Snippet: Propofol significantly inhibits CML growth in vivo and augments dasatinib’s inhibitor effect. Propofol dose-dependently decreases tumor size ( a ) and weight ( b ) in a CML xenograft mouse model. Combination of propofol and dasatinib is more effective in decreasing tumor size ( c ) and weight ( d ) than propofol or dasatinib alone. In combination studies, 20 mg/kg propofol and 5 mg/kg dasatinib was given to mice by intraperitoneal and oral administration, respectively. DMSO/Saline (20%/80%) was used as vehicle control. Tumour volume was calculated using the formula: length × width 2 /2. * p

    Techniques Used: In Vivo, Mouse Assay

    Propofol enhances imatinib’s effects in CML cells by suppressing Akt/mTOR signaling pathway. Representative western blot images showing the inhibitory effects of propofol alone ( a ) and combination of propofol and imatinib ( b ) on phosphorylation of Akt, mTOR and S6 in K562 cells. Propofol at 5 μM and imatinib at 1 μM were used for combination studies. Overexpression of constitutively active Akt (myr Akt) significantly reverses the effects of propofol in inhibiting proliferation ( c ) and inducing apoptosis ( d ) in K562 cells. DMSO (final concentration 0.5%) was used as control. * p
    Figure Legend Snippet: Propofol enhances imatinib’s effects in CML cells by suppressing Akt/mTOR signaling pathway. Representative western blot images showing the inhibitory effects of propofol alone ( a ) and combination of propofol and imatinib ( b ) on phosphorylation of Akt, mTOR and S6 in K562 cells. Propofol at 5 μM and imatinib at 1 μM were used for combination studies. Overexpression of constitutively active Akt (myr Akt) significantly reverses the effects of propofol in inhibiting proliferation ( c ) and inducing apoptosis ( d ) in K562 cells. DMSO (final concentration 0.5%) was used as control. * p

    Techniques Used: Western Blot, Over Expression, Concentration Assay

    Propofol is active against CML cells and enhances imatinib’s inhibitory effects. Propofol significantly inhibits proliferation ( a ), induces apoptosis ( b ) and increases caspase 3 activities ( c ) in K562, KBM7 and KU812 cells in a dose-dependent manner. Combination of propofol and imatinib results in significant more proliferation inhibition and apoptosis induction than propofol or imatinib alone in CML cells. Propofol at 5 μM and imatinib at 1 μM were used for combination studies. DMSO (final concentration 0.5%) was used as control. * p
    Figure Legend Snippet: Propofol is active against CML cells and enhances imatinib’s inhibitory effects. Propofol significantly inhibits proliferation ( a ), induces apoptosis ( b ) and increases caspase 3 activities ( c ) in K562, KBM7 and KU812 cells in a dose-dependent manner. Combination of propofol and imatinib results in significant more proliferation inhibition and apoptosis induction than propofol or imatinib alone in CML cells. Propofol at 5 μM and imatinib at 1 μM were used for combination studies. DMSO (final concentration 0.5%) was used as control. * p

    Techniques Used: Inhibition, Concentration Assay

    24) Product Images from "Semisynthetic flavonoid 7-O-galloylquercetin activates Nrf2 and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells"

    Article Title: Semisynthetic flavonoid 7-O-galloylquercetin activates Nrf2 and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells

    Journal: Chemico-Biological Interactions

    doi: 10.1016/j.cbi.2016.10.015

    Effect of MAPK inhibitors and N -acetyl- l -cysteine (NAC) on 7- O -galloylquercetin (compound 3 )-induced Hmox1 gene expression in RAW264.7 cells. (A) Cells were pretreated for 30 min with 0.1% DMSO (control), 15 μM PD98059, 15 μM SB203580 or 30 μM SP600125 and then incubated in the absence or presence of 15 μM 3 for an additional 6 h. (B) Cells were pretreated for 30 min with 2.5 or 5 mM NAC and then incubated in the absence or presence of 15 μM 3 for an additional 6 h. The levels of Hmox1 mRNA were determined by quantitative real-time PCR and normalized to Gapdh mRNA. Results are expressed as the percentage of compound 3 -induced Hmox1 mRNA expression. Data are means ± SD of three experiments. ** p
    Figure Legend Snippet: Effect of MAPK inhibitors and N -acetyl- l -cysteine (NAC) on 7- O -galloylquercetin (compound 3 )-induced Hmox1 gene expression in RAW264.7 cells. (A) Cells were pretreated for 30 min with 0.1% DMSO (control), 15 μM PD98059, 15 μM SB203580 or 30 μM SP600125 and then incubated in the absence or presence of 15 μM 3 for an additional 6 h. (B) Cells were pretreated for 30 min with 2.5 or 5 mM NAC and then incubated in the absence or presence of 15 μM 3 for an additional 6 h. The levels of Hmox1 mRNA were determined by quantitative real-time PCR and normalized to Gapdh mRNA. Results are expressed as the percentage of compound 3 -induced Hmox1 mRNA expression. Data are means ± SD of three experiments. ** p

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction

    7- O -Galloylquercetin ( 3 ) induces NQO1 activity and Nrf2 accumulation in Hepa1c1c7 cells. (A) Cells were treated for 48 h with 0.1% DMSO (control), 5 μM sulforaphane (SFN; positive control) or with 3.75–15 μM 3 , quercetin (QUE) or methyl gallate (MG). After treatment, the activity of NQO1 was determined using the NQO1 assay. Data are means ± SD of three experiments. * p
    Figure Legend Snippet: 7- O -Galloylquercetin ( 3 ) induces NQO1 activity and Nrf2 accumulation in Hepa1c1c7 cells. (A) Cells were treated for 48 h with 0.1% DMSO (control), 5 μM sulforaphane (SFN; positive control) or with 3.75–15 μM 3 , quercetin (QUE) or methyl gallate (MG). After treatment, the activity of NQO1 was determined using the NQO1 assay. Data are means ± SD of three experiments. * p

    Techniques Used: Activity Assay, Positive Control

    Biotransformation and uptake of 7- O -galloylquercetin ( 3 ) by RAW264.7 cells. Cells were treated for 0–6 h with 15 μM 3 or 0.1% DMSO (control), and cell extracts and culture medium were analyzed by HPLC with negative ESI-MS detection. (A) HPLC/MS chromatograms of 3 (inset) and of its metabolites found in cells after 2 h of treatment. (B) Time course of distribution of 3 , quercetin (QUE) and gallic acid (GA) in cells and medium. Data are means ± SD of three experiments.
    Figure Legend Snippet: Biotransformation and uptake of 7- O -galloylquercetin ( 3 ) by RAW264.7 cells. Cells were treated for 0–6 h with 15 μM 3 or 0.1% DMSO (control), and cell extracts and culture medium were analyzed by HPLC with negative ESI-MS detection. (A) HPLC/MS chromatograms of 3 (inset) and of its metabolites found in cells after 2 h of treatment. (B) Time course of distribution of 3 , quercetin (QUE) and gallic acid (GA) in cells and medium. Data are means ± SD of three experiments.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry

    7- O -Galloylquercetin ( 3 ) induces ARE-driven gene expression. AREc32 reporter cells were treated for 24 h with 0.1% DMSO (control), 5 μM sulforaphane (SFN; positive control) or with 3.75–15 μM 3 , quercetin (QUE) or methyl gallate (MG). After treatment, luciferase reporter activity was determined luminometrically and normalized to protein content. Data are means ± SD of three experiments. * p
    Figure Legend Snippet: 7- O -Galloylquercetin ( 3 ) induces ARE-driven gene expression. AREc32 reporter cells were treated for 24 h with 0.1% DMSO (control), 5 μM sulforaphane (SFN; positive control) or with 3.75–15 μM 3 , quercetin (QUE) or methyl gallate (MG). After treatment, luciferase reporter activity was determined luminometrically and normalized to protein content. Data are means ± SD of three experiments. * p

    Techniques Used: Expressing, Positive Control, Luciferase, Activity Assay

    25) Product Images from "HDAC8 Inhibitor WK2-16 Therapeutically Targets Lipopolysaccharide-Induced Mouse Model of Neuroinflammation and Microglial Activation"

    Article Title: HDAC8 Inhibitor WK2-16 Therapeutically Targets Lipopolysaccharide-Induced Mouse Model of Neuroinflammation and Microglial Activation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20020410

    WK2-16 decreased inflammatory enzymes and TNF-α production in LPS-stimulated BV-2 microglial cells. ( A ) BV-2 cells were incubated with WK2-16 for 2 h, and the acetylation level of SMC3 was determined using Western blotting. ( B , C ) BV-2 cells were pretreated with vehicle (DMSO) or WK2-16 (0.5, 1, and 2 µM) for 30 min followed by LPS (150 ng/mL) stimulation for 24 h. The expression of COX-2 and iNOS in LPS-stimulated BV-2 cells in the presence or absence of WK2-16 was examined using Western blotting. β-actin was used as the internal control. ( D ) TNF-α levels in conditioned media of cultured BV-2 microglial cells were measured using ELISA. Values are presented as the means ± SD from three independent experiments. R: resting; V: vehicle. ## p
    Figure Legend Snippet: WK2-16 decreased inflammatory enzymes and TNF-α production in LPS-stimulated BV-2 microglial cells. ( A ) BV-2 cells were incubated with WK2-16 for 2 h, and the acetylation level of SMC3 was determined using Western blotting. ( B , C ) BV-2 cells were pretreated with vehicle (DMSO) or WK2-16 (0.5, 1, and 2 µM) for 30 min followed by LPS (150 ng/mL) stimulation for 24 h. The expression of COX-2 and iNOS in LPS-stimulated BV-2 cells in the presence or absence of WK2-16 was examined using Western blotting. β-actin was used as the internal control. ( D ) TNF-α levels in conditioned media of cultured BV-2 microglial cells were measured using ELISA. Values are presented as the means ± SD from three independent experiments. R: resting; V: vehicle. ## p

    Techniques Used: Incubation, Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

    The effects of WK2-16 on LPS-activated pathway signaling in microglial BV-2 cells. Microglial BV-2 cells were pretreated with vehicle (DMSO) or WK2-16 (1 and 2 μM) followed by LPS (150 ng/mL) stimulation for the indicated time. Phosphorylated levels of p65 (A), p38 (B), ERK (C), and Akt (D) in LPS-stimulated BV-2 cells treated with various concentration of WK2-16 were examined using Western blotting. Values are presented as the means ± SD from 3 independent experiments. R: resting; V: vehicle. p-p65: phosphorylated p65; p-p38: phosphorylated p38; p-ERK: phosphorylated ERK; p-AKT: phosphorylated AKT. ## p
    Figure Legend Snippet: The effects of WK2-16 on LPS-activated pathway signaling in microglial BV-2 cells. Microglial BV-2 cells were pretreated with vehicle (DMSO) or WK2-16 (1 and 2 μM) followed by LPS (150 ng/mL) stimulation for the indicated time. Phosphorylated levels of p65 (A), p38 (B), ERK (C), and Akt (D) in LPS-stimulated BV-2 cells treated with various concentration of WK2-16 were examined using Western blotting. Values are presented as the means ± SD from 3 independent experiments. R: resting; V: vehicle. p-p65: phosphorylated p65; p-p38: phosphorylated p38; p-ERK: phosphorylated ERK; p-AKT: phosphorylated AKT. ## p

    Techniques Used: Concentration Assay, Western Blot

    WK2-16 suppressed LPS-induced microglial activation and proliferation. ( A ) Schematic illustration of the site of stereotaxic injection in the caudate/putamen region (red arrow). ( B , C , D ) Brain sections (50 μm thick) were prepared eight hours after intrastriatal normal saline or LPS (4 μg/3μL) injection in the presence or absence of WK2-16 (30 mg/kg). Microglia were visualized using fluorescent immunostaining with an anti-Iba-1 antibody (red). ( E , F , G ) Higher magnification fluorescence microscopy of the rectangular area in Figure 3 A. Figure 3 E,F,G are magnified from Figure 3 B, C, and D in the striatum, respectively. Iba-1 immunostaining was counterstained with DAPI (blue). Scale bar = 75 μm. ( H ) Quantification of cells positive for Iba-1. The data are presented as the means ± SD from 4 animals. (I) BV-2 microglia were pretreated with WK2-16 at varying concentrations followed by LPS (150 ng/mL) incubation for 48 h. The relative cell proliferation was determined using the SRB assay in vitro ( n = 3). R: resting; V: vehicle (DMSO). # p
    Figure Legend Snippet: WK2-16 suppressed LPS-induced microglial activation and proliferation. ( A ) Schematic illustration of the site of stereotaxic injection in the caudate/putamen region (red arrow). ( B , C , D ) Brain sections (50 μm thick) were prepared eight hours after intrastriatal normal saline or LPS (4 μg/3μL) injection in the presence or absence of WK2-16 (30 mg/kg). Microglia were visualized using fluorescent immunostaining with an anti-Iba-1 antibody (red). ( E , F , G ) Higher magnification fluorescence microscopy of the rectangular area in Figure 3 A. Figure 3 E,F,G are magnified from Figure 3 B, C, and D in the striatum, respectively. Iba-1 immunostaining was counterstained with DAPI (blue). Scale bar = 75 μm. ( H ) Quantification of cells positive for Iba-1. The data are presented as the means ± SD from 4 animals. (I) BV-2 microglia were pretreated with WK2-16 at varying concentrations followed by LPS (150 ng/mL) incubation for 48 h. The relative cell proliferation was determined using the SRB assay in vitro ( n = 3). R: resting; V: vehicle (DMSO). # p

    Techniques Used: Activation Assay, Injection, Immunostaining, Fluorescence, Microscopy, Incubation, Sulforhodamine B Assay, In Vitro

    WK2-16 inhibited the activation of STAT-1/-3 in LPS-stimulated microglial BV-2 cells. Microglial BV-2 cells were pretreated with the vehicle (DMSO) or WK2-16 (1 and 2 μM) followed by stimulation with LPS (150 ng/mL) for 3 h. Phosphorylated STAT-1 ( n = 3) and STAT-3 ( n = 4) were evaluated using Western blotting. The detection of STAT-1 and β-actin were used as the internal controls. R: resting; V: vehicle; p-STAT1: phosphorylated STAT-1; p-STAT3: phosphorylated STAT-3. ### p
    Figure Legend Snippet: WK2-16 inhibited the activation of STAT-1/-3 in LPS-stimulated microglial BV-2 cells. Microglial BV-2 cells were pretreated with the vehicle (DMSO) or WK2-16 (1 and 2 μM) followed by stimulation with LPS (150 ng/mL) for 3 h. Phosphorylated STAT-1 ( n = 3) and STAT-3 ( n = 4) were evaluated using Western blotting. The detection of STAT-1 and β-actin were used as the internal controls. R: resting; V: vehicle; p-STAT1: phosphorylated STAT-1; p-STAT3: phosphorylated STAT-3. ### p

    Techniques Used: Activation Assay, Western Blot

    26) Product Images from "Analysis of toxicity effects of Di-(2-ethylhexyl) phthalate exposure on human bronchial epithelial 16HBE cells"

    Article Title: Analysis of toxicity effects of Di-(2-ethylhexyl) phthalate exposure on human bronchial epithelial 16HBE cells

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0111-6

    Effects of DEHP on cell cycle progression in 16HBE cells. The 16HBE cells were treated with different doses of DEHP (0.125, 0.5 and 2 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. a , b Show the cell cycle distribution examined by flow cytometry. Results were expressed as the percentage of cells in G0/G1, S and G2/M phase of the cell cycle. Values are presented as mean ± SD of three independent experiments. The asterisk indicates a significant difference between DMSO control and DEHP-treated cells. * P
    Figure Legend Snippet: Effects of DEHP on cell cycle progression in 16HBE cells. The 16HBE cells were treated with different doses of DEHP (0.125, 0.5 and 2 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. a , b Show the cell cycle distribution examined by flow cytometry. Results were expressed as the percentage of cells in G0/G1, S and G2/M phase of the cell cycle. Values are presented as mean ± SD of three independent experiments. The asterisk indicates a significant difference between DMSO control and DEHP-treated cells. * P

    Techniques Used: Flow Cytometry, Cytometry

    Effects of DEHP on cell apoptosis in 16HBE cells. 16HBE cells were treated with different doses of DEHP (0.125, 0.5 and 2 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. a Show the flow cytometric plots of normal cells and apoptotic cells stained by Annexin V-FITC/PI. Q1, necrotic cells; Q2, late apoptotic cells; Q3, normal cells; Q4 early apoptotic cells. b–d Show the proportion of apoptotic cells and normal cells. Values are presented as mean ± SD of three independent experiments. The asterisks indicate a significant difference between DMSO control and DEHP-treated cells. * P
    Figure Legend Snippet: Effects of DEHP on cell apoptosis in 16HBE cells. 16HBE cells were treated with different doses of DEHP (0.125, 0.5 and 2 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. a Show the flow cytometric plots of normal cells and apoptotic cells stained by Annexin V-FITC/PI. Q1, necrotic cells; Q2, late apoptotic cells; Q3, normal cells; Q4 early apoptotic cells. b–d Show the proportion of apoptotic cells and normal cells. Values are presented as mean ± SD of three independent experiments. The asterisks indicate a significant difference between DMSO control and DEHP-treated cells. * P

    Techniques Used: Flow Cytometry, Staining

    Effects of DEHP on the global DNA methylation and mRNA expression levels of Dnmt1 , Dnmt3a and Dnmt3b in 16HBE cells. 16HBE cells were treated with different doses of DEHP (0.125, 0.5 and 2 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. a Show the global DNA methylation levels of 16HBE cells. b Show the mRNA expression levels of Dnmt1 , Dnmt3a and Dnmt3b in 16HBE cells. Values are presented as mean ± SD of three independent experiments. The asterisk s indicate a significant difference between DMSO control and DEHP-treated cells. * P
    Figure Legend Snippet: Effects of DEHP on the global DNA methylation and mRNA expression levels of Dnmt1 , Dnmt3a and Dnmt3b in 16HBE cells. 16HBE cells were treated with different doses of DEHP (0.125, 0.5 and 2 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. a Show the global DNA methylation levels of 16HBE cells. b Show the mRNA expression levels of Dnmt1 , Dnmt3a and Dnmt3b in 16HBE cells. Values are presented as mean ± SD of three independent experiments. The asterisk s indicate a significant difference between DMSO control and DEHP-treated cells. * P

    Techniques Used: DNA Methylation Assay, Expressing

    Morphometric analysis of the response of 16HBE cells exposed to DEHP and effects of DEHP on cell viability. a Typical images used for analysis the effect of DEHP exposure on 16HBE cell number. Images of live cells were captured at ×200. b Cell counts determined using manual tagging in ImagePro Plus 6.1 for cells exposed to vehicle control and different doses of DEHP. c Cell viability was determined by MTT assay after treatment with different concentrations of DEHP (0.25, 0.5, 1, 2, 4, 8, 10, 12, 14 and 16 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. The value of DMSO control was set to 100%.Values are mean ± SD. The asterisks indicate a significant difference between DMSO control and DEHP-treated cells. * P
    Figure Legend Snippet: Morphometric analysis of the response of 16HBE cells exposed to DEHP and effects of DEHP on cell viability. a Typical images used for analysis the effect of DEHP exposure on 16HBE cell number. Images of live cells were captured at ×200. b Cell counts determined using manual tagging in ImagePro Plus 6.1 for cells exposed to vehicle control and different doses of DEHP. c Cell viability was determined by MTT assay after treatment with different concentrations of DEHP (0.25, 0.5, 1, 2, 4, 8, 10, 12, 14 and 16 mmol/L) or vehicle (DMSO, 0.1%) for 48 h. The value of DMSO control was set to 100%.Values are mean ± SD. The asterisks indicate a significant difference between DMSO control and DEHP-treated cells. * P

    Techniques Used: MTT Assay

    27) Product Images from "Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R"

    Article Title: Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R

    Journal: mBio

    doi: 10.1128/mBio.00121-17

    HCV replication inhibits p53 activation following DNA damage. (A) Immunofluorescence confocal microscopy for p53 and HCV core protein in HepG2/miR-122 cells electroporated with genome-length HCV RNA (JFH1-QL) or a nonreplicating control RNA (JFH1/GND) and treated 72 h later with 100 μM etoposide (ETOP) or DMSO for 2 h. Nuclei were labeled with DAPI. Bars, 50 μm. (B) Immunoblots of p53, p21, PUMA, and HCV core protein in HepG2/miR-122 cells electroporated with JFH1-QL or JFH1/GND RNA and treated 72 h later with 50 μM ETOP, 10 μM MDM2 inhibitor (nutlin-3), or DMSO for 6 h. β-Tubulin was used as a loading control. (C) Flow cytometric analysis of p53 and HCV core protein levels in cells treated as described for panel A. Quadrants are based on staining with isotype control antibodies. The frequency of events in each quadrant is represented as the percentage of total gated events. (D) p53 accumulation in cell populations from panel C that do not express HCV core [HCV core (-)] versus cell populations that express HCV core [HCV core (+)]. The numbers indicate the percentages of p53-positive cells following etoposide treatment. (E) Median fluorescence intensity (MFI) values for p53 for the indicated populations are shown normalized to JFH1/GND-electroporated, DMSO-treated controls. Relative MFI values represent the means plus standard errors of the means (SEM) (error bars) from three independent experiments. (F) p21 upregulation in HCV core (-) versus HCV core (+) cells treated with 50 μM ETOP or DMSO for 6 h. The numbers indicate the percentages of p21-positive cells following etoposide treatment. (G) MFI values for p21 are shown normalized to JFH1/GND-electroporated, DMSO-treated controls. Relative MFI values represent the means plus SEM from three independent experiments. Values that are significantly different ( P
    Figure Legend Snippet: HCV replication inhibits p53 activation following DNA damage. (A) Immunofluorescence confocal microscopy for p53 and HCV core protein in HepG2/miR-122 cells electroporated with genome-length HCV RNA (JFH1-QL) or a nonreplicating control RNA (JFH1/GND) and treated 72 h later with 100 μM etoposide (ETOP) or DMSO for 2 h. Nuclei were labeled with DAPI. Bars, 50 μm. (B) Immunoblots of p53, p21, PUMA, and HCV core protein in HepG2/miR-122 cells electroporated with JFH1-QL or JFH1/GND RNA and treated 72 h later with 50 μM ETOP, 10 μM MDM2 inhibitor (nutlin-3), or DMSO for 6 h. β-Tubulin was used as a loading control. (C) Flow cytometric analysis of p53 and HCV core protein levels in cells treated as described for panel A. Quadrants are based on staining with isotype control antibodies. The frequency of events in each quadrant is represented as the percentage of total gated events. (D) p53 accumulation in cell populations from panel C that do not express HCV core [HCV core (-)] versus cell populations that express HCV core [HCV core (+)]. The numbers indicate the percentages of p53-positive cells following etoposide treatment. (E) Median fluorescence intensity (MFI) values for p53 for the indicated populations are shown normalized to JFH1/GND-electroporated, DMSO-treated controls. Relative MFI values represent the means plus standard errors of the means (SEM) (error bars) from three independent experiments. (F) p21 upregulation in HCV core (-) versus HCV core (+) cells treated with 50 μM ETOP or DMSO for 6 h. The numbers indicate the percentages of p21-positive cells following etoposide treatment. (G) MFI values for p21 are shown normalized to JFH1/GND-electroporated, DMSO-treated controls. Relative MFI values represent the means plus SEM from three independent experiments. Values that are significantly different ( P

    Techniques Used: Activation Assay, Immunofluorescence, Confocal Microscopy, Labeling, Western Blot, Flow Cytometry, Staining, Fluorescence

    p53 activation is impaired in cells inoculated with infectious HCV. (A) Flow cytometric analysis of HepG2-HFL cells 72 h after mock infection or inoculation with JFH1-QL virus (MOI of 0.5). Cells were gated into mock-infected (-) versus virus-infected (+) populations based on HCV core protein staining in mock-infected cells. The percentage of cells in each population is indicated. (B) p53 accumulation in cells that do not express HCV core [HCV core (-)] versus cells that express HCV core [HCV core (+)]. The cells were treated with 50 μM etoposide or DMSO control for 6 h. Numbers indicate the percentages of p53-positive cells following etoposide treatment. (C) p21 upregulation in HCV core (-) cells versus HCV core (+) cells treated as described for panel B. Numbers indicate the percentages of p21-positive cells following etoposide treatment.
    Figure Legend Snippet: p53 activation is impaired in cells inoculated with infectious HCV. (A) Flow cytometric analysis of HepG2-HFL cells 72 h after mock infection or inoculation with JFH1-QL virus (MOI of 0.5). Cells were gated into mock-infected (-) versus virus-infected (+) populations based on HCV core protein staining in mock-infected cells. The percentage of cells in each population is indicated. (B) p53 accumulation in cells that do not express HCV core [HCV core (-)] versus cells that express HCV core [HCV core (+)]. The cells were treated with 50 μM etoposide or DMSO control for 6 h. Numbers indicate the percentages of p53-positive cells following etoposide treatment. (C) p21 upregulation in HCV core (-) cells versus HCV core (+) cells treated as described for panel B. Numbers indicate the percentages of p21-positive cells following etoposide treatment.

    Techniques Used: Activation Assay, Flow Cytometry, Infection, Staining

    HCV inhibition of p53 activation is independent of MDM2 and proteasome activity. (A) p53 accumulation in HCV core (-) versus HCV core (+) populations of HepG2/miR-122 cells electroporated with indicated HCV RNA genomes and after 72 h, treated with 10 μM MDM2 inhibitor (nutlin-3) or DMSO for an additional 6 h. The numbers indicate the percentages of p53-positive cells following nutlin-3 treatment. (B) p53 accumulation in HCV core (-) versus HCV core (+) populations treated with 250 nM proteasome inhibitor (epoxomycin [EPX]) or DMSO for 6 h. The numbers indicate the percentages of p53-positive cells following EPX treatment.
    Figure Legend Snippet: HCV inhibition of p53 activation is independent of MDM2 and proteasome activity. (A) p53 accumulation in HCV core (-) versus HCV core (+) populations of HepG2/miR-122 cells electroporated with indicated HCV RNA genomes and after 72 h, treated with 10 μM MDM2 inhibitor (nutlin-3) or DMSO for an additional 6 h. The numbers indicate the percentages of p53-positive cells following nutlin-3 treatment. (B) p53 accumulation in HCV core (-) versus HCV core (+) populations treated with 250 nM proteasome inhibitor (epoxomycin [EPX]) or DMSO for 6 h. The numbers indicate the percentages of p53-positive cells following EPX treatment.

    Techniques Used: Inhibition, Activation Assay, Activity Assay

    Comparison of p53 responses in HepG2 and Huh7 cells. (A) Schematic representation of p53, including key functional domains. HepG2 cells express functional, wild-type p53, whereas Huh7 cells express Y220C-mutated p53 that is aberrantly stable and transcriptionally inactive ( 12 , 13 ). (B) Immunoblots of p53 and its transcriptional targets, p21 and PUMA, in lysates of HepG2 and Huh7 cells treated with increasing concentrations of etoposide (ETOP) or DMSO control for 6 h. β-Actin was used as a loading control.
    Figure Legend Snippet: Comparison of p53 responses in HepG2 and Huh7 cells. (A) Schematic representation of p53, including key functional domains. HepG2 cells express functional, wild-type p53, whereas Huh7 cells express Y220C-mutated p53 that is aberrantly stable and transcriptionally inactive ( 12 , 13 ). (B) Immunoblots of p53 and its transcriptional targets, p21 and PUMA, in lysates of HepG2 and Huh7 cells treated with increasing concentrations of etoposide (ETOP) or DMSO control for 6 h. β-Actin was used as a loading control.

    Techniques Used: Functional Assay, Western Blot

    28) Product Images from "Selective inhibition of liver X receptor α-mediated lipogenesis in primary hepatocytes by licochalcone A"

    Article Title: Selective inhibition of liver X receptor α-mediated lipogenesis in primary hepatocytes by licochalcone A

    Journal: Chinese Medicine

    doi: 10.1186/s13020-015-0037-x

    Effects of LicA on (A) the LXRE-containing promoter activity of ABCA1 and ABCG1 genes and (B) the T0901317-stimulated mRNA levels of ABCA1 and ABCG1. (A) The activity of ABCA1 LXRE-luciferase or ABCG1 LXRE-luciferase was determined, following treatment with 1 μM T0901317 and 10 μg/mL LicA, in HepG2 cells and normalized to cotransfected β-galactosidase activity. Data were presented as means (SD) from four independent experiments with duplicate determinations. (B) The mRNA levels of ABCA1 and ABCG1 were measured in primary hepatocytes treated with 1 μM T0901317 and 10 μg/mL LicA by qRT-PCR. Data were presented as means (SD) from 4 independent experiments with triplicate determinations. Statistical analysis was performed by one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.
    Figure Legend Snippet: Effects of LicA on (A) the LXRE-containing promoter activity of ABCA1 and ABCG1 genes and (B) the T0901317-stimulated mRNA levels of ABCA1 and ABCG1. (A) The activity of ABCA1 LXRE-luciferase or ABCG1 LXRE-luciferase was determined, following treatment with 1 μM T0901317 and 10 μg/mL LicA, in HepG2 cells and normalized to cotransfected β-galactosidase activity. Data were presented as means (SD) from four independent experiments with duplicate determinations. (B) The mRNA levels of ABCA1 and ABCG1 were measured in primary hepatocytes treated with 1 μM T0901317 and 10 μg/mL LicA by qRT-PCR. Data were presented as means (SD) from 4 independent experiments with triplicate determinations. Statistical analysis was performed by one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.

    Techniques Used: Activity Assay, Luciferase, Quantitative RT-PCR

    Effects of LicA on the T0901317-stimulated activation of LXRE-containing promoters. LXRα-mediated transcriptional activity was determined on (A) 3 × LXRE-luciferase or (B) SREBP-1c LXRE-luciferase reporters in the presence of 1 μM T0901317 and 10 μg/mL LicA in HepG2 cells. Luciferase activity was normalized to cotransfected β-galactosidase activity. Data were presented as means (SD) from four independent experiments with duplicate determinations. Statistical analysis was performed using one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.
    Figure Legend Snippet: Effects of LicA on the T0901317-stimulated activation of LXRE-containing promoters. LXRα-mediated transcriptional activity was determined on (A) 3 × LXRE-luciferase or (B) SREBP-1c LXRE-luciferase reporters in the presence of 1 μM T0901317 and 10 μg/mL LicA in HepG2 cells. Luciferase activity was normalized to cotransfected β-galactosidase activity. Data were presented as means (SD) from four independent experiments with duplicate determinations. Statistical analysis was performed using one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.

    Techniques Used: Activation Assay, Activity Assay, Luciferase

    Effects of LicA on (A) the LXRα-stimulated expression of lipogenic genes and (B) TG accumulation in mouse primary hepatocytes. (A) T0901317-induced expression of SREBP-1c, SCD1, FAS, and GPAT was determined in the presence or absence of LicA by qRT-PCR. Data were presented as means (SD) from four independent experiments with triplicate determinations. (B) TG accumulation in response to cotreatment with T0901317 and LicA. Intracellular TG levels were measured enzymatically after treatment of primary hepatocytes with 1 μM T0901317 and 10 μg/mL LicA and normalized to total protein levels. Data were presented as means (SD) from four independent experiments with duplicate determinations. Statistical analysis was performed by one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.
    Figure Legend Snippet: Effects of LicA on (A) the LXRα-stimulated expression of lipogenic genes and (B) TG accumulation in mouse primary hepatocytes. (A) T0901317-induced expression of SREBP-1c, SCD1, FAS, and GPAT was determined in the presence or absence of LicA by qRT-PCR. Data were presented as means (SD) from four independent experiments with triplicate determinations. (B) TG accumulation in response to cotreatment with T0901317 and LicA. Intracellular TG levels were measured enzymatically after treatment of primary hepatocytes with 1 μM T0901317 and 10 μg/mL LicA and normalized to total protein levels. Data were presented as means (SD) from four independent experiments with duplicate determinations. Statistical analysis was performed by one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.

    Techniques Used: Expressing, Quantitative RT-PCR

    Effects of LicA on the recruitments of RNA polymerase II to the LXRE regions of SREBP-1c (A) and ABCA1 (B) genes. After treatment of primary hepatocytes with 1 μM T0901317 and 10 μg/mL LicA, ChIP assays were performed using anti-RNA polymerase II or IgG antibodies. Immunoprecipitated LXRE-containing DNA levels were determined by qRT-PCR and normalized to input DNA. Data were presented as means (SD) from four independent experiments with triplicate determinations. Statistical analysis was performed by one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.
    Figure Legend Snippet: Effects of LicA on the recruitments of RNA polymerase II to the LXRE regions of SREBP-1c (A) and ABCA1 (B) genes. After treatment of primary hepatocytes with 1 μM T0901317 and 10 μg/mL LicA, ChIP assays were performed using anti-RNA polymerase II or IgG antibodies. Immunoprecipitated LXRE-containing DNA levels were determined by qRT-PCR and normalized to input DNA. Data were presented as means (SD) from four independent experiments with triplicate determinations. Statistical analysis was performed by one-way ANOVA. P * = P value for Bonferroni correction. DMSO, dimethyl sulfoxide; T1317, T0901317.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR

    29) Product Images from "Transcriptional Regulation of Human Cytosolic Sulfotransferase 1C3 by Peroxisome Proliferator-Activated Receptor γ in LS180 Human Colorectal Adenocarcinoma Cells"

    Article Title: Transcriptional Regulation of Human Cytosolic Sulfotransferase 1C3 by Peroxisome Proliferator-Activated Receptor γ in LS180 Human Colorectal Adenocarcinoma Cells

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.116.106005

    Effect of PPAR knockdowns on SULT1C3 transcriptional activation by different classes of PPAR agonist. LS180 cells were transiently cotransfected with either the SULT1C3 1-kb or the CYP4A1 PPRE reporter plasmid (positive control) and 20 pmol of either nontargeting (NT) siRNA or siRNA targeting PPAR γ (A), PPAR α (B), or PPAR δ (C). Twenty-four hours after transfection cells were treated with DMSO (0.1%), rosiglitazone (1 μ M), GW7647 (10 μ M), or GW0742 (10 μ M) for 48 hours and then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements (three wells per treatment). Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated controls at P
    Figure Legend Snippet: Effect of PPAR knockdowns on SULT1C3 transcriptional activation by different classes of PPAR agonist. LS180 cells were transiently cotransfected with either the SULT1C3 1-kb or the CYP4A1 PPRE reporter plasmid (positive control) and 20 pmol of either nontargeting (NT) siRNA or siRNA targeting PPAR γ (A), PPAR α (B), or PPAR δ (C). Twenty-four hours after transfection cells were treated with DMSO (0.1%), rosiglitazone (1 μ M), GW7647 (10 μ M), or GW0742 (10 μ M) for 48 hours and then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements (three wells per treatment). Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated controls at P

    Techniques Used: Activation Assay, Plasmid Preparation, Positive Control, Transfection, Luciferase

    Effects of PPAR agonists on transcription of SULT1C3 reporter constructs. (A) LS180 cells were transiently transfected with a luciferase reporter plasmid containing either the 2.8-kb or 1.9-kb SULT1C3 5′-flanking region fragment, with the 1-kb reporter containing the deleted region or with control empty vector. Twenty-four hours after transfection, the cells were treated with DMSO (0.1%) or rosiglitazone (10 µ M) for 48 hours. (B) LS180 cells transfected with the 1-kb reporter were treated with DMSO (0.1%) or with 0.1–10 µ M rosiglitazone (PPAR γ agonist), GW7647 (PPAR α ), or GW0742 (PPAR δ ) for 48 hours. (C) LS180 cells transfected with the 2.8-kb, 1.9-kb, or 1-kb reporter or with control empty vector were treated with DMSO (0.1%), GW7647 (10 µ M), or GW0742 (10 µ M). The cells were then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements relative to the corresponding DMSO control ( n = 3 wells per treatment) from one cell culture experiment. Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated control at P
    Figure Legend Snippet: Effects of PPAR agonists on transcription of SULT1C3 reporter constructs. (A) LS180 cells were transiently transfected with a luciferase reporter plasmid containing either the 2.8-kb or 1.9-kb SULT1C3 5′-flanking region fragment, with the 1-kb reporter containing the deleted region or with control empty vector. Twenty-four hours after transfection, the cells were treated with DMSO (0.1%) or rosiglitazone (10 µ M) for 48 hours. (B) LS180 cells transfected with the 1-kb reporter were treated with DMSO (0.1%) or with 0.1–10 µ M rosiglitazone (PPAR γ agonist), GW7647 (PPAR α ), or GW0742 (PPAR δ ) for 48 hours. (C) LS180 cells transfected with the 2.8-kb, 1.9-kb, or 1-kb reporter or with control empty vector were treated with DMSO (0.1%), GW7647 (10 µ M), or GW0742 (10 µ M). The cells were then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements relative to the corresponding DMSO control ( n = 3 wells per treatment) from one cell culture experiment. Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated control at P

    Techniques Used: Construct, Transfection, Luciferase, Plasmid Preparation, Cell Culture

    Impact of a deletion in the 5′-flanking region of the SULT1C3 gene on its regulation by transcription factor activators. (A) PCR was performed with primers designed to amplify a 2.8-kb fragment of the SULT1C3 5′-flanking region using genomic DNA from MCF10A or LS180 cells. The PCR products were resolved on a 1% agarose gel (1-kb ladder also shown). A schematic representation of the 2.8-, 1.9-, and 1-kb SULT1C3 5′-flanking fragments is shown adjacent to the gel image. (B) LS180 cells were transiently transfected with the SULT1C3-2.8-kb or 1.9-kb luciferase reporter plasmid or with the pGL4.10 [luc2] empty reporter vector and then treated with DMSO (0.1%), ciprofibrate (PPAR α agonist, 100 μ M), GW3965 (LXR, 10 μ M), GW4064 (FXR, 1 μ M), rifampicin (PXR, 30 μ M), rosiglitazone (PPAR γ , 10 μ M), or TCDD (AhR, 0.01 μ M) for 48 hours. The cells were then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements relative to DMSO control ( n = 3 wells per treatment) from one cell culture experiment. Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated controls at P
    Figure Legend Snippet: Impact of a deletion in the 5′-flanking region of the SULT1C3 gene on its regulation by transcription factor activators. (A) PCR was performed with primers designed to amplify a 2.8-kb fragment of the SULT1C3 5′-flanking region using genomic DNA from MCF10A or LS180 cells. The PCR products were resolved on a 1% agarose gel (1-kb ladder also shown). A schematic representation of the 2.8-, 1.9-, and 1-kb SULT1C3 5′-flanking fragments is shown adjacent to the gel image. (B) LS180 cells were transiently transfected with the SULT1C3-2.8-kb or 1.9-kb luciferase reporter plasmid or with the pGL4.10 [luc2] empty reporter vector and then treated with DMSO (0.1%), ciprofibrate (PPAR α agonist, 100 μ M), GW3965 (LXR, 10 μ M), GW4064 (FXR, 1 μ M), rifampicin (PXR, 30 μ M), rosiglitazone (PPAR γ , 10 μ M), or TCDD (AhR, 0.01 μ M) for 48 hours. The cells were then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements relative to DMSO control ( n = 3 wells per treatment) from one cell culture experiment. Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated controls at P

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Luciferase, Plasmid Preparation, Cell Culture

    Evaluation of three computationally predicted PPREs within the deleted region of the SULT1C3 5′-flanking region. LS180 cells were transiently transfected with a series of reporter plasmids with progressive 5′-deletions from the 1-kb SULT1C3 reporter plasmid that were designed based on the positions of computationally predicted PPREs (A) or with the 1-kb SULT1C3 reporter plasmid containing either the wild-type sequence or site-directed mutations at one or two of the predicted PPREs (B). Transfected cells were treated with DMSO (0.1%), rosiglitazone (1 μ M), GW7647 (10 μ M), or GW0742 (10 μ M) for 48 hours and then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements relative to the DMSO-treated, empty vector-transfected group ( n = 3 wells per treatment) from one cell culture experiment. Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated controls at P
    Figure Legend Snippet: Evaluation of three computationally predicted PPREs within the deleted region of the SULT1C3 5′-flanking region. LS180 cells were transiently transfected with a series of reporter plasmids with progressive 5′-deletions from the 1-kb SULT1C3 reporter plasmid that were designed based on the positions of computationally predicted PPREs (A) or with the 1-kb SULT1C3 reporter plasmid containing either the wild-type sequence or site-directed mutations at one or two of the predicted PPREs (B). Transfected cells were treated with DMSO (0.1%), rosiglitazone (1 μ M), GW7647 (10 μ M), or GW0742 (10 μ M) for 48 hours and then harvested for measurement of luciferase activities. Each column represents the mean ± S.D. of normalized (Firefly/ Renilla ) luciferase measurements relative to the DMSO-treated, empty vector-transfected group ( n = 3 wells per treatment) from one cell culture experiment. Similar data were obtained in two additional independent experiments. * , ** , ***Significantly different from DMSO-treated controls at P

    Techniques Used: Transfection, Plasmid Preparation, Sequencing, Luciferase, Cell Culture

    30) Product Images from "A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis"

    Article Title: A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3329

    JAK2 blockade ameliorates collagen antibody-induced arthritis (CAIA) paw inflammation . Female DBA/1 mice were injected intravenously (i.v.) with purified anti-collagen type II (anti-CII) antibodies, and then 25 μg of lipopolysaccharide (LPS) was administered intraperitoneally (i.p.) 3 days later to induce arthritis by day 5. Mice that met the arthritis criteria score of 1 per paw were entered into the study. CEP-33779 was administered p.o. twice daily (b.i.d.) throughout the experiment, 1.5 mg/kg dexamethasone (Dex) was administered i.p. three times weekly and vehicle (PEG400 + 1% dimethyl sulfoxide (DMSO)) was administered p.o. (A) Mean paw size (individual paws measured for thickness) measured over time (top) and total clinical score from each paw were recorded (bottom). * P
    Figure Legend Snippet: JAK2 blockade ameliorates collagen antibody-induced arthritis (CAIA) paw inflammation . Female DBA/1 mice were injected intravenously (i.v.) with purified anti-collagen type II (anti-CII) antibodies, and then 25 μg of lipopolysaccharide (LPS) was administered intraperitoneally (i.p.) 3 days later to induce arthritis by day 5. Mice that met the arthritis criteria score of 1 per paw were entered into the study. CEP-33779 was administered p.o. twice daily (b.i.d.) throughout the experiment, 1.5 mg/kg dexamethasone (Dex) was administered i.p. three times weekly and vehicle (PEG400 + 1% dimethyl sulfoxide (DMSO)) was administered p.o. (A) Mean paw size (individual paws measured for thickness) measured over time (top) and total clinical score from each paw were recorded (bottom). * P

    Techniques Used: Mouse Assay, Injection, Purification

    Disease treatment reflected in local paw environment supports the use of a selective JAK2 inhibitor for suppressing joint inflammation . DBA/1 female mice were injected i.v. with purified anti-CII antibodies, then administered 25 μg of LPS i.p. 3 days later to induce arthritis by day 5. Mice that met the arthritis criteria score of 1 per paw were entered into the study. CEP-33779 was administered p.o., b.i.d., throughout the experiment, Dex was administered at 1.5 mg/kg three times weekly i.p., and vehicle (PEG400 + 1% DMSO) was administered p.o. (A) Spleen and paw phospho-STAT3 concentrations were determined by using Luminex bead kits, with the concentrations shown along the y -axis in units per milliliter per 3 mg of total protein as determined by using a bovine serum albumin (BSA) assays. Tissues were removed 2 and 6 hours after administration of CEP-33779 p.o. at 55 mg/kg. Graph shows means ± SEM. * P
    Figure Legend Snippet: Disease treatment reflected in local paw environment supports the use of a selective JAK2 inhibitor for suppressing joint inflammation . DBA/1 female mice were injected i.v. with purified anti-CII antibodies, then administered 25 μg of LPS i.p. 3 days later to induce arthritis by day 5. Mice that met the arthritis criteria score of 1 per paw were entered into the study. CEP-33779 was administered p.o., b.i.d., throughout the experiment, Dex was administered at 1.5 mg/kg three times weekly i.p., and vehicle (PEG400 + 1% DMSO) was administered p.o. (A) Spleen and paw phospho-STAT3 concentrations were determined by using Luminex bead kits, with the concentrations shown along the y -axis in units per milliliter per 3 mg of total protein as determined by using a bovine serum albumin (BSA) assays. Tissues were removed 2 and 6 hours after administration of CEP-33779 p.o. at 55 mg/kg. Graph shows means ± SEM. * P

    Techniques Used: Mouse Assay, Injection, Purification, Luminex

    JAK2 blockade reduces several disease correlates in a model of chronic degenerative arthritis . Female DBA/1 mice were injected with purified CII in Complete Freund's Adjuvant (CFA) i.d., then boosted with CII in Incomplete Freund's Adjuvant (IFA) s.c. on day 21 followed by a day 28 LPS injection i.p. to induce collagen-induced arthritis (CIA). Mice that scored 1 or better for each paw were considered arthritic and entered into the study. Treatments started after 7 to 10 days of full arthritis. CEP-33779 was administered p.o., b.i.d., throughout the remainder of the experiment. Dex was administered i.p. at 1.5 mg/kg three times weekly, and vehicle was PEG400 + 1% DMSO p.o., b.i.d. (A) Mean paw size (individual paws measured) and clinical scores for each mouse are graphed. (B) Phospho-STAT3 expression in the paws of treated CIA mice. (C) Serum cytokines (IL-12, IL-2, IL-10 and TNFα) from treated CIA mice. (D) Representative ELISPOT images showing IFNγ enzyme-linked immunosorbent spot (ELISPOT) assay results from stimulated splenocytes from treated mice challenged with CII fragment (CB11), chicken ovalbumin (OVA) or media alone. Graph was constructed using data from five mice tested from each group. Spots were counted using a CTL ImmunoSpot ELISPOT scanner and spot-counting software. (E) Hematoxylin and eosin-stained, paraffin wax-embedded sections from treated CIA mice showing joint infiltrates and bone resorption. Representative (pathologist-selected) images are shown at × 10 original magnification. Safranin O-stain sections are not shown but were used for scoring. All graphs show means ± SEM, and all statistics were calculated using Prism software and two-way ANOVA. * P
    Figure Legend Snippet: JAK2 blockade reduces several disease correlates in a model of chronic degenerative arthritis . Female DBA/1 mice were injected with purified CII in Complete Freund's Adjuvant (CFA) i.d., then boosted with CII in Incomplete Freund's Adjuvant (IFA) s.c. on day 21 followed by a day 28 LPS injection i.p. to induce collagen-induced arthritis (CIA). Mice that scored 1 or better for each paw were considered arthritic and entered into the study. Treatments started after 7 to 10 days of full arthritis. CEP-33779 was administered p.o., b.i.d., throughout the remainder of the experiment. Dex was administered i.p. at 1.5 mg/kg three times weekly, and vehicle was PEG400 + 1% DMSO p.o., b.i.d. (A) Mean paw size (individual paws measured) and clinical scores for each mouse are graphed. (B) Phospho-STAT3 expression in the paws of treated CIA mice. (C) Serum cytokines (IL-12, IL-2, IL-10 and TNFα) from treated CIA mice. (D) Representative ELISPOT images showing IFNγ enzyme-linked immunosorbent spot (ELISPOT) assay results from stimulated splenocytes from treated mice challenged with CII fragment (CB11), chicken ovalbumin (OVA) or media alone. Graph was constructed using data from five mice tested from each group. Spots were counted using a CTL ImmunoSpot ELISPOT scanner and spot-counting software. (E) Hematoxylin and eosin-stained, paraffin wax-embedded sections from treated CIA mice showing joint infiltrates and bone resorption. Representative (pathologist-selected) images are shown at × 10 original magnification. Safranin O-stain sections are not shown but were used for scoring. All graphs show means ± SEM, and all statistics were calculated using Prism software and two-way ANOVA. * P

    Techniques Used: Mouse Assay, Injection, Purification, Immunofluorescence, Expressing, Enzyme-linked Immunospot, ELISpot Assay, Construct, CTL Assay, Software, Staining

    31) Product Images from "Zebrafish Cardiotoxicity: The Effects of CYP1A Inhibition and AHR2 Knockdown Following Exposure to Weak Aryl Hydrocarbon Receptor Agonists"

    Article Title: Zebrafish Cardiotoxicity: The Effects of CYP1A Inhibition and AHR2 Knockdown Following Exposure to Weak Aryl Hydrocarbon Receptor Agonists

    Journal: Environmental science and pollution research international

    doi: 10.1007/s11356-014-3969-2

    Mean pericardial edema in larval zebrafish following exposure to weak agonists: Carb, 2MI, 3MI, Phe, indigo, and indirubin (white bars, numbers preceding treatments are μM concentrations) The black bars represent agonists combined with 500 μg/L fluoranthene (FL). Values are expressed as percent DMSO pericardial edema ± SEM. The graph represents three experimental replicates with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Each n represents the average of five embryos. Asterisks indicate that the treatment is statistically different than DMSO controls ( p ≤ 0.05).
    Figure Legend Snippet: Mean pericardial edema in larval zebrafish following exposure to weak agonists: Carb, 2MI, 3MI, Phe, indigo, and indirubin (white bars, numbers preceding treatments are μM concentrations) The black bars represent agonists combined with 500 μg/L fluoranthene (FL). Values are expressed as percent DMSO pericardial edema ± SEM. The graph represents three experimental replicates with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Each n represents the average of five embryos. Asterisks indicate that the treatment is statistically different than DMSO controls ( p ≤ 0.05).

    Techniques Used:

    Dose response EROD expression in larval zebrafish following exposure to weak agonists: carbaryl (Carb), 2-methylindole (2MI), 3-methylindole (3MI), phenanthrene (Phe), indigo, and indirubin at 3 equimolar concentrations (0.1, 1, and 10 μM, the white, grey, and black bars respectively) The EROD assay was performed at 96 hpf to compare cytochrome P450 activity in response to weak agonist exposure (measured by fluorescence and compared to DMSO controls). The EROD assay was repeated three times with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Each n represents the average of five embryos. Asterisk indicates significantly different than DMSO EROD activity ( p ≤ 0.05).
    Figure Legend Snippet: Dose response EROD expression in larval zebrafish following exposure to weak agonists: carbaryl (Carb), 2-methylindole (2MI), 3-methylindole (3MI), phenanthrene (Phe), indigo, and indirubin at 3 equimolar concentrations (0.1, 1, and 10 μM, the white, grey, and black bars respectively) The EROD assay was performed at 96 hpf to compare cytochrome P450 activity in response to weak agonist exposure (measured by fluorescence and compared to DMSO controls). The EROD assay was repeated three times with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Each n represents the average of five embryos. Asterisk indicates significantly different than DMSO EROD activity ( p ≤ 0.05).

    Techniques Used: Expressing, Activity Assay, Fluorescence

    Mean pericardial edema in larval zebrafish following exposure to weak agonists: Carb, 2MI, 3MI, Phe, indigo, and indirubin White bars are non-injected (NI) embryos treated with isolated weak agonists. The grey bars are control morpholino (Co-mo) injected embryos. Black bars represent agonists combined with 500 μg/L fluoranthene (FL). Patterned bars are CYP1A-mo injected embryos that received exposure to individual agonists. Values are expressed as percent DMSO pericardial edema ± SEM. The graph represents three experimental replicates with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Groups not sharing common letters are statistically different ( p ≤ 0.05).
    Figure Legend Snippet: Mean pericardial edema in larval zebrafish following exposure to weak agonists: Carb, 2MI, 3MI, Phe, indigo, and indirubin White bars are non-injected (NI) embryos treated with isolated weak agonists. The grey bars are control morpholino (Co-mo) injected embryos. Black bars represent agonists combined with 500 μg/L fluoranthene (FL). Patterned bars are CYP1A-mo injected embryos that received exposure to individual agonists. Values are expressed as percent DMSO pericardial edema ± SEM. The graph represents three experimental replicates with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Groups not sharing common letters are statistically different ( p ≤ 0.05).

    Techniques Used: Injection, Isolation

    Mean pericardial edema in larval zebrafish following exposure to weak agonists: Carb, 2MI, 3MI, Phe, indigo, and indirubin Numbers preceding treatments are μM concentrations. White bars are non-injected (NI) embryos treated with isolated weak agonists. The grey bars are control morpholino (Co) injected embryos. Black bars are AHR2-mo knockdown embryos. Values are expressed as percent DMSO pericardial edema ± SEM. The graph represents three experimental replicates with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Groups not sharing common letters are statistically different ( p ≤ 0.05).
    Figure Legend Snippet: Mean pericardial edema in larval zebrafish following exposure to weak agonists: Carb, 2MI, 3MI, Phe, indigo, and indirubin Numbers preceding treatments are μM concentrations. White bars are non-injected (NI) embryos treated with isolated weak agonists. The grey bars are control morpholino (Co) injected embryos. Black bars are AHR2-mo knockdown embryos. Values are expressed as percent DMSO pericardial edema ± SEM. The graph represents three experimental replicates with 3 vials of 5 embryos per experiment (n = 3 per experiment, experiment total n = 9). Groups not sharing common letters are statistically different ( p ≤ 0.05).

    Techniques Used: Injection, Isolation

    32) Product Images from "Bisphenol A Impairs Follicle Growth, Inhibits Steroidogenesis, and Downregulates Rate-Limiting Enzymes in the Estradiol Biosynthesis Pathway"

    Article Title: Bisphenol A Impairs Follicle Growth, Inhibits Steroidogenesis, and Downregulates Rate-Limiting Enzymes in the Estradiol Biosynthesis Pathway

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfq319

    Effect of BPA exposure on StAR, 3β-HSD, and Cyp17α mRNA expression levels. After exposure of antral follicles to DMSO control of BPA (4.4–440μM) for 120 h in vitro , the follicles were collected and subjected to qPCR analysis
    Figure Legend Snippet: Effect of BPA exposure on StAR, 3β-HSD, and Cyp17α mRNA expression levels. After exposure of antral follicles to DMSO control of BPA (4.4–440μM) for 120 h in vitro , the follicles were collected and subjected to qPCR analysis

    Techniques Used: Expressing, In Vitro, Real-time Polymerase Chain Reaction

    Effect of pregnenolone cotreatment with BPA on antral follicle hormone production. After exposure of antral follicles to DMSO control, BPA (4.4–440μM) pregnenolone (10 μg/ml) or pregnenolone, and BPA (44–440μM)
    Figure Legend Snippet: Effect of pregnenolone cotreatment with BPA on antral follicle hormone production. After exposure of antral follicles to DMSO control, BPA (4.4–440μM) pregnenolone (10 μg/ml) or pregnenolone, and BPA (44–440μM)

    Techniques Used:

    Effect of BPA exposure on antral follicle hormone production. After exposure of antral follicles to DMSO control or BPA (4.4–440μM) for 120 h in vitro , media was collected and subjected to various hormone measurements by ELISA. These graphs
    Figure Legend Snippet: Effect of BPA exposure on antral follicle hormone production. After exposure of antral follicles to DMSO control or BPA (4.4–440μM) for 120 h in vitro , media was collected and subjected to various hormone measurements by ELISA. These graphs

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay

    Effect of pregnenolone cotreatment with BPA on steroidogenic enzyme mRNA expression levels. After exposure of antral follicles to DMSO control, BPA (4.4–440μM) pregnenolone (10 μg/ml) or pregnenolone, and BPA (44–440μM)
    Figure Legend Snippet: Effect of pregnenolone cotreatment with BPA on steroidogenic enzyme mRNA expression levels. After exposure of antral follicles to DMSO control, BPA (4.4–440μM) pregnenolone (10 μg/ml) or pregnenolone, and BPA (44–440μM)

    Techniques Used: Expressing

    33) Product Images from "Profiling of Biomarkers for the Exposure of Polycyclic Aromatic Hydrocarbons: Lamin-A/C Isoform 3, Poly[ADP-ribose] Polymerase 1, and Mitochondria Copy Number Are Identified as Universal Biomarkers"

    Article Title: Profiling of Biomarkers for the Exposure of Polycyclic Aromatic Hydrocarbons: Lamin-A/C Isoform 3, Poly[ADP-ribose] Polymerase 1, and Mitochondria Copy Number Are Identified as Universal Biomarkers

    Journal: BioMed Research International

    doi: 10.1155/2014/605135

    mRNA expression study of candidate biomarker genes. mRNA expression of PARP-1 and LMNA gene was generally increased in THP-1 and h-TERT cell lines after exposure of PAHs with different pattern. Normal, no treatment group; DMSO, only DMSO (0.1%) treated group.
    Figure Legend Snippet: mRNA expression study of candidate biomarker genes. mRNA expression of PARP-1 and LMNA gene was generally increased in THP-1 and h-TERT cell lines after exposure of PAHs with different pattern. Normal, no treatment group; DMSO, only DMSO (0.1%) treated group.

    Techniques Used: Expressing, Biomarker Assay

    The change of cell count and viability after PAHs exposure in THP-1 and Molt-4 cell line. Depending on the type of PAHs, each cell count showed different aspects. In comparison to DMSO treated (0.1%) group, fluoranthene displayed profound significant reduction in cell count, especially in THP-1 and Molt-4 cell line ((a) and (b)). Viability was significantly decreased after fluoranthene exposure for two days. On the third day of PAHs exposure, viability was reduced remarkably in both cell lines ((c) and (d)).
    Figure Legend Snippet: The change of cell count and viability after PAHs exposure in THP-1 and Molt-4 cell line. Depending on the type of PAHs, each cell count showed different aspects. In comparison to DMSO treated (0.1%) group, fluoranthene displayed profound significant reduction in cell count, especially in THP-1 and Molt-4 cell line ((a) and (b)). Viability was significantly decreased after fluoranthene exposure for two days. On the third day of PAHs exposure, viability was reduced remarkably in both cell lines ((c) and (d)).

    Techniques Used: Cell Counting

    The change of mtDNA copy number after PAHs exposure. mtDNA copy number was increased after exposure of PAHs with different pattern in THP-1 cell line (a) and in vivo zebrafish model (b). mtDNA copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene in both THP-1 cell line and in vivo zebrafish model. hpf, hours per fertilization in zebrafish; normal, no treatment group; and DMSO, only DMSO (0.1%) treated group.
    Figure Legend Snippet: The change of mtDNA copy number after PAHs exposure. mtDNA copy number was increased after exposure of PAHs with different pattern in THP-1 cell line (a) and in vivo zebrafish model (b). mtDNA copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene in both THP-1 cell line and in vivo zebrafish model. hpf, hours per fertilization in zebrafish; normal, no treatment group; and DMSO, only DMSO (0.1%) treated group.

    Techniques Used: In Vivo

    Morphological change of human mesenchymal stem (h-TERT) cells after PAHs exposure. PAH-untreated cells (DMSO and normal) showed compact cellularity with spindle shape. h-TERT cells were tightly attached to each other and to the substrate. Generally, direct exposure of PAHs depressed the proliferative capacity of h-TERT cells with a thread-like or round shape and loose cell-to-cell attachment. Each PAHs compound showed different cytotoxic effect. DMSO and normal indicated only DMSO-treatment and culture solution itself (no treatment of PAHs and DMSO), respectively.
    Figure Legend Snippet: Morphological change of human mesenchymal stem (h-TERT) cells after PAHs exposure. PAH-untreated cells (DMSO and normal) showed compact cellularity with spindle shape. h-TERT cells were tightly attached to each other and to the substrate. Generally, direct exposure of PAHs depressed the proliferative capacity of h-TERT cells with a thread-like or round shape and loose cell-to-cell attachment. Each PAHs compound showed different cytotoxic effect. DMSO and normal indicated only DMSO-treatment and culture solution itself (no treatment of PAHs and DMSO), respectively.

    Techniques Used: Cell Attachment Assay

    34) Product Images from "Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2 *"

    Article Title: Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2 *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M114.043331

    ( A ) TPP2 inhibition does not affect ERK2 cellular protein level. Median relative ERK2 protein level normalized to DMSO treated cells as determined by SILAC ( n = 4 biological replicates, ±S.D. (butabindide n = 3, B6 n = 4 quantified replicates).
    Figure Legend Snippet: ( A ) TPP2 inhibition does not affect ERK2 cellular protein level. Median relative ERK2 protein level normalized to DMSO treated cells as determined by SILAC ( n = 4 biological replicates, ±S.D. (butabindide n = 3, B6 n = 4 quantified replicates).

    Techniques Used: Inhibition

    ( A ) and ( B ) Inhibition of TPP2 induces neurite outgrowth. ( A ) Graph shows mean total neurite length/cell ± S.D. ( n = 6) of SH-SY5Y cells treated for 4 h with 1 μ m butabindide, 1 μ m B6 or DMSO, and as positive control SH-SY5Y cells
    Figure Legend Snippet: ( A ) and ( B ) Inhibition of TPP2 induces neurite outgrowth. ( A ) Graph shows mean total neurite length/cell ± S.D. ( n = 6) of SH-SY5Y cells treated for 4 h with 1 μ m butabindide, 1 μ m B6 or DMSO, and as positive control SH-SY5Y cells

    Techniques Used: Inhibition, Positive Control

    B6 specificity and effects in cellular application. ( A ) Optimum concentration to inhibit TPP2 is 1 μ m B6. SH-SY5Y cells were incubated for 4 h with different concentrations of B6, butabindide or respective amounts of the inhibitor solvent DMSO.
    Figure Legend Snippet: B6 specificity and effects in cellular application. ( A ) Optimum concentration to inhibit TPP2 is 1 μ m B6. SH-SY5Y cells were incubated for 4 h with different concentrations of B6, butabindide or respective amounts of the inhibitor solvent DMSO.

    Techniques Used: Concentration Assay, Incubation

    ( A ) Only SOX11 mRNA is increased by 4 h TPP2 inhibition with butabindide but not with B6. Mean relative change in mRNA level (qPCR, n = 3, ±S.D.) of B6/DMSO; butabindide/DMSO; TPP2/mock shRNA stable cell lines. ( B ) and ( C ) SOX11 protein level
    Figure Legend Snippet: ( A ) Only SOX11 mRNA is increased by 4 h TPP2 inhibition with butabindide but not with B6. Mean relative change in mRNA level (qPCR, n = 3, ±S.D.) of B6/DMSO; butabindide/DMSO; TPP2/mock shRNA stable cell lines. ( B ) and ( C ) SOX11 protein level

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction, shRNA, Stable Transfection

    35) Product Images from "Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules"

    Article Title: Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules

    Journal: Nature

    doi: 10.1038/nature13889

    Auxin effect on fast responsiveness of MT rearrangement is dependent on ABP1 (a-b) Projections of EB1b-GFP in WT (a) or SS12S (b) roots (left panels) and quantification (right charts) from every 15 sec acquisitions during 10 min ( Supplementary Video 1 , 2 , 3 , 5 ) following DMSO or 100 nM NAA application (n=10). Blue and red strips represent transversal (90±30°) and oblique/longitudinal (0-60°/120-180°) directions, respectively. In all panels, error bars are s.e.m. determined by student’s T-test (*p
    Figure Legend Snippet: Auxin effect on fast responsiveness of MT rearrangement is dependent on ABP1 (a-b) Projections of EB1b-GFP in WT (a) or SS12S (b) roots (left panels) and quantification (right charts) from every 15 sec acquisitions during 10 min ( Supplementary Video 1 , 2 , 3 , 5 ) following DMSO or 100 nM NAA application (n=10). Blue and red strips represent transversal (90±30°) and oblique/longitudinal (0-60°/120-180°) directions, respectively. In all panels, error bars are s.e.m. determined by student’s T-test (*p

    Techniques Used: Size-exclusion Chromatography

    ABP1 is required for auxin regulation of MT reorientation (a-b) MAP4-GFP visualization of MTs reorientation in WT, abp1-5 , SS12S/K root (a) and hypocotyl (b) induced with ethanol vapors for 48h (a) and 8h (b) and following 60 min treatment with DMSO, 100 nM NAA (a) or 10 μM IAA (b). The ratio of transversal MTs in DMSO-versus NAA-treated is indicated above the charts (a, b). In all panels, error bars are s.e.m. and student’s T-test was calculated for transversal MTs (** p
    Figure Legend Snippet: ABP1 is required for auxin regulation of MT reorientation (a-b) MAP4-GFP visualization of MTs reorientation in WT, abp1-5 , SS12S/K root (a) and hypocotyl (b) induced with ethanol vapors for 48h (a) and 8h (b) and following 60 min treatment with DMSO, 100 nM NAA (a) or 10 μM IAA (b). The ratio of transversal MTs in DMSO-versus NAA-treated is indicated above the charts (a, b). In all panels, error bars are s.e.m. and student’s T-test was calculated for transversal MTs (** p

    Techniques Used:

    Auxin-ABP1 control MT arrangement through downstream ROP6-RIC1 and involvement of KTN1 (a) MTs orientation and quantification in roots of WT, rop6-1, ric1-1 , SS12S/K ric1-1 following 60 min of DMSO or 100 nM NAA application. (b) MTs orientation and quantification in 24h ethanol induced hypocotyls of WT, SS12K, ktn1 and SS12K ktn1 following 60 min of DMSO or 10 μM IAA application. The ratio of transversal MTs in DMSO-versus NAA/IAA-treated is indicated above the charts (a, b). In all panels, error bars are s.e.m. and student’s T-test was calculated for transversal MTs (** p
    Figure Legend Snippet: Auxin-ABP1 control MT arrangement through downstream ROP6-RIC1 and involvement of KTN1 (a) MTs orientation and quantification in roots of WT, rop6-1, ric1-1 , SS12S/K ric1-1 following 60 min of DMSO or 100 nM NAA application. (b) MTs orientation and quantification in 24h ethanol induced hypocotyls of WT, SS12K, ktn1 and SS12K ktn1 following 60 min of DMSO or 10 μM IAA application. The ratio of transversal MTs in DMSO-versus NAA/IAA-treated is indicated above the charts (a, b). In all panels, error bars are s.e.m. and student’s T-test was calculated for transversal MTs (** p

    Techniques Used:

    Auxin-ABP1 controls MT arrangement through the downstream ROP6-RIC1-KTN1 signaling (a) MAP4-GFP visualization of MTs orientation in the root of WT, rop6-1, ric1-1 , SS12S ric1-1 , SS12K ric1-1 following DMSO application for 60 min. Corresponding to quantifications in Fig. 4a . (b-c) MTs reorientation patterns were visualized by MAP4-GFP in the roots of WT and rop6-1 +/− following DMSO or 100nM NAA application for 60 min (Student’s T-test, p > 0.05). (d) The transcript level of the scFv12 coding the recombinant antibody responsible for ABP1 knockdown in WT, ric1-1, ktn1 , SS12S, SS12K, SS12S ric1-1 , SS12K ric1-1 , SS12S ktn1 and SS12K ktn1 after 48h ethanol induction. The transcript level of the scFv12 in SS12S was standardized as “1” (Student’s T-test, p > 0.05). (e) MTs orientation by MAP4-GFP in dark grown hypocotyls of WT, SS12K , ktn1 , SS12K ktn1 (with 24h ethanol induction) following DMSO application for 60 min. Corresponding to Fig. 4b . In all panels, error bars are s.e.m. Scale bars: 5μm (a-b) and 10μm (d).
    Figure Legend Snippet: Auxin-ABP1 controls MT arrangement through the downstream ROP6-RIC1-KTN1 signaling (a) MAP4-GFP visualization of MTs orientation in the root of WT, rop6-1, ric1-1 , SS12S ric1-1 , SS12K ric1-1 following DMSO application for 60 min. Corresponding to quantifications in Fig. 4a . (b-c) MTs reorientation patterns were visualized by MAP4-GFP in the roots of WT and rop6-1 +/− following DMSO or 100nM NAA application for 60 min (Student’s T-test, p > 0.05). (d) The transcript level of the scFv12 coding the recombinant antibody responsible for ABP1 knockdown in WT, ric1-1, ktn1 , SS12S, SS12K, SS12S ric1-1 , SS12K ric1-1 , SS12S ktn1 and SS12K ktn1 after 48h ethanol induction. The transcript level of the scFv12 in SS12S was standardized as “1” (Student’s T-test, p > 0.05). (e) MTs orientation by MAP4-GFP in dark grown hypocotyls of WT, SS12K , ktn1 , SS12K ktn1 (with 24h ethanol induction) following DMSO application for 60 min. Corresponding to Fig. 4b . In all panels, error bars are s.e.m. Scale bars: 5μm (a-b) and 10μm (d).

    Techniques Used: Recombinant

    36) Product Images from "Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin"

    Article Title: Efficacy of glycogen synthase kinase-3β targeting against osteosarcoma via activation of β-catenin

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12781

    Effect of GSK-3β inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.
    Figure Legend Snippet: Effect of GSK-3β inhibition on the expression, phosphorylation, subcellular localization and co-transcriptional activity of β-catenin in osteosarcoma and osteoblast cells ( A ) Western-blotting analysis was used to compare the expression and phosphorylation of β-catenin between cells treated with DMSO and either GSK-3β inhibitor. Expression of β-actin was monitored as a loading control. ( B ) The left panels show representative immunofluorescence microscopic findings of expression and subcellular localization of β-catenin in osteosarcoma (143B, MG-63) and osteoblast (hFOB1.19) cells. The scale bar in each panel indicates 25 μm. The number shown below each panel indicates the percentage of nuclear β-catenin-positive cells among the total number of cells. The bar graphs on the right shows the effects of DMSO and AR-A014418 on the incidence of nuclear localization of β-catenin in osteosarcoma and osteoblast cells. In each assay, the mean percentage of nuclear β-catenin-positive cells in 3 microscopic fields was evaluated with standard deviation. ( C ) Relative co-transcriptional activity of β-catenin was measured by the TOP/FOP flash assay and compared between cells treated with DMSO, AR-A014418 and SB-216763, respectively. (B, C) Asterisks denote a statistically-significant difference between the data after administration of vehicle and GSK-3β inhibitors.

    Techniques Used: Inhibition, Expressing, Activity Assay, Western Blot, Immunofluorescence, Standard Deviation

    Effect of small-molecule GSK-3β inhibitors on the survival of osteosarcoma cells The osteosarcoma cells were treated with DMSO or the indicated concentrations of AR-A014418 or SB-216763 for the designated times. The relative number of viable cells at each time point was measured by the WST-8 assay. Values shown are the means ± SD of six separate experiments.
    Figure Legend Snippet: Effect of small-molecule GSK-3β inhibitors on the survival of osteosarcoma cells The osteosarcoma cells were treated with DMSO or the indicated concentrations of AR-A014418 or SB-216763 for the designated times. The relative number of viable cells at each time point was measured by the WST-8 assay. Values shown are the means ± SD of six separate experiments.

    Techniques Used:

    Effects of GSK-3β inhibitors on the proliferation and apoptosis of osteosarcoma cells and osteoblasts ( A ) The indicated cells were treated with DMSO or 25 μmol/L each of either AR-A014418 or SB-216763 for 72 hours. The relative number of proliferating cells was evaluated by measuring the amount of BrdU incorporation. ( B ) Relative numbers of TUNEL-positive apoptotic cells were scored for the indicated cells at 12 hours after treatment with DMSO or 25 μmol/L each of either GSK-3β inhibitor. (A, B) Values shown are the means ± SD of six separate experiments. Asterisks denote a statistically-significant difference ( p
    Figure Legend Snippet: Effects of GSK-3β inhibitors on the proliferation and apoptosis of osteosarcoma cells and osteoblasts ( A ) The indicated cells were treated with DMSO or 25 μmol/L each of either AR-A014418 or SB-216763 for 72 hours. The relative number of proliferating cells was evaluated by measuring the amount of BrdU incorporation. ( B ) Relative numbers of TUNEL-positive apoptotic cells were scored for the indicated cells at 12 hours after treatment with DMSO or 25 μmol/L each of either GSK-3β inhibitor. (A, B) Values shown are the means ± SD of six separate experiments. Asterisks denote a statistically-significant difference ( p

    Techniques Used: BrdU Incorporation Assay, TUNEL Assay

    ( A, B ) Efficacy of GSK-3β inhibitors on the size and weight of orthotopic 143B tumors (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in Supplementary Figure S7 . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in Supplementary Figure S8 . Higher resolution versions of the lower six panels are shown in Supplementary Figure S9 . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.
    Figure Legend Snippet: ( A, B ) Efficacy of GSK-3β inhibitors on the size and weight of orthotopic 143B tumors (A) Tumor size was measured weekly and the volume calculated. (B) Mean weight of the tumors removed at necropsy. Asterisks denote a statistically-significant difference in tumor volume and weight compared to mice treated with DMSO. The scatter plots corresponding to the data in (A) and (B) are shown in Supplementary Figure S7 . ( C ) Histological and immunohistochemical findings for orthotopic tumors in mice treated with DMSO or with GSK-3β inhibitors. Representative paraffin-embedded sections of tumors were stained with H E or immunostained for β-catenin. Magnified images of the sections (upper three panels) are shown in Supplementary Figure S8 . Higher resolution versions of the lower six panels are shown in Supplementary Figure S9 . Scale bar in each of the upper three panels indicates 5 mm and that of the middle and lower six panels indicates 100 μm.

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    37) Product Images from "Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo"

    Article Title: Assessment of Drug Sensitivity in Hematopoietic Stem and Progenitor Cells from Acute Myelogenous Leukemia and Myelodysplastic Syndrome Ex Vivo

    Journal: Stem Cells Translational Medicine

    doi: 10.5966/sctm.2016-0034

    Survival of AML hematopoietic stem and progenitor cells in MLN4924 or cytarabine versus diluent. Aliquots of bone marrow mononuclear cells from AML patients were treated for 24 hours with increasing concentrations of MLN4924, 100 nM AraC, or diluent (0.1% DMSO). Cells were harvested, stained using the fluorescent antibody panel for identification of live stem and progenitor cells (see Methods), and analyzed by flow cytometry. HSC and progenitor populations were quantified using the gating scheme outlined in Figure 1A . Summary of survival of AML stem and progenitor cells relative to populations in diluent treated samples from seven AML patient BMs. Patient data and cell count in each population are reported in Table 1 and supplemental online Table 1 (de novo AML, n = 5, patients 1 through 5; post‐MDS AML, n = 2, patients 6 and 7). Several populations could not be gated for quantification from patient 4 nor could the MPP from patient 3 ( Table 1 ). CD34 + CD45 dim = all stem and myeloid progenitor populations; stem cells = HSC + MPP; myeloid progenitors = CMP + GMP + MEP. Abbreviations: AraC, cytarabine; AML, acute myelogenous leukemia; CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MDS, myelodysplastic syndrome; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor.
    Figure Legend Snippet: Survival of AML hematopoietic stem and progenitor cells in MLN4924 or cytarabine versus diluent. Aliquots of bone marrow mononuclear cells from AML patients were treated for 24 hours with increasing concentrations of MLN4924, 100 nM AraC, or diluent (0.1% DMSO). Cells were harvested, stained using the fluorescent antibody panel for identification of live stem and progenitor cells (see Methods), and analyzed by flow cytometry. HSC and progenitor populations were quantified using the gating scheme outlined in Figure 1A . Summary of survival of AML stem and progenitor cells relative to populations in diluent treated samples from seven AML patient BMs. Patient data and cell count in each population are reported in Table 1 and supplemental online Table 1 (de novo AML, n = 5, patients 1 through 5; post‐MDS AML, n = 2, patients 6 and 7). Several populations could not be gated for quantification from patient 4 nor could the MPP from patient 3 ( Table 1 ). CD34 + CD45 dim = all stem and myeloid progenitor populations; stem cells = HSC + MPP; myeloid progenitors = CMP + GMP + MEP. Abbreviations: AraC, cytarabine; AML, acute myelogenous leukemia; CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MDS, myelodysplastic syndrome; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Cell Counting

    Quantification of hematopoietic stem and progenitor cell populations in cord blood units and relative survival of populations in MLN4924‐treated versus control samples. (A): Gating scheme for identification of hematopoietic stem and progenitor cell populations from initial CD34 + CD45 dim population. See text for details. (B): Antigen profiles for each population of interest. (C): Survival of CD34 + CD45 dim cord blood populations in MLN4924 or AraC compared with diluent control. Kruskal‐Wallis test, 6 sample groups, p value = NS (.71). Error bars represent ± SEM from four independent cord blood samples. Abbreviations: AraC, cytarabine; CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor; NS, not significant.
    Figure Legend Snippet: Quantification of hematopoietic stem and progenitor cell populations in cord blood units and relative survival of populations in MLN4924‐treated versus control samples. (A): Gating scheme for identification of hematopoietic stem and progenitor cell populations from initial CD34 + CD45 dim population. See text for details. (B): Antigen profiles for each population of interest. (C): Survival of CD34 + CD45 dim cord blood populations in MLN4924 or AraC compared with diluent control. Kruskal‐Wallis test, 6 sample groups, p value = NS (.71). Error bars represent ± SEM from four independent cord blood samples. Abbreviations: AraC, cytarabine; CMP, common myeloid progenitor; DMSO, dimethyl sulfoxide; GMP, granulocyte‐monocyte progenitor; HSC, hematopoietic stem cell; MEP, megakaryocyte‐erythroid progenitor; MPP, multipotent progenitor; NS, not significant.

    Techniques Used:

    MLN4924 induces apoptosis in post‐MDS AML. (A–C): Bone marrow mononuclear cells from a patient with post‐MDS AML were treated with increasing concentrations of MLN4924 versus diluent (0.1% DMSO) for 24 hours. Parallel samples were assayed for subdiploid (apoptotic) cells by propidium iodide staining (A) ; Cullin1, c‐Myc, and Bcl‐2 family member expression by immunoblotting (B) ; and Noxa mRNA levels (expressed relative to diluent control) by quantitative RT‐PCR (C) . Note the increase in c‐Myc and Noxa as previously seen in de novo AML [ 27 ]. Abbreviations: AML, acute myelogenous leukemia; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MDS, myelodysplastic syndrome.
    Figure Legend Snippet: MLN4924 induces apoptosis in post‐MDS AML. (A–C): Bone marrow mononuclear cells from a patient with post‐MDS AML were treated with increasing concentrations of MLN4924 versus diluent (0.1% DMSO) for 24 hours. Parallel samples were assayed for subdiploid (apoptotic) cells by propidium iodide staining (A) ; Cullin1, c‐Myc, and Bcl‐2 family member expression by immunoblotting (B) ; and Noxa mRNA levels (expressed relative to diluent control) by quantitative RT‐PCR (C) . Note the increase in c‐Myc and Noxa as previously seen in de novo AML [ 27 ]. Abbreviations: AML, acute myelogenous leukemia; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MDS, myelodysplastic syndrome.

    Techniques Used: Staining, Expressing, Quantitative RT-PCR

    38) Product Images from "A novel prostate cancer therapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variants"

    Article Title: A novel prostate cancer therapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variants

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgv040

    Effect of ICT on AR- or ARvs-signaling pathway. ( A ) AR transcriptional activity on consensus ARE was assessed in HeLa and LNCaP cells transfected with AR and ARE-Luc reporter genes. Luciferase activity was measured after exposure to either DMSO (Veh)
    Figure Legend Snippet: Effect of ICT on AR- or ARvs-signaling pathway. ( A ) AR transcriptional activity on consensus ARE was assessed in HeLa and LNCaP cells transfected with AR and ARE-Luc reporter genes. Luciferase activity was measured after exposure to either DMSO (Veh)

    Techniques Used: Activity Assay, Transfection, Luciferase

    Effect of ICT on the stability of AR and AR splice variant proteins in PCa cells. ( A ) Androgen-sensitive (LNCaP) and castration-resistant (CWR22Rv1 and C4-2) PCa cells were treated with DMSO (Veh); 10, 30 and 50 µmol/l of ICT and BIC in the presence
    Figure Legend Snippet: Effect of ICT on the stability of AR and AR splice variant proteins in PCa cells. ( A ) Androgen-sensitive (LNCaP) and castration-resistant (CWR22Rv1 and C4-2) PCa cells were treated with DMSO (Veh); 10, 30 and 50 µmol/l of ICT and BIC in the presence

    Techniques Used: Variant Assay

    ICT promotes AR and AR splice variant protein degradation through AhR-mediated ubiquitin proteasome pathway. ( A ) LNCaP and CWR22Rv1 cells were treated with DMSO (Veh), 3MC (5 μmol/l) and ICT for 24h. CYP1A1 mRNA levels were analyzed using quantitative
    Figure Legend Snippet: ICT promotes AR and AR splice variant protein degradation through AhR-mediated ubiquitin proteasome pathway. ( A ) LNCaP and CWR22Rv1 cells were treated with DMSO (Veh), 3MC (5 μmol/l) and ICT for 24h. CYP1A1 mRNA levels were analyzed using quantitative

    Techniques Used: Variant Assay

    ICT inhibits growth and induces apoptosis in human AR-positive PCa cells largely via AhR-mediated AR destabilization. ( A ) PCa cells were treated with DMSO (Veh); 10, 30 and 50 µmol/l of ICT; BIC or MDV, in the presence of DHT (10 nmol/l DHT for
    Figure Legend Snippet: ICT inhibits growth and induces apoptosis in human AR-positive PCa cells largely via AhR-mediated AR destabilization. ( A ) PCa cells were treated with DMSO (Veh); 10, 30 and 50 µmol/l of ICT; BIC or MDV, in the presence of DHT (10 nmol/l DHT for

    Techniques Used:

    39) Product Images from "Transcriptional Regulation of the Human Tumor Suppressor DOK1 by E2F1"

    Article Title: Transcriptional Regulation of the Human Tumor Suppressor DOK1 by E2F1

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01050-12

    DOK1 acts as a mediator of etoposide-induced apoptosis. HEK293 cells stably expressing scramble shRNA, DOK1 -shRNA1, or DOK1 -shRNA2 were treated with 50 μM etoposide or an equivalent volume of DMSO (mock control) for 24 h. (A) DOK1 expression level
    Figure Legend Snippet: DOK1 acts as a mediator of etoposide-induced apoptosis. HEK293 cells stably expressing scramble shRNA, DOK1 -shRNA1, or DOK1 -shRNA2 were treated with 50 μM etoposide or an equivalent volume of DMSO (mock control) for 24 h. (A) DOK1 expression level

    Techniques Used: Stable Transfection, Expressing, shRNA

    Effects of etoposide on DOK1 expression in HNC cells. HEK293 cells and HNC-41 and HNC-124 cells were treated with 25, 50, or 100 μM etoposide or an equivalent volume of DMSO (as a control) for 24 h, and cells were then harvested for analysis.
    Figure Legend Snippet: Effects of etoposide on DOK1 expression in HNC cells. HEK293 cells and HNC-41 and HNC-124 cells were treated with 25, 50, or 100 μM etoposide or an equivalent volume of DMSO (as a control) for 24 h, and cells were then harvested for analysis.

    Techniques Used: Expressing

    Etoposide treatment upregulates DOK1 gene expression via E2F1. HEK293 cells were treated with 25, 50, or 100 μM etoposide or an equivalent volume of DMSO (as a control) for 24 h, and cells were then harvested for analysis. (A) mRNA levels of
    Figure Legend Snippet: Etoposide treatment upregulates DOK1 gene expression via E2F1. HEK293 cells were treated with 25, 50, or 100 μM etoposide or an equivalent volume of DMSO (as a control) for 24 h, and cells were then harvested for analysis. (A) mRNA levels of

    Techniques Used: Expressing

    40) Product Images from "Anti-inflammatory and utero-relaxant effect of α-bisabolol on the pregnant human uterus"

    Article Title: Anti-inflammatory and utero-relaxant effect of α-bisabolol on the pregnant human uterus

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2018.22.4.391

    Effects of α-bisabolol and forskolin on the secretion of TNFα induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml), in the presence of solely α-bisabolol at 560, 860 and 1,200 µM, and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p
    Figure Legend Snippet: Effects of α-bisabolol and forskolin on the secretion of TNFα induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml), in the presence of solely α-bisabolol at 560, 860 and 1,200 µM, and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p

    Techniques Used: Cell Culture

    Effects of α-bisabolol and forskolin on the secretion of IL-1β induced by LPS in the pregnant human myometrium. Explants were cultured both without and with LPS (10 µg/ml) and in the presence of solely α-bisabolol at 560, 860 and 1,200 µM and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS, or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p
    Figure Legend Snippet: Effects of α-bisabolol and forskolin on the secretion of IL-1β induced by LPS in the pregnant human myometrium. Explants were cultured both without and with LPS (10 µg/ml) and in the presence of solely α-bisabolol at 560, 860 and 1,200 µM and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS, or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p

    Techniques Used: Cell Culture

    Effects of α-bisabolol and forskolin on the secretion of IL-10 induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml) and in presence of solely α-bisabolol using the concentration of 860 µM both on its own and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p
    Figure Legend Snippet: Effects of α-bisabolol and forskolin on the secretion of IL-10 induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml) and in presence of solely α-bisabolol using the concentration of 860 µM both on its own and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p

    Techniques Used: Cell Culture, Concentration Assay

    41) Product Images from "Anti-inflammatory and utero-relaxant effect of α-bisabolol on the pregnant human uterus"

    Article Title: Anti-inflammatory and utero-relaxant effect of α-bisabolol on the pregnant human uterus

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2018.22.4.391

    Effects of α-bisabolol and forskolin on the secretion of TNFα induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml), in the presence of solely α-bisabolol at 560, 860 and 1,200 µM, and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p
    Figure Legend Snippet: Effects of α-bisabolol and forskolin on the secretion of TNFα induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml), in the presence of solely α-bisabolol at 560, 860 and 1,200 µM, and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p

    Techniques Used: Cell Culture

    Effects of α-bisabolol and forskolin on the secretion of IL-1β induced by LPS in the pregnant human myometrium. Explants were cultured both without and with LPS (10 µg/ml) and in the presence of solely α-bisabolol at 560, 860 and 1,200 µM and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS, or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p
    Figure Legend Snippet: Effects of α-bisabolol and forskolin on the secretion of IL-1β induced by LPS in the pregnant human myometrium. Explants were cultured both without and with LPS (10 µg/ml) and in the presence of solely α-bisabolol at 560, 860 and 1,200 µM and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS, or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p

    Techniques Used: Cell Culture

    Effects of α-bisabolol and forskolin on the secretion of IL-10 induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml) and in presence of solely α-bisabolol using the concentration of 860 µM both on its own and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p
    Figure Legend Snippet: Effects of α-bisabolol and forskolin on the secretion of IL-10 induced by LPS in the pregnant human myometrium. Explants were cultured both with and without LPS (10 µg/ml) and in presence of solely α-bisabolol using the concentration of 860 µM both on its own and combined with FSK (10 µM) for 24 h. Control experiments, both basal and DMSO, were run without the addition of LPS or any other compound. Each column represents the mean of the results of 4 experiments (n=4) conducted on both compounds tested, while the vertical bars indicate the standard error of the mean (±SEM). † Difference vs . Basal; ‡ Difference vs . LPS (10 µg/ml); § Difference vs . LPS+FSK, p

    Techniques Used: Cell Culture, Concentration Assay

    42) Product Images from "New therapeutic strategy of hinokitiol in haemorrhagic shock‐induced liver injury, et al. New therapeutic strategy of hinokitiol in haemorrhagic shock‐induced liver injury"

    Article Title: New therapeutic strategy of hinokitiol in haemorrhagic shock‐induced liver injury, et al. New therapeutic strategy of hinokitiol in haemorrhagic shock‐induced liver injury

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14070

    Effects of hinokitiol on H1/R6‐induce apoptosis of primary hepatocytes. Primary hepatocytes were incubated with DMSO or hinokitiol (5 or 10 μmol/L) prior to H1/R6. The total RNA was extracted and converted to cDNA . Specific primers were used to detect the gene expression of (A) Bax and (B) Bcl‐2. Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to GAPDH , as an internal control. The data are presented as the means ± SEM (n = 4). * P
    Figure Legend Snippet: Effects of hinokitiol on H1/R6‐induce apoptosis of primary hepatocytes. Primary hepatocytes were incubated with DMSO or hinokitiol (5 or 10 μmol/L) prior to H1/R6. The total RNA was extracted and converted to cDNA . Specific primers were used to detect the gene expression of (A) Bax and (B) Bcl‐2. Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to GAPDH , as an internal control. The data are presented as the means ± SEM (n = 4). * P

    Techniques Used: Incubation, Expressing, Polymerase Chain Reaction, Produced

    Effects of hinokitiol on H1/R6‐induced inflammation in primary hepatocytes. Primary hepatocytes were incubated with DMSO or hinokitiol (5 or 10 μmol/L) prior to H1/R6. Cells were collected, and subcellular extracts were analysed through immunoblotting. Specific antibodies were used to dectect (A) p65, and (B) IκBα expression. The mRNA levels of (C) TNF ‐α and (D) IL ‐6 were determined through real‐time PCR . Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to GAPDH , as an internal control. Data are presented as the means ± SEM (n = 4). * P
    Figure Legend Snippet: Effects of hinokitiol on H1/R6‐induced inflammation in primary hepatocytes. Primary hepatocytes were incubated with DMSO or hinokitiol (5 or 10 μmol/L) prior to H1/R6. Cells were collected, and subcellular extracts were analysed through immunoblotting. Specific antibodies were used to dectect (A) p65, and (B) IκBα expression. The mRNA levels of (C) TNF ‐α and (D) IL ‐6 were determined through real‐time PCR . Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to GAPDH , as an internal control. Data are presented as the means ± SEM (n = 4). * P

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Produced

    Effects of hinokitiol on HS /R‐induced hepatic injury. HS mice were treated with DMSO (solvent control) or 1.7 mg/kg hinokitiol on resuscitation. (A, B and C) Blood were collected at 2, 6 and 24 h after resuscitation ( HS 1/R2, HS 1/R6 and HS 1/R24), respectively and serum ALT , AST , and LDH were determined. (D) After being subjected to HS 1/R6 and HS 1/R24, the mice were sacrificed and photomicrographs of liver sections were stained with haematoxylin and eosin (H E). (E and F) Liver section and tissue were collected, and subcellular extracts were analysed through immunohistochemical staining and immunoblotting. Specific antibodies were used to detect cleaved caspase‐3, PARP , Bax and Bcl‐2 expression. (G and H) Total RNA was extracted from liver tissue, and gene expression of Bax and Bcl‐2 was quantified by real‐time PCR . Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ), as an internal control. Data (A, B, C, G and H) are presented as the means ± SEM (n = 4). * P
    Figure Legend Snippet: Effects of hinokitiol on HS /R‐induced hepatic injury. HS mice were treated with DMSO (solvent control) or 1.7 mg/kg hinokitiol on resuscitation. (A, B and C) Blood were collected at 2, 6 and 24 h after resuscitation ( HS 1/R2, HS 1/R6 and HS 1/R24), respectively and serum ALT , AST , and LDH were determined. (D) After being subjected to HS 1/R6 and HS 1/R24, the mice were sacrificed and photomicrographs of liver sections were stained with haematoxylin and eosin (H E). (E and F) Liver section and tissue were collected, and subcellular extracts were analysed through immunohistochemical staining and immunoblotting. Specific antibodies were used to detect cleaved caspase‐3, PARP , Bax and Bcl‐2 expression. (G and H) Total RNA was extracted from liver tissue, and gene expression of Bax and Bcl‐2 was quantified by real‐time PCR . Each sample was examined in triplicate, and the amounts of the PCR products produced were normalized to glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ), as an internal control. Data (A, B, C, G and H) are presented as the means ± SEM (n = 4). * P

    Techniques Used: Mouse Assay, AST Assay, Staining, Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Produced

    43) Product Images from "The Phytosterol Peniocerol Inhibits Cell Proliferation and Tumor Growth in a Colon Cancer Xenograft Model"

    Article Title: The Phytosterol Peniocerol Inhibits Cell Proliferation and Tumor Growth in a Colon Cancer Xenograft Model

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2019.01341

    Antitumor activity evaluation of peniocerol three times a week for 21 day administration in nu/nu mice. Treatment scheme: Three times a week for 21 days, a total of nine administrations were performed, when the tumor reached an approximate volume of 50 mm 3 , on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. The animals were sacrificed on day 21. (A) Antitumor activity graph. Groups of six nu/nu mice inoculated with 1.5 × 10 6 HCT-116 cells were treated three times a week for 21 days, the doses tested were 30 mg/kg and 15 mg/kg of peniocerol, 2 mg/kg of cisplatin and as a negative control the vehicle was used (sesame oil + 5% DMSO). The size of the tumors was measured three times per week. The bars indicate the standard deviation of the mean * p
    Figure Legend Snippet: Antitumor activity evaluation of peniocerol three times a week for 21 day administration in nu/nu mice. Treatment scheme: Three times a week for 21 days, a total of nine administrations were performed, when the tumor reached an approximate volume of 50 mm 3 , on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. The animals were sacrificed on day 21. (A) Antitumor activity graph. Groups of six nu/nu mice inoculated with 1.5 × 10 6 HCT-116 cells were treated three times a week for 21 days, the doses tested were 30 mg/kg and 15 mg/kg of peniocerol, 2 mg/kg of cisplatin and as a negative control the vehicle was used (sesame oil + 5% DMSO). The size of the tumors was measured three times per week. The bars indicate the standard deviation of the mean * p

    Techniques Used: Activity Assay, Mouse Assay, Negative Control, Standard Deviation

    Toxicity evaluation of peniocerol in nu/nu mice. (A) Treatment scheme: Once a week for 3 weeks, the administrations were performed on days 0, 7, and 14; Three times a week for 3 weeks, administrations were performed, on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. In both schemes, the animals were slaughtered on day 21. (B) Toxicity graphs: Once a week for 3 weeks, doses 30 mg/kg and 15 mg/kg of peniocerol, 4 mg/kg of cisplatin and as a negative control (sesame oil + DMSO 5%); three times a week for 3 weeks, doses 30 mg/kg and 15 mg/kg of peniocerol, 2 mg/kg of cisplatin and as a negative control the vehicle (sesame oil + 5% DMSO). The weight of the mice was monitored three times a week for 21 days. The results shown are the mean ± standard deviation of the monitoring of three mice. The significant difference **** p
    Figure Legend Snippet: Toxicity evaluation of peniocerol in nu/nu mice. (A) Treatment scheme: Once a week for 3 weeks, the administrations were performed on days 0, 7, and 14; Three times a week for 3 weeks, administrations were performed, on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. In both schemes, the animals were slaughtered on day 21. (B) Toxicity graphs: Once a week for 3 weeks, doses 30 mg/kg and 15 mg/kg of peniocerol, 4 mg/kg of cisplatin and as a negative control (sesame oil + DMSO 5%); three times a week for 3 weeks, doses 30 mg/kg and 15 mg/kg of peniocerol, 2 mg/kg of cisplatin and as a negative control the vehicle (sesame oil + 5% DMSO). The weight of the mice was monitored three times a week for 21 days. The results shown are the mean ± standard deviation of the monitoring of three mice. The significant difference **** p

    Techniques Used: Mouse Assay, Negative Control, Standard Deviation

    Antitumor activity evaluation of peniocerol once a week for 21 days administration in nu/nu mice. Treatment scheme: Once a week for 21 days, treatment started when the tumors reached a volume of approximate 50 mm 3 , on days 0, 7, and 14. The animals were sacrificed on day 21. (A) Antitumor activity graphs. Groups of six nu/nu mice inoculated with 1.5 × 10 6 HCT-116 cells were treated once a week for 21 days with 30 mg/kg and 15 mg/kg of peniocerol, 4 mg/kg of cisplatin and the vehicle (sesame oil + 5% DMSO), on days 0, 7, and 14 (black arrows). The tumors were measured three times per week. The bars indicate the SD of the mean ** p
    Figure Legend Snippet: Antitumor activity evaluation of peniocerol once a week for 21 days administration in nu/nu mice. Treatment scheme: Once a week for 21 days, treatment started when the tumors reached a volume of approximate 50 mm 3 , on days 0, 7, and 14. The animals were sacrificed on day 21. (A) Antitumor activity graphs. Groups of six nu/nu mice inoculated with 1.5 × 10 6 HCT-116 cells were treated once a week for 21 days with 30 mg/kg and 15 mg/kg of peniocerol, 4 mg/kg of cisplatin and the vehicle (sesame oil + 5% DMSO), on days 0, 7, and 14 (black arrows). The tumors were measured three times per week. The bars indicate the SD of the mean ** p

    Techniques Used: Activity Assay, Mouse Assay

    44) Product Images from "Antineoplastic effects of histone deacetylase inhibitors in neuroendocrine cancer cells are mediated through transcriptional regulation of Notch1 by activator protein 1"

    Article Title: Antineoplastic effects of histone deacetylase inhibitors in neuroendocrine cancer cells are mediated through transcriptional regulation of Notch1 by activator protein 1

    Journal: Cancer Medicine

    doi: 10.1002/cam4.1151

    Effect of AP ‐1/c‐Jun knockdown and overexpression on c‐Jun and Notch1 mRNA transcription. (A) H727 and MZ cells were transiently transfected with either c‐Jun or nonspecific ( NS ) control si RNA . Twenty‐four hours later, the cells were then treated with TDP ‐A (10 nmol/L in H727, 5 nmol/L in MZ ) and VPA (5 mmol/L in H727, 3 mmol/L in MZ ) close to their IC 50 for 24 h. The data were plotted relative to the mRNA expression levels measured by RT ‐ PCR in cells treated with NS control si RNA . The mRNA expression of cells treated with DMSO only is also shown. Statistical significance was noted with (* P
    Figure Legend Snippet: Effect of AP ‐1/c‐Jun knockdown and overexpression on c‐Jun and Notch1 mRNA transcription. (A) H727 and MZ cells were transiently transfected with either c‐Jun or nonspecific ( NS ) control si RNA . Twenty‐four hours later, the cells were then treated with TDP ‐A (10 nmol/L in H727, 5 nmol/L in MZ ) and VPA (5 mmol/L in H727, 3 mmol/L in MZ ) close to their IC 50 for 24 h. The data were plotted relative to the mRNA expression levels measured by RT ‐ PCR in cells treated with NS control si RNA . The mRNA expression of cells treated with DMSO only is also shown. Statistical significance was noted with (* P

    Techniques Used: Over Expression, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Notch1 mRNA expression after HDAC inhibitor treatment in neuroendocrine ( NE ) cell lines. Three NE cell lines BON , H727, and MZ ‐ CRC ‐1 were treated with HDAC inhibitors TDP ‐A and VPA in increasing concentrations close to their IC 50 . The data were plotted relative to the mRNA expression levels in cells treated with DMSO vehicle control. All values were presented as mean relative fold ± SEM (* P
    Figure Legend Snippet: Notch1 mRNA expression after HDAC inhibitor treatment in neuroendocrine ( NE ) cell lines. Three NE cell lines BON , H727, and MZ ‐ CRC ‐1 were treated with HDAC inhibitors TDP ‐A and VPA in increasing concentrations close to their IC 50 . The data were plotted relative to the mRNA expression levels in cells treated with DMSO vehicle control. All values were presented as mean relative fold ± SEM (* P

    Techniques Used: Expressing

    Deletion analysis of the human Notch1 promoter. BON (A) and H727 (B) cells were treated with TDP ‐A and VPA after the cells were transfected with fragments of the Notch1 promoter region joined to a luciferase reporter. A schematic of the individual constructs is shown on the left. The promoter activities of the different deletion fragments were normalized to the relative light unit in cells treated with DMSO vehicle control for each individual constructs. All values were presented as mean relative fold ± SEM . (C) The active DNA sequence of the Notch1 promoter fragment (−80/−1) with the potential transcription factor binding site. The proposed noncanonical AP ‐1 binding site is bolded and underlined. Sequences that are different from the canonical AP ‐1 binding site are denoted with *.
    Figure Legend Snippet: Deletion analysis of the human Notch1 promoter. BON (A) and H727 (B) cells were treated with TDP ‐A and VPA after the cells were transfected with fragments of the Notch1 promoter region joined to a luciferase reporter. A schematic of the individual constructs is shown on the left. The promoter activities of the different deletion fragments were normalized to the relative light unit in cells treated with DMSO vehicle control for each individual constructs. All values were presented as mean relative fold ± SEM . (C) The active DNA sequence of the Notch1 promoter fragment (−80/−1) with the potential transcription factor binding site. The proposed noncanonical AP ‐1 binding site is bolded and underlined. Sequences that are different from the canonical AP ‐1 binding site are denoted with *.

    Techniques Used: Transfection, Luciferase, Construct, Sequencing, Binding Assay

    45) Product Images from "Chemotherapeutic Potential of 17-AAG against Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis"

    Article Title: Chemotherapeutic Potential of 17-AAG against Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003275

    Treatment with 17-AAG reduces intracellular L. (V.) braziliensis viability. L. (V.) braziliensis - infected macrophages were treated for 24 h with increasing concentrations of 17-AAG or with vehicle alone (DMSO). The number of viable parasites was evaluated by further culture (5 days) in Schneider medium, free of 17-AAG (A). Infected macrophages were treated with 220 nM (IC 50 ) of 17-AAG for 24–72 h. The number of viable parasites was evaluated by further culture for five days in Schneider medium, free of 17-AAG (B). Data, shown as mean ±SEM, are from one of two independent repeats (** p
    Figure Legend Snippet: Treatment with 17-AAG reduces intracellular L. (V.) braziliensis viability. L. (V.) braziliensis - infected macrophages were treated for 24 h with increasing concentrations of 17-AAG or with vehicle alone (DMSO). The number of viable parasites was evaluated by further culture (5 days) in Schneider medium, free of 17-AAG (A). Infected macrophages were treated with 220 nM (IC 50 ) of 17-AAG for 24–72 h. The number of viable parasites was evaluated by further culture for five days in Schneider medium, free of 17-AAG (B). Data, shown as mean ±SEM, are from one of two independent repeats (** p

    Techniques Used: Infection

    In vivo treatment with 17-AAG decreases L. (V.) braziliensis infection. Mice were infected with L. (V.) braziliensis and four weeks later, mice were treated with 17-AAG, 3x a week for 3 weeks (boxed area) or with vehicle (DMSO) alone. (A) The course of lesion development was monitored weekly. (B) Disease burden [shown as Area Under the Curves (AUC) depicted in (A)] in mice treated with 17-AAG or injected with DMSO. Parasite load was determined at the infection site (C) and at the dLN (D), 6 weeks later, by limiting dilution analysis. Data, shown as mean ±SEM, are from one of two independent repeats, each performed with 10 mice in each group (** p
    Figure Legend Snippet: In vivo treatment with 17-AAG decreases L. (V.) braziliensis infection. Mice were infected with L. (V.) braziliensis and four weeks later, mice were treated with 17-AAG, 3x a week for 3 weeks (boxed area) or with vehicle (DMSO) alone. (A) The course of lesion development was monitored weekly. (B) Disease burden [shown as Area Under the Curves (AUC) depicted in (A)] in mice treated with 17-AAG or injected with DMSO. Parasite load was determined at the infection site (C) and at the dLN (D), 6 weeks later, by limiting dilution analysis. Data, shown as mean ±SEM, are from one of two independent repeats, each performed with 10 mice in each group (** p

    Techniques Used: In Vivo, Infection, Mouse Assay, Injection

    Treatment with 17-AAG controls L. (V.) braziliensis replication inside macrophages. L. (V.) braziliensis- infected macrophages were treated with increasing concentrations of 17-AAG or with vehicle alone (DMSO). After 12–72 h, glass coverslips were stained with H E and assessed for the percentage of infected macrophages (A) and the number of amastigotes per 100 macrophages (B) by light microscopy. Data, shown as mean ±SEM, are shown as the percentage of DMSO -treated cultures, from one of three independent repeats (* p
    Figure Legend Snippet: Treatment with 17-AAG controls L. (V.) braziliensis replication inside macrophages. L. (V.) braziliensis- infected macrophages were treated with increasing concentrations of 17-AAG or with vehicle alone (DMSO). After 12–72 h, glass coverslips were stained with H E and assessed for the percentage of infected macrophages (A) and the number of amastigotes per 100 macrophages (B) by light microscopy. Data, shown as mean ±SEM, are shown as the percentage of DMSO -treated cultures, from one of three independent repeats (* p

    Techniques Used: Infection, Staining, Light Microscopy

    17-AAG induces killing of Leishmania (V.) braziliensis promastigotes in a dose-dependent and irreversible manner. L. (V.) braziliensis promastigotes were exposed to increasing concentrations of 17-AAG, to vehicle alone (DMSO) or were left unexposed (Lb) for 48 h. (A) The number of viable parasites was evaluated by direct counting. L.(V) braziliensis promastigotes were treated with 65 nM (IC 50 ) of 17-AAG for 24 h (B) and (C) 48 h. After washing, promastigotes were cultured for additional 48 h and the number of viable parasites was evaluated. Data, shown as mean ±SEM, are from one of two independent repeats (** p
    Figure Legend Snippet: 17-AAG induces killing of Leishmania (V.) braziliensis promastigotes in a dose-dependent and irreversible manner. L. (V.) braziliensis promastigotes were exposed to increasing concentrations of 17-AAG, to vehicle alone (DMSO) or were left unexposed (Lb) for 48 h. (A) The number of viable parasites was evaluated by direct counting. L.(V) braziliensis promastigotes were treated with 65 nM (IC 50 ) of 17-AAG for 24 h (B) and (C) 48 h. After washing, promastigotes were cultured for additional 48 h and the number of viable parasites was evaluated. Data, shown as mean ±SEM, are from one of two independent repeats (** p

    Techniques Used: Cell Culture

    46) Product Images from "Effects of Resveratrol on Crosstalk between Canonical Β-Catenin/Wnt and FOXO Pathways in Coronary Artery Disease Patients with Metabolic Syndrome: A Case Control Study"

    Article Title: Effects of Resveratrol on Crosstalk between Canonical Β-Catenin/Wnt and FOXO Pathways in Coronary Artery Disease Patients with Metabolic Syndrome: A Case Control Study

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    doi:

    Pearson correlation coefficients of total β-catenin protein and MnSOD enzyme activity. a) In blank group; total β-catenin protein and MnSOD enzyme activity were significantly correlated in healthy subjects, while they were not significantly correlated in CAD patients. b) After treatment with resveratrol (RES), total β-catenin protein and MnSOD enzyme activity showed a significant positive correlation in healthy subjects, while no significant correlation was observed in CAD patients. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%)
    Figure Legend Snippet: Pearson correlation coefficients of total β-catenin protein and MnSOD enzyme activity. a) In blank group; total β-catenin protein and MnSOD enzyme activity were significantly correlated in healthy subjects, while they were not significantly correlated in CAD patients. b) After treatment with resveratrol (RES), total β-catenin protein and MnSOD enzyme activity showed a significant positive correlation in healthy subjects, while no significant correlation was observed in CAD patients. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%)

    Techniques Used: Activity Assay, Concentration Assay

    Effects of resveratrol (RES) on β-catenin, MnSOD, and PPAR-δ gene expression by real-time PCR. PBMCs were treated with 50 µM RES for 12 h incubation. a) A non-significant change in MnSOD mRNA was observed in healthy subjects after treatment with RES as compared to blank. Treatment with RES could significantly increase the mRNA level of MnSOD in CAD patients compared to blank. b) No significant differences were observed for β-catenin mRNA in healthy subjects and CAD patients after treatment with RES compared to their blank. c) No significant differences in PPAR-δ mRNA were observed for both healthy subjects and CAD patients after treatment with RES compared to their blanks. d) Between-group analysis showed non-significant higher levels of MnSOD, β-catenin and PPAR-δ in healthy subjects than CAD patients in both blank and RES treatment. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%). All gene expression tests were performed in triplicate in each experiment, and the mean of duplicates were used for statistical analyses. Data are expressed as means ± SEM.
    Figure Legend Snippet: Effects of resveratrol (RES) on β-catenin, MnSOD, and PPAR-δ gene expression by real-time PCR. PBMCs were treated with 50 µM RES for 12 h incubation. a) A non-significant change in MnSOD mRNA was observed in healthy subjects after treatment with RES as compared to blank. Treatment with RES could significantly increase the mRNA level of MnSOD in CAD patients compared to blank. b) No significant differences were observed for β-catenin mRNA in healthy subjects and CAD patients after treatment with RES compared to their blank. c) No significant differences in PPAR-δ mRNA were observed for both healthy subjects and CAD patients after treatment with RES compared to their blanks. d) Between-group analysis showed non-significant higher levels of MnSOD, β-catenin and PPAR-δ in healthy subjects than CAD patients in both blank and RES treatment. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%). All gene expression tests were performed in triplicate in each experiment, and the mean of duplicates were used for statistical analyses. Data are expressed as means ± SEM.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay

    Pearson correlation coefficients of mRNA expression of genes (β-catenin, MnSOD and PPAR-δ). a) In blank groups; β-catenin and MnSOD mRNA expressions were significantly correlated in healthy subjects, while they were not significantly correlated in CAD patients. b) β-catenin and PPAR-δ mRNA showed significant positive correlations in both healthy subjects and CAD patients in blank groups. c) After treatment with resveratrol (RES) a significant positive correlation was found between β-catenin and MnSOD mRNA expressions in healthy subjects, but it was not observed in CAD patients. d) Also β-catenin and PPAR-δ mRNA showed significant positive correlations in both healthy subjects and CAD patients after treatment with RES. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%).
    Figure Legend Snippet: Pearson correlation coefficients of mRNA expression of genes (β-catenin, MnSOD and PPAR-δ). a) In blank groups; β-catenin and MnSOD mRNA expressions were significantly correlated in healthy subjects, while they were not significantly correlated in CAD patients. b) β-catenin and PPAR-δ mRNA showed significant positive correlations in both healthy subjects and CAD patients in blank groups. c) After treatment with resveratrol (RES) a significant positive correlation was found between β-catenin and MnSOD mRNA expressions in healthy subjects, but it was not observed in CAD patients. d) Also β-catenin and PPAR-δ mRNA showed significant positive correlations in both healthy subjects and CAD patients after treatment with RES. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%).

    Techniques Used: Expressing, Concentration Assay

    Effects of resveratrol (RES) on MnSOD enzyme activity and total β-catenin protein. PBMCs were treated with 50 µM RES for 12 h incubation. a) MnSOD enzyme activity was non-significantly increased in healthy subjects after treatment with RES. Also it was significantly increased in CAD patients after treatment with RES in comparison with blank. b) After treatment with RES a non-significant increasing trend was found for total β-catenin protein of healthy subjects compared to blank. Total β-catenin protein of CAD patients did not significantly increase after treatment with RES. c) Between-group differences showed higher MnSOD enzyme activitiy and total β-catenin protein levels for healthy subjects in comparison with CAD patients. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%). All experiments were performed in duplicate, and the mean of duplicates were used for statistical analyses. Data are expressed as means ± SEM
    Figure Legend Snippet: Effects of resveratrol (RES) on MnSOD enzyme activity and total β-catenin protein. PBMCs were treated with 50 µM RES for 12 h incubation. a) MnSOD enzyme activity was non-significantly increased in healthy subjects after treatment with RES. Also it was significantly increased in CAD patients after treatment with RES in comparison with blank. b) After treatment with RES a non-significant increasing trend was found for total β-catenin protein of healthy subjects compared to blank. Total β-catenin protein of CAD patients did not significantly increase after treatment with RES. c) Between-group differences showed higher MnSOD enzyme activitiy and total β-catenin protein levels for healthy subjects in comparison with CAD patients. RES was dissolved in DMSO, and blank groups (untreated cells) were containing only DMSO. In both blank and RES treated cells, DMSO was present at equal concentration (0.025%). All experiments were performed in duplicate, and the mean of duplicates were used for statistical analyses. Data are expressed as means ± SEM

    Techniques Used: Activity Assay, Incubation, Concentration Assay

    47) Product Images from "CD45RA Distinguishes CD4+CD25+CD127−/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression"

    Article Title: CD45RA Distinguishes CD4+CD25+CD127−/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression

    Journal: Transplantation

    doi: 10.1097/TP.0000000000001278

    Expansion capacity of Treg cell populations. A, Fold expansion in each Treg cell population was measured after in vitro expansion in the presence of DMSO or TAC (Wilcoxon-matched pair test), comparing day 7 with day 14 cell numbers. B, Ratio of proliferation between each Treg cell population incubated with DMSO or TAC (Mann-Whitney U test). n = 12 donors are shown (* P
    Figure Legend Snippet: Expansion capacity of Treg cell populations. A, Fold expansion in each Treg cell population was measured after in vitro expansion in the presence of DMSO or TAC (Wilcoxon-matched pair test), comparing day 7 with day 14 cell numbers. B, Ratio of proliferation between each Treg cell population incubated with DMSO or TAC (Mann-Whitney U test). n = 12 donors are shown (* P

    Techniques Used: In Vitro, Incubation, MANN-WHITNEY

    Foxp3 expression and demethylation status of the TSDR region of expanded Treg cell subsets. A, Percentages of TSDR demethylation (n = 8 donors); B, cells expressing Foxp3 (n = 8 donors); C, cells expressing CD27 (n = 7 donors); and D, ratio of cells that express FOXP3 and with a demethylated TSDR were measured in each Treg cell population before and after in vitro expansion in the presence of DMSO or TAC. For statistical analysis, Wilcoxon-matched pair test was performed comparing, CD27 expression, Foxp3 expression, TSDR demethylation or FOXP3 + /demethylated TSDR ratio between the respective Treg population after being exposing to DMSO or TAC, and the Mann-Whitney U test was used to compare between cell populations (* P
    Figure Legend Snippet: Foxp3 expression and demethylation status of the TSDR region of expanded Treg cell subsets. A, Percentages of TSDR demethylation (n = 8 donors); B, cells expressing Foxp3 (n = 8 donors); C, cells expressing CD27 (n = 7 donors); and D, ratio of cells that express FOXP3 and with a demethylated TSDR were measured in each Treg cell population before and after in vitro expansion in the presence of DMSO or TAC. For statistical analysis, Wilcoxon-matched pair test was performed comparing, CD27 expression, Foxp3 expression, TSDR demethylation or FOXP3 + /demethylated TSDR ratio between the respective Treg population after being exposing to DMSO or TAC, and the Mann-Whitney U test was used to compare between cell populations (* P

    Techniques Used: Expressing, In Vitro, MANN-WHITNEY

    In vitro suppressive capacity of Treg cell populations. A, Suppressive capacity of CD25 int mTreg and naive Treg (n = 6 donors), and total Treg (n = 5 donors) after in vitro expansion in DMSO control media. B, Suppressive capacity of CD25 int mTreg and naive Treg (n = 6 and n = 5 donors, respectively), and total Treg cells (n = 5 donors) after in vitro expansion in the presence of TAC. Percent suppression of CD4 + effector cell proliferation based on division index of PBMCs compared with the proliferation of PBMCs cultured in the absence of suppressor cells. Each data point is the average of 3 replicate wells in the suppression assay of each donor. Mann-Whitney U test used to compare suppression between Treg subpopulations (* P
    Figure Legend Snippet: In vitro suppressive capacity of Treg cell populations. A, Suppressive capacity of CD25 int mTreg and naive Treg (n = 6 donors), and total Treg (n = 5 donors) after in vitro expansion in DMSO control media. B, Suppressive capacity of CD25 int mTreg and naive Treg (n = 6 and n = 5 donors, respectively), and total Treg cells (n = 5 donors) after in vitro expansion in the presence of TAC. Percent suppression of CD4 + effector cell proliferation based on division index of PBMCs compared with the proliferation of PBMCs cultured in the absence of suppressor cells. Each data point is the average of 3 replicate wells in the suppression assay of each donor. Mann-Whitney U test used to compare suppression between Treg subpopulations (* P

    Techniques Used: In Vitro, Cell Culture, Suppression Assay, MANN-WHITNEY

    Cytokine production of Treg cell subpopulations. IL-10, IFN-γ, and IL-17A production by CD25 int mTreg and naive Treg cells after in vitro expansion in the presence of TAC or DMSO was determined in triplicate wells in 2 different donors (A and B). Bars represent means with SEM (*** P
    Figure Legend Snippet: Cytokine production of Treg cell subpopulations. IL-10, IFN-γ, and IL-17A production by CD25 int mTreg and naive Treg cells after in vitro expansion in the presence of TAC or DMSO was determined in triplicate wells in 2 different donors (A and B). Bars represent means with SEM (*** P

    Techniques Used: In Vitro

    48) Product Images from ""

    Article Title:

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.111.077446

    Effect of the IQ - 1 on human PBMC cytokine production. PBMCs were pretreated with 20 μM IQ - 1 or DMSO for 30 min, followed by addition of 200 ng/ml LPS or buffer for 24 h. Production of cytokines in the supernatants was evaluated using a semiquantity
    Figure Legend Snippet: Effect of the IQ - 1 on human PBMC cytokine production. PBMCs were pretreated with 20 μM IQ - 1 or DMSO for 30 min, followed by addition of 200 ng/ml LPS or buffer for 24 h. Production of cytokines in the supernatants was evaluated using a semiquantity

    Techniques Used:

    Structure of and activity of IQ - 1 . THP1-Blue monocytes (2 × 10 5 cells/well) were pretreated with the indicated concentrations of IQ - 1 or DMSO for 30 min, followed by addition of 200 ng/ml LPS or buffer for 24 h. Secreted alkaline phosphatase activity
    Figure Legend Snippet: Structure of and activity of IQ - 1 . THP1-Blue monocytes (2 × 10 5 cells/well) were pretreated with the indicated concentrations of IQ - 1 or DMSO for 30 min, followed by addition of 200 ng/ml LPS or buffer for 24 h. Secreted alkaline phosphatase activity

    Techniques Used: Activity Assay

    Effect of the IQ - 1 and IQ - 3 on TNF-α production by human PBMCs. PBMCs were pretreated with the indicated compounds or DMSO for 30 min, followed by addition of 200 ng/ml LPS or buffer for 24 h. Production of TNF-α in the supernatants was
    Figure Legend Snippet: Effect of the IQ - 1 and IQ - 3 on TNF-α production by human PBMCs. PBMCs were pretreated with the indicated compounds or DMSO for 30 min, followed by addition of 200 ng/ml LPS or buffer for 24 h. Production of TNF-α in the supernatants was

    Techniques Used:

    49) Product Images from "Enoxacin inhibits growth of prostate cancer cells and effectively restores microRNA processing"

    Article Title: Enoxacin inhibits growth of prostate cancer cells and effectively restores microRNA processing

    Journal: Epigenetics

    doi: 10.4161/epi.24519

    Figure 5. Effect of enoxacin on the invasive potential of PCa cells. Relative invasion was evaluated by Oris™ Cell Invasion Assay in DU145 cell line after exposure to enoxacin 40 μg/mL or DMSO (vehicle) at day eight. Data are presented
    Figure Legend Snippet: Figure 5. Effect of enoxacin on the invasive potential of PCa cells. Relative invasion was evaluated by Oris™ Cell Invasion Assay in DU145 cell line after exposure to enoxacin 40 μg/mL or DMSO (vehicle) at day eight. Data are presented

    Techniques Used: Invasion Assay

    Figure 4. Effect of enoxacin on PCa cell cycle distribution. Cell cycle distribution was assessed by flow cytometry in LNCaP, 22Rv1, VCaP, DU145 and PC-3 cell lines after exposure to enoxacin 40 μg/mL or DMSO (vehicle) at day five. The
    Figure Legend Snippet: Figure 4. Effect of enoxacin on PCa cell cycle distribution. Cell cycle distribution was assessed by flow cytometry in LNCaP, 22Rv1, VCaP, DU145 and PC-3 cell lines after exposure to enoxacin 40 μg/mL or DMSO (vehicle) at day five. The

    Techniques Used: Flow Cytometry, Cytometry

    Figure 2. ( A ) Effect of enoxacin on the expression of TRBP and DICER. Protein expression of TRBP and DICER was analyzed by Western Blot in LNCaP, 22Rv1, VCaP, DU145 and PC-3 cell lines after exposure to enoxacin 40 μg/mL or DMSO (vehicle)
    Figure Legend Snippet: Figure 2. ( A ) Effect of enoxacin on the expression of TRBP and DICER. Protein expression of TRBP and DICER was analyzed by Western Blot in LNCaP, 22Rv1, VCaP, DU145 and PC-3 cell lines after exposure to enoxacin 40 μg/mL or DMSO (vehicle)

    Techniques Used: Expressing, Western Blot

    Figure 3. Effect of enoxacin on PCa cell apoptosis. ( A ) Apoptosis was analyzed by APOPercentage assay at days two and five in LNCaP, 22Rv1, VCaP, DU145 and PC-3 cell lines after exposure to enoxacin 40 μg/mL or DMSO (vehicle) at days two
    Figure Legend Snippet: Figure 3. Effect of enoxacin on PCa cell apoptosis. ( A ) Apoptosis was analyzed by APOPercentage assay at days two and five in LNCaP, 22Rv1, VCaP, DU145 and PC-3 cell lines after exposure to enoxacin 40 μg/mL or DMSO (vehicle) at days two

    Techniques Used:

    50) Product Images from "Flavonolignan 2,3-dehydrosilydianin activates Nrf2 and upregulates NAD(P)H:quinone oxidoreductase 1 in Hepa1c1c7 cells"

    Article Title: Flavonolignan 2,3-dehydrosilydianin activates Nrf2 and upregulates NAD(P)H:quinone oxidoreductase 1 in Hepa1c1c7 cells

    Journal: Fitoterapia

    doi: 10.1016/j.fitote.2017.04.012

    Effect of 2,3-dehydrosilydianin (DHSD) on ARE-driven gene expression in AREc32 reporter cells. Cells were treated for 24 h with 0.1% DMSO (control), 2.5 μM sulforaphane (SFN; positive control) or with 12.5–50 μM DHSD. After treatment, luciferase reporter activity was determined luminometrically and normalized to protein content. Data are means ± standard deviation of three experiments. * p
    Figure Legend Snippet: Effect of 2,3-dehydrosilydianin (DHSD) on ARE-driven gene expression in AREc32 reporter cells. Cells were treated for 24 h with 0.1% DMSO (control), 2.5 μM sulforaphane (SFN; positive control) or with 12.5–50 μM DHSD. After treatment, luciferase reporter activity was determined luminometrically and normalized to protein content. Data are means ± standard deviation of three experiments. * p

    Techniques Used: Expressing, Positive Control, Luciferase, Activity Assay, Standard Deviation

    Effect of tested flavonolignans on NQO1 activity in Hepa1c1c7 cells. Cells were treated for 48 h with 0.1% DMSO (control), 2.5 μM sulforaphane (SFN; positive control) or with indicated concentrations of silybin (SB), 2,3-dehydrosilybin (DHSB), silychristin (SC), 2,3-dehydrosilychristin (DHSC), silydianin (SD) or 2,3-dehydrosilydianin (DHSD). After treatment, the activity of NQO1 was determined using the NQO1 assay. Data are means ± standard deviation of three experiments. * p
    Figure Legend Snippet: Effect of tested flavonolignans on NQO1 activity in Hepa1c1c7 cells. Cells were treated for 48 h with 0.1% DMSO (control), 2.5 μM sulforaphane (SFN; positive control) or with indicated concentrations of silybin (SB), 2,3-dehydrosilybin (DHSB), silychristin (SC), 2,3-dehydrosilychristin (DHSC), silydianin (SD) or 2,3-dehydrosilydianin (DHSD). After treatment, the activity of NQO1 was determined using the NQO1 assay. Data are means ± standard deviation of three experiments. * p

    Techniques Used: Activity Assay, Positive Control, Standard Deviation

    Effect of 2,3-dehydrosilydianin (DHSD) on the levels of Nrf2 and Nrf2-regulated proteins in Hepa1c1c7 cells. Cells were treated for 3 or 24 h (as indicated) with 0.1% DMSO (control), 5 μM sulforaphane (SFN; positive control) or 50 μM DHSD. After treatment, the protein levels of Nrf2, NQO1, HMOX1, GCLC, GCLM, and actin in the whole cell lysates (30 μg/lane) were analyzed in duplicate by Western blotting. Representative Western blots are shown. Relative band intensities were determined densitometrically and normalized to actin. Data expressed as folds of control are means of three experiments.
    Figure Legend Snippet: Effect of 2,3-dehydrosilydianin (DHSD) on the levels of Nrf2 and Nrf2-regulated proteins in Hepa1c1c7 cells. Cells were treated for 3 or 24 h (as indicated) with 0.1% DMSO (control), 5 μM sulforaphane (SFN; positive control) or 50 μM DHSD. After treatment, the protein levels of Nrf2, NQO1, HMOX1, GCLC, GCLM, and actin in the whole cell lysates (30 μg/lane) were analyzed in duplicate by Western blotting. Representative Western blots are shown. Relative band intensities were determined densitometrically and normalized to actin. Data expressed as folds of control are means of three experiments.

    Techniques Used: Positive Control, Western Blot

    51) Product Images from "Identification of Chemosensitivity Nodes for Vinblastine through Small Interfering RNA High-Throughput Screens S⃞"

    Article Title: Identification of Chemosensitivity Nodes for Vinblastine through Small Interfering RNA High-Throughput Screens S⃞

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.111.184879

    ABT-263-dependent caspase-3/7 induction of intrinsic apoptosis. A, T98G cells were treated with decreasing concentrations of ABT-263 (3.13–0.31 μM), and the levels of cytochrome c in the mitochondrial fractions were visualized by Western blot analyses. ERAB, a mitochondria-specific marker, was used as a loading control. B, quantification of cytochrome c localization to the mitochondrial fraction relative to ERAB expression levels. All of the values were normalized to the DMSO control. C, levels of cytochrome c in the mitochondrial fractions in the presence of DMSO, 1.56 μM ABT-263, 12.5 nM VBL, and ABT-263 plus VBL. D, quantification of localization of cytochrome c to the mitochondrial fraction in the presence of ABT-263, VBL, or ABT-263 plus VBL. Expression levels were normalized to ERAB expression levels as well as the DMSO control. Blots are representative of three independent experiments. Bars, S.E.M.
    Figure Legend Snippet: ABT-263-dependent caspase-3/7 induction of intrinsic apoptosis. A, T98G cells were treated with decreasing concentrations of ABT-263 (3.13–0.31 μM), and the levels of cytochrome c in the mitochondrial fractions were visualized by Western blot analyses. ERAB, a mitochondria-specific marker, was used as a loading control. B, quantification of cytochrome c localization to the mitochondrial fraction relative to ERAB expression levels. All of the values were normalized to the DMSO control. C, levels of cytochrome c in the mitochondrial fractions in the presence of DMSO, 1.56 μM ABT-263, 12.5 nM VBL, and ABT-263 plus VBL. D, quantification of localization of cytochrome c to the mitochondrial fraction in the presence of ABT-263, VBL, or ABT-263 plus VBL. Expression levels were normalized to ERAB expression levels as well as the DMSO control. Blots are representative of three independent experiments. Bars, S.E.M.

    Techniques Used: Western Blot, Marker, Expressing

    BCL-xL siRNA sensitization of T98G cells to VBL. Three siRNAs targeting BCL-xL were pooled and tested as sensitizers of T98G cells to VBL. A–E, cells were transfected with negative control siRNAs (SCR), positive control siRNAs (cell death), and BCL-xL siRNAs at decreasing concentrations: (A) 10 nM, (B) 5 nM, (C) 2.5 nM, (D) 1.25 nM, and (E) 0.63 nM. Forty-eight hours post-transfection, the cells were treated with increasing concentrations of VBL ranging from 25 pM to 25 nM. After 48 h, cell viability was determined by CellTiter-Blue fluorometric assay as described under Materials and Methods . All of the data points were normalized to a SCR/DMSO control to allow for well to-well comparisons. The effect of different concentrations of VBL on cell viability in cells treated with SCR or BCL-xL siRNA was determined. ○, T98G cells treated with VBL in combination with siRNA negative control (SCR). □, T98G cells treated with VBL in combination with pooled BCL-xL siRNAs. ▴, T98G cells transfected with cell death negative control siRNA. F, SCR and BCL-xL siRNAs were nontoxic to cells. Each value is the mean of four independent experiments. Bars represent S.E.M. *, p ≤ 0.05.
    Figure Legend Snippet: BCL-xL siRNA sensitization of T98G cells to VBL. Three siRNAs targeting BCL-xL were pooled and tested as sensitizers of T98G cells to VBL. A–E, cells were transfected with negative control siRNAs (SCR), positive control siRNAs (cell death), and BCL-xL siRNAs at decreasing concentrations: (A) 10 nM, (B) 5 nM, (C) 2.5 nM, (D) 1.25 nM, and (E) 0.63 nM. Forty-eight hours post-transfection, the cells were treated with increasing concentrations of VBL ranging from 25 pM to 25 nM. After 48 h, cell viability was determined by CellTiter-Blue fluorometric assay as described under Materials and Methods . All of the data points were normalized to a SCR/DMSO control to allow for well to-well comparisons. The effect of different concentrations of VBL on cell viability in cells treated with SCR or BCL-xL siRNA was determined. ○, T98G cells treated with VBL in combination with siRNA negative control (SCR). □, T98G cells treated with VBL in combination with pooled BCL-xL siRNAs. ▴, T98G cells transfected with cell death negative control siRNA. F, SCR and BCL-xL siRNAs were nontoxic to cells. Each value is the mean of four independent experiments. Bars represent S.E.M. *, p ≤ 0.05.

    Techniques Used: Transfection, Negative Control, Positive Control

    Sensitization of T98G and A549 cells to VBL by ABT-263. A, T98G cells were treated with increasing concentrations of VBL (25 pM to 25 nM) in the presence and absence of a nontoxic concentration of ABT-263 (1.56 μM). B, A549 cells were treated with increasing concentrations of VBL (25 pM to 25 nM) in the presence and absence of a nontoxic concentration of ABT-263 (12.5 μM). At higher concentrations of VBL (1.56–25 nM for T98G and 3.13–25 nM for A549), ABT-263 significantly decreases the cell viability relative to VBL alone in both T98G and A549 cancer cell lines. Cell viability was determined using a CellTiter-Blue assay. All of the values were normalized to the DMSO control (no ABT-263). ○, Cells treated with increasing concentrations of VBL. ■, Cells treated with increasing concentrations of VBL in the presence of nontoxic ABT-263 concentrations. Each value is the mean of four independent experiments. Bars represent S.E.M. *, p ≤ 0.05.
    Figure Legend Snippet: Sensitization of T98G and A549 cells to VBL by ABT-263. A, T98G cells were treated with increasing concentrations of VBL (25 pM to 25 nM) in the presence and absence of a nontoxic concentration of ABT-263 (1.56 μM). B, A549 cells were treated with increasing concentrations of VBL (25 pM to 25 nM) in the presence and absence of a nontoxic concentration of ABT-263 (12.5 μM). At higher concentrations of VBL (1.56–25 nM for T98G and 3.13–25 nM for A549), ABT-263 significantly decreases the cell viability relative to VBL alone in both T98G and A549 cancer cell lines. Cell viability was determined using a CellTiter-Blue assay. All of the values were normalized to the DMSO control (no ABT-263). ○, Cells treated with increasing concentrations of VBL. ■, Cells treated with increasing concentrations of VBL in the presence of nontoxic ABT-263 concentrations. Each value is the mean of four independent experiments. Bars represent S.E.M. *, p ≤ 0.05.

    Techniques Used: Concentration Assay, CtB Assay

    52) Product Images from "Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo"

    Article Title: Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19030912

    Astaxanthin inhibits the expression of the genes involved in osteoclastogenesis. BMMs were treated with 0.1% DMSO or Astaxanthin (30 μM) and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/ml) for the indicated number of days. The expressed mRNA levels of ( A ) NFATc1 , ( B ) TRAP , ( C ) DC-STAMP , and ( D ) cathepsin K were analyzed by real-time PCR compared with the DMSO control. * p
    Figure Legend Snippet: Astaxanthin inhibits the expression of the genes involved in osteoclastogenesis. BMMs were treated with 0.1% DMSO or Astaxanthin (30 μM) and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/ml) for the indicated number of days. The expressed mRNA levels of ( A ) NFATc1 , ( B ) TRAP , ( C ) DC-STAMP , and ( D ) cathepsin K were analyzed by real-time PCR compared with the DMSO control. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    BMMs were pretreated with 0.1%DMSO or Astaxanthin (30 μM) for 1 h and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) for the indicated time. Cell lysates were resolved by SDS-PAGE, and western blotting was performed with anti-NFATc1 and actin antibodies as indicated.
    Figure Legend Snippet: BMMs were pretreated with 0.1%DMSO or Astaxanthin (30 μM) for 1 h and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) for the indicated time. Cell lysates were resolved by SDS-PAGE, and western blotting was performed with anti-NFATc1 and actin antibodies as indicated.

    Techniques Used: SDS Page, Western Blot

    Astaxanthin suppresses osteoclastogenesis. ( A ) Chemical structure of Astaxanthin; ( B ) BMMs prepared from bone marrow cells were cultured for 4 days with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence of the indicated concentrations of Astaxanthin or 0.1% DMSO (control vehicle). The cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained for TRAP, a marker enzyme of osteoclasts; ( C ) TRAP-positive multinuclear cells (nuclei ≥ 3) were counted as osteoclasts. * p
    Figure Legend Snippet: Astaxanthin suppresses osteoclastogenesis. ( A ) Chemical structure of Astaxanthin; ( B ) BMMs prepared from bone marrow cells were cultured for 4 days with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence of the indicated concentrations of Astaxanthin or 0.1% DMSO (control vehicle). The cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained for TRAP, a marker enzyme of osteoclasts; ( C ) TRAP-positive multinuclear cells (nuclei ≥ 3) were counted as osteoclasts. * p

    Techniques Used: Cell Culture, Staining, Marker

    53) Product Images from "Effects of β2 Agonists, Corticosteroids, and Novel Therapies on Rhinovirus-Induced Cytokine Release and Rhinovirus Replication in Primary Airway Fibroblasts"

    Article Title: Effects of β2 Agonists, Corticosteroids, and Novel Therapies on Rhinovirus-Induced Cytokine Release and Rhinovirus Replication in Primary Airway Fibroblasts

    Journal: Journal of Allergy

    doi: 10.1155/2011/457169

    (a–d) Effect of BAY and DMF on RV-induced IL-6 and IL-8. Concentration of IL-6 and IL-8 release from noninfected fibroblast (constitutive release), UVi-RV-(UVi-) or RV-16-infected fibroblasts (RV) (MOI = 0.1), highest concentration of vehicle (0.1% DMSO) and RV infected fibroblasts in the presence of 10 −8 –10 −6 M BAY ( n = 10) and DMF ( n = 9) measured 48 hrs post infection by ELISA. All IL-6 and IL-8 concentrations were compared to their respective RV-induced values (in the absence of drug and vehicle), using a 1-way ANOVA. All data are presented as mean ± SEM. Significance is represented as ** P
    Figure Legend Snippet: (a–d) Effect of BAY and DMF on RV-induced IL-6 and IL-8. Concentration of IL-6 and IL-8 release from noninfected fibroblast (constitutive release), UVi-RV-(UVi-) or RV-16-infected fibroblasts (RV) (MOI = 0.1), highest concentration of vehicle (0.1% DMSO) and RV infected fibroblasts in the presence of 10 −8 –10 −6 M BAY ( n = 10) and DMF ( n = 9) measured 48 hrs post infection by ELISA. All IL-6 and IL-8 concentrations were compared to their respective RV-induced values (in the absence of drug and vehicle), using a 1-way ANOVA. All data are presented as mean ± SEM. Significance is represented as ** P

    Techniques Used: Concentration Assay, Infection, Enzyme-linked Immunosorbent Assay

    Effect of DMF on RV replication. Concentration of virus from RV-infected fibroblasts ± vehicle (0.1% DMSO); or 10 −8 –10 −6 M DMF ( n = 14) was measured 24 hrs post infection by RV titration. All RV concentrations were compared to RV concentration in the absence of drug and vehicle by 1-way ANOVA. All data are presented as mean ±SEM. Significance is represented as * P
    Figure Legend Snippet: Effect of DMF on RV replication. Concentration of virus from RV-infected fibroblasts ± vehicle (0.1% DMSO); or 10 −8 –10 −6 M DMF ( n = 14) was measured 24 hrs post infection by RV titration. All RV concentrations were compared to RV concentration in the absence of drug and vehicle by 1-way ANOVA. All data are presented as mean ±SEM. Significance is represented as * P

    Techniques Used: Concentration Assay, Infection, Titration

    (a–f) Effect of dexamethasone (Dex), fluticasone (Flut) and salmeterol (Sal) on RV-induced IL-6 and IL-8. Concentration of IL-6 and IL-8 release from noninfected fibroblasts (constitutive release), UVi-RV-(UVi-) or RV-16-infected fibroblasts (RV) (MOI = 0.1), highest concentration of vehicle (Dex Sal: 0.1% DMSO; Flut: 0.001% DMSO) and RV infected fibroblasts in the presence of Dex: 10 −12 –10 −7 M ( n = 7), Flut: 10 −10 –10 −8 M ( n = 7) and Sal: 10 −8 –10 −6 M ( n = 9) were measured 48 hrs post infection by ELISA. All IL-6 and IL-8 concentrations were compared to their respective RV-induced values (in the absence of drug and vehicle), using a 1-way ANOVA. All data are presented as mean ± SEM. Significance is represented as * P
    Figure Legend Snippet: (a–f) Effect of dexamethasone (Dex), fluticasone (Flut) and salmeterol (Sal) on RV-induced IL-6 and IL-8. Concentration of IL-6 and IL-8 release from noninfected fibroblasts (constitutive release), UVi-RV-(UVi-) or RV-16-infected fibroblasts (RV) (MOI = 0.1), highest concentration of vehicle (Dex Sal: 0.1% DMSO; Flut: 0.001% DMSO) and RV infected fibroblasts in the presence of Dex: 10 −12 –10 −7 M ( n = 7), Flut: 10 −10 –10 −8 M ( n = 7) and Sal: 10 −8 –10 −6 M ( n = 9) were measured 48 hrs post infection by ELISA. All IL-6 and IL-8 concentrations were compared to their respective RV-induced values (in the absence of drug and vehicle), using a 1-way ANOVA. All data are presented as mean ± SEM. Significance is represented as * P

    Techniques Used: Concentration Assay, Infection, Enzyme-linked Immunosorbent Assay

    54) Product Images from "Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo"

    Article Title: Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19030912

    Astaxanthin inhibits the expression of the genes involved in osteoclastogenesis. BMMs were treated with 0.1% DMSO or Astaxanthin (30 μM) and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/ml) for the indicated number of days. The expressed mRNA levels of ( A ) NFATc1 , ( B ) TRAP , ( C ) DC-STAMP , and ( D ) cathepsin K were analyzed by real-time PCR compared with the DMSO control. * p
    Figure Legend Snippet: Astaxanthin inhibits the expression of the genes involved in osteoclastogenesis. BMMs were treated with 0.1% DMSO or Astaxanthin (30 μM) and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/ml) for the indicated number of days. The expressed mRNA levels of ( A ) NFATc1 , ( B ) TRAP , ( C ) DC-STAMP , and ( D ) cathepsin K were analyzed by real-time PCR compared with the DMSO control. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    BMMs were pretreated with 0.1%DMSO or Astaxanthin (30 μM) for 1 h and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) for the indicated time. Cell lysates were resolved by SDS-PAGE, and western blotting was performed with anti-NFATc1 and actin antibodies as indicated.
    Figure Legend Snippet: BMMs were pretreated with 0.1%DMSO or Astaxanthin (30 μM) for 1 h and then stimulated with RANKL (10 ng/mL) and M-CSF (30 ng/mL) for the indicated time. Cell lysates were resolved by SDS-PAGE, and western blotting was performed with anti-NFATc1 and actin antibodies as indicated.

    Techniques Used: SDS Page, Western Blot

    Astaxanthin suppresses osteoclastogenesis. ( A ) Chemical structure of Astaxanthin; ( B ) BMMs prepared from bone marrow cells were cultured for 4 days with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence of the indicated concentrations of Astaxanthin or 0.1% DMSO (control vehicle). The cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained for TRAP, a marker enzyme of osteoclasts; ( C ) TRAP-positive multinuclear cells (nuclei ≥ 3) were counted as osteoclasts. * p
    Figure Legend Snippet: Astaxanthin suppresses osteoclastogenesis. ( A ) Chemical structure of Astaxanthin; ( B ) BMMs prepared from bone marrow cells were cultured for 4 days with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in the presence of the indicated concentrations of Astaxanthin or 0.1% DMSO (control vehicle). The cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained for TRAP, a marker enzyme of osteoclasts; ( C ) TRAP-positive multinuclear cells (nuclei ≥ 3) were counted as osteoclasts. * p

    Techniques Used: Cell Culture, Staining, Marker

    55) Product Images from "Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors"

    Article Title: Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154919

    CK2 inhibition in proliferating cells resulted in accelerated expression of differentiation markers. 24 h after isolation, mouse primary myoblasts were incubated with 100 μM TBB or DMSO for 6 hours before fixation. Indirect immunofluorescence for Pax7, Syndecan-4 (A, B), MyoD (A), Myogenin (B) was performed as indicated. TBB treatment results in a significant increase in the percentage of cells expressing reduced Pax7 levels, while expressing high levels of myogenin (arrows). (C) Quantification of cell subpopulations present in (B), shows a ~10-fold increase in the percentage of Pax7(-)/ myogenin (+) cells (i.e. differentiating cells) with a concomitant decrease in the percentage of proliferating Pax7(+)/myogenin(-) sub population. mean±SEM, n = 3. (D) qPCR analysis determining relative Pax7 mRNA expression upon CK2 inhibition (sample pool obtained from one experiment performed as in A and B).
    Figure Legend Snippet: CK2 inhibition in proliferating cells resulted in accelerated expression of differentiation markers. 24 h after isolation, mouse primary myoblasts were incubated with 100 μM TBB or DMSO for 6 hours before fixation. Indirect immunofluorescence for Pax7, Syndecan-4 (A, B), MyoD (A), Myogenin (B) was performed as indicated. TBB treatment results in a significant increase in the percentage of cells expressing reduced Pax7 levels, while expressing high levels of myogenin (arrows). (C) Quantification of cell subpopulations present in (B), shows a ~10-fold increase in the percentage of Pax7(-)/ myogenin (+) cells (i.e. differentiating cells) with a concomitant decrease in the percentage of proliferating Pax7(+)/myogenin(-) sub population. mean±SEM, n = 3. (D) qPCR analysis determining relative Pax7 mRNA expression upon CK2 inhibition (sample pool obtained from one experiment performed as in A and B).

    Techniques Used: Inhibition, Expressing, Isolation, Incubation, Immunofluorescence, Real-time Polymerase Chain Reaction

    CK2 regulates Pax7 stability in proliferating myoblasts. (A) C3H10T1/2 cells transfected with myc-Pax7-WT or mutants were treated with DMSO or 100 μM of CK2 inhibitor (TBB) for 6 hours prior to lysis and Western Blot analysis. GFP was used as transfection/loading control. Right panels show quantification of fold reduction in myc-Pax7 levels (myc/GFP) for each treatment compared to vehicle (DMSO); mean±SEM, n = 5 (upper), n = 4 (lower). Pax7-DS and Pax7-DD phospho-mimetics exhibit enhanced stability upon CK2 inhibition compared to other phospho-mutants. Proliferating C2C12 cells were incubated with DMSO, TBB (B) or TBCA (C) at the indicated concentration for 6 hours. Endogenous Pax7 levels were analyzed by Western Blot using GAPDH as loading control. Anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. (B)-(C), Lower panels show quantification of Pax7/GAPDH ratio in relative units; mean±SEM, n = 4; ANOVA, * p
    Figure Legend Snippet: CK2 regulates Pax7 stability in proliferating myoblasts. (A) C3H10T1/2 cells transfected with myc-Pax7-WT or mutants were treated with DMSO or 100 μM of CK2 inhibitor (TBB) for 6 hours prior to lysis and Western Blot analysis. GFP was used as transfection/loading control. Right panels show quantification of fold reduction in myc-Pax7 levels (myc/GFP) for each treatment compared to vehicle (DMSO); mean±SEM, n = 5 (upper), n = 4 (lower). Pax7-DS and Pax7-DD phospho-mimetics exhibit enhanced stability upon CK2 inhibition compared to other phospho-mutants. Proliferating C2C12 cells were incubated with DMSO, TBB (B) or TBCA (C) at the indicated concentration for 6 hours. Endogenous Pax7 levels were analyzed by Western Blot using GAPDH as loading control. Anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. (B)-(C), Lower panels show quantification of Pax7/GAPDH ratio in relative units; mean±SEM, n = 4; ANOVA, * p

    Techniques Used: Transfection, Lysis, Western Blot, Inhibition, Incubation, Concentration Assay

    CK2 inhibition in proliferating myoblasts increases Pax7 ubiquitination and its proteasome-dependent degradation. (A) TBB-induced Pax7 decline is prevented by concomitant proteasome inhibition. Proliferating C2C12 cells were treated as indicated with TBB 125 μM and/or 1 μM of the proteasome inhibitor epoxomicin (Epo) for 6 hours and analyzed by Western blotting. GAPDH was used as loading control and anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. Right panel shows quantification of protein levels (Pax7/GAPDH) normalized to control (DMSO); mean±SEM, n = 3; ANOVA, * p
    Figure Legend Snippet: CK2 inhibition in proliferating myoblasts increases Pax7 ubiquitination and its proteasome-dependent degradation. (A) TBB-induced Pax7 decline is prevented by concomitant proteasome inhibition. Proliferating C2C12 cells were treated as indicated with TBB 125 μM and/or 1 μM of the proteasome inhibitor epoxomicin (Epo) for 6 hours and analyzed by Western blotting. GAPDH was used as loading control and anti-phospho-CK2 substrate antibody was used as a control of TBB treatment. Right panel shows quantification of protein levels (Pax7/GAPDH) normalized to control (DMSO); mean±SEM, n = 3; ANOVA, * p

    Techniques Used: Inhibition, Western Blot

    56) Product Images from "Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene"

    Article Title: Pharmacological inhibition of c-Jun N-terminal kinase signaling prevents cardiomyopathy caused by mutation in LMNA gene

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbadis.2010.04.001

    Treatment with SP600125 prevents cardiac fibrosis in Lmna H222P/H222P mice. (A) Representative heart tissue sections from Lmna H222P/H222P mice treated with SP600125 or DMSO stained with Gomori’s Trichrome are shown. Heart tissue section from Lmna +/+ mice is shown for comparison. Scale bar: 50 μm. Bar graph represents the quantification of fibrotic area in hearts from mice. Micrographs (n=3) for each heart were processed (JMicroVision software) and blue-staining fibrotic tissue was quantified (ImageJ64 software). Bars indicate the percentage of fibrosis in heart from Lmna +/+ and Lmna H222P/H222P mice treated with SP600125 or DMSO. Values are means ± standard errors for n=3 mice per group. Comparison between DMSO-treated Lmna H222P/H222P and Lmna +/+ mice was performed using Student unpaired t -test; *** P
    Figure Legend Snippet: Treatment with SP600125 prevents cardiac fibrosis in Lmna H222P/H222P mice. (A) Representative heart tissue sections from Lmna H222P/H222P mice treated with SP600125 or DMSO stained with Gomori’s Trichrome are shown. Heart tissue section from Lmna +/+ mice is shown for comparison. Scale bar: 50 μm. Bar graph represents the quantification of fibrotic area in hearts from mice. Micrographs (n=3) for each heart were processed (JMicroVision software) and blue-staining fibrotic tissue was quantified (ImageJ64 software). Bars indicate the percentage of fibrosis in heart from Lmna +/+ and Lmna H222P/H222P mice treated with SP600125 or DMSO. Values are means ± standard errors for n=3 mice per group. Comparison between DMSO-treated Lmna H222P/H222P and Lmna +/+ mice was performed using Student unpaired t -test; *** P

    Techniques Used: Mouse Assay, Staining, Software

    Treatment of male Lmna H222P/H222P mice with SP600125 inhibits expression of natriuretic peptides and myosin chains. Quantitative real-time RT-PCR showing the expression of (A) mRNAs from NppA and NppB genes encoding natriuretic peptide precursors A and B, respectively and (B) mRNAs from Myh7 encoding myosing heavy chain and Myl4 and Myl7 encoding myosin light chain. Results from hearts from Lmna +/+ mice are shown for comparison. Bars indicate the fold over-expression of the indicated mRNA calculated by the ΔΔC T method. Values are means ± standard errors for n=5 samples from different animals per group. Reactions were performed in triplicate for each different RNA sample. Comparison between DMSO-treated Lmna H222P/H222P and Lmna +/+ mice was performed using Student unpaired t -test; * P
    Figure Legend Snippet: Treatment of male Lmna H222P/H222P mice with SP600125 inhibits expression of natriuretic peptides and myosin chains. Quantitative real-time RT-PCR showing the expression of (A) mRNAs from NppA and NppB genes encoding natriuretic peptide precursors A and B, respectively and (B) mRNAs from Myh7 encoding myosing heavy chain and Myl4 and Myl7 encoding myosin light chain. Results from hearts from Lmna +/+ mice are shown for comparison. Bars indicate the fold over-expression of the indicated mRNA calculated by the ΔΔC T method. Values are means ± standard errors for n=5 samples from different animals per group. Reactions were performed in triplicate for each different RNA sample. Comparison between DMSO-treated Lmna H222P/H222P and Lmna +/+ mice was performed using Student unpaired t -test; * P

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Over Expression

    Treatment of male Lmna H222P/H222P mice with SP600125 inhibits phosphorylation of JNK signaling. (A) Representative immunoblots using antibodies against phophorylated JNK (p-JNK), total JNK, phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 to probe proteins extracted from hearts from Lmna H222P/H222P mice treated with SP600125 or DMSO. Blots of proteins extracted from hearts of Lmna +/+ mice are shown for comparison. Data in bar graphs show quantification of phosphorylated JNK compared with total JNK measured by scanning immunoblots and using ImageJ64 software. Values are means ± standard errors for n=5 samples from different animals per group. Comparison between DMSO-treated Lmna H222P/H222P and Lmna +/+ mice was performed using Student unpaired t -test; *** P
    Figure Legend Snippet: Treatment of male Lmna H222P/H222P mice with SP600125 inhibits phosphorylation of JNK signaling. (A) Representative immunoblots using antibodies against phophorylated JNK (p-JNK), total JNK, phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 to probe proteins extracted from hearts from Lmna H222P/H222P mice treated with SP600125 or DMSO. Blots of proteins extracted from hearts of Lmna +/+ mice are shown for comparison. Data in bar graphs show quantification of phosphorylated JNK compared with total JNK measured by scanning immunoblots and using ImageJ64 software. Values are means ± standard errors for n=5 samples from different animals per group. Comparison between DMSO-treated Lmna H222P/H222P and Lmna +/+ mice was performed using Student unpaired t -test; *** P

    Techniques Used: Mouse Assay, Western Blot, Software

    57) Product Images from "Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines"

    Article Title: Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075198

    11a induces G 1 /S phase cell cycle arrest in a dose dependent manner in HCT116 isogenic cell lines. (A) Western blot against p53, Cyclin D1, p21 and Hsp90 as the loading control. (B) Histograms of the DNA FACS analysis of the cultures from panel A. (C) Quantification of the FACS analysis and cell cycle distribution of the two cell lines treated with 0, 5, 10, 50, or 100 nM 11a in 0.1% DMSO for 12 hours.
    Figure Legend Snippet: 11a induces G 1 /S phase cell cycle arrest in a dose dependent manner in HCT116 isogenic cell lines. (A) Western blot against p53, Cyclin D1, p21 and Hsp90 as the loading control. (B) Histograms of the DNA FACS analysis of the cultures from panel A. (C) Quantification of the FACS analysis and cell cycle distribution of the two cell lines treated with 0, 5, 10, 50, or 100 nM 11a in 0.1% DMSO for 12 hours.

    Techniques Used: Western Blot, FACS

    The effect of 11a on PNR, TLX, COUP-TFI and COUP-TFII activation of the DR2-luciferase reporter. (A) Chemical structure of 11a. HEK293T cells transfected with the indicated constructs were treated in triplicate with 0.1% DMSO, 15 nM, 30 nM, 60 nM, 120 nM or 150 nM 11a. Data are expressed as relative luciferase units normalized to the DMSO control ± SD. (B) Comparison between different nuclear receptors with increasing 11a concentrations. (C) Comparison between various doses of 11a with different nuclear receptors.
    Figure Legend Snippet: The effect of 11a on PNR, TLX, COUP-TFI and COUP-TFII activation of the DR2-luciferase reporter. (A) Chemical structure of 11a. HEK293T cells transfected with the indicated constructs were treated in triplicate with 0.1% DMSO, 15 nM, 30 nM, 60 nM, 120 nM or 150 nM 11a. Data are expressed as relative luciferase units normalized to the DMSO control ± SD. (B) Comparison between different nuclear receptors with increasing 11a concentrations. (C) Comparison between various doses of 11a with different nuclear receptors.

    Techniques Used: Activation Assay, Luciferase, Transfection, Construct

    11a induces G 1 /S phase cell cycle arrest in a time dependent manner in HCT116 isogenic cell lines. (A) Western blot against p53, Cyclin D1, p21 and Hsp90, which was used as the loading control (B) Histograms of the FACS analysis (C) Quantification of the FACS analysis and cell cycle distribution of the two cell lines treated with 0.1% DMSO or 50 nM 11a.
    Figure Legend Snippet: 11a induces G 1 /S phase cell cycle arrest in a time dependent manner in HCT116 isogenic cell lines. (A) Western blot against p53, Cyclin D1, p21 and Hsp90, which was used as the loading control (B) Histograms of the FACS analysis (C) Quantification of the FACS analysis and cell cycle distribution of the two cell lines treated with 0.1% DMSO or 50 nM 11a.

    Techniques Used: Western Blot, FACS

    58) Product Images from "Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells"

    Article Title: Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074968

    Effects of PFOS on cell viability and morphology in mESCs. (A and B) Cell viability was determined by MTT assay after exposure to various concentrations of PFOS for 24 h and 48 h. (C) D3 mESCs was exposed to PFOS for 24 h, cell morphology was observed. Magnification, 100×. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control. (D) Effects of PFOS on alkaline phosphatase staining in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control. Scale bar = 25 µm. Values of the experiment were represented as the percentages of cell viability compared with that of the control and expressed as means ± S.E. from five separate experiments in which treatments were performed in quadruplicate. *indicates significant difference when the values were compared to that of the control at p
    Figure Legend Snippet: Effects of PFOS on cell viability and morphology in mESCs. (A and B) Cell viability was determined by MTT assay after exposure to various concentrations of PFOS for 24 h and 48 h. (C) D3 mESCs was exposed to PFOS for 24 h, cell morphology was observed. Magnification, 100×. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control. (D) Effects of PFOS on alkaline phosphatase staining in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control. Scale bar = 25 µm. Values of the experiment were represented as the percentages of cell viability compared with that of the control and expressed as means ± S.E. from five separate experiments in which treatments were performed in quadruplicate. *indicates significant difference when the values were compared to that of the control at p

    Techniques Used: MTT Assay, Cell Culture, Staining

    Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of Oct4, Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p
    Figure Legend Snippet: Effects of PFOS on pluripotency and expressions of miR-145, miR-490-3p in mESCs. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (A) Oct-4/Sox-2/Nanog mRNA levels were determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. (B) The protein levels of Oct-4/Sox-2/Nanog were determined by Western blot analysis using GAPDH as an internal control. (C) miRNA levels( miR-145 , miR-490-3p) were determined by quantitative real-time PCR and were normalized to U6 as an internal control. Each data point was normalized to the control (DMSO) and represented the means ± S.E. from three independent experiments. (D) Relative protein levels of Oct4, Sox2 and Nanog. *indicates significant difference when the values were compared to that of the control ( p

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

    Over-expression of miR-490-3p reduced Nanog expression. (A)The expression of its host gene Chrm2 mRNA levels was determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (B) Cells were transfected with 50 nM miR-490 mimics or 100 nM miR-490 inhibitor for 24 h. qRT-PCR was performed to evaluate the mRNA level of Nanog. (C) The relative expression levels of miR-490-3p after transfection. (D) Cells were co-transfected with miR-490-3p mimics and negative control, renilla luciferase vector pRL-SV40 and Nanog 3′UTR luciferase reporters for 24 h. Both firefly and Renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with Renilla luciferase signals. (E) Sequence alignment of miR-490-3p with 3′ UTR of Nanog. Bottom: mutations in the 3′UTR of Nanog in order to create the mutant luciferase reporter constructs. *indicates significant difference compared with that of control cells (P
    Figure Legend Snippet: Over-expression of miR-490-3p reduced Nanog expression. (A)The expression of its host gene Chrm2 mRNA levels was determined by quantitative real-time PCR using a housekeeping gene GAPDH as an internal control. Cells were cultured with various concentrations of PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) or DMSO as control for 24 h. (B) Cells were transfected with 50 nM miR-490 mimics or 100 nM miR-490 inhibitor for 24 h. qRT-PCR was performed to evaluate the mRNA level of Nanog. (C) The relative expression levels of miR-490-3p after transfection. (D) Cells were co-transfected with miR-490-3p mimics and negative control, renilla luciferase vector pRL-SV40 and Nanog 3′UTR luciferase reporters for 24 h. Both firefly and Renilla luciferase activities are measured in the same sample. Firefly luciferase signals were normalized with Renilla luciferase signals. (E) Sequence alignment of miR-490-3p with 3′ UTR of Nanog. Bottom: mutations in the 3′UTR of Nanog in order to create the mutant luciferase reporter constructs. *indicates significant difference compared with that of control cells (P

    Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Quantitative RT-PCR, Negative Control, Luciferase, Plasmid Preparation, Sequencing, Mutagenesis, Construct

    59) Product Images from "In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes"

    Article Title: In Vitro Influence of Mycophenolic Acid on Selected Parameters of Stimulated Peripheral Canine Lymphocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154429

    The percentage of CD4 + CD25 + (A), CD4 + CD25 + FoxP3 + (B) lymphocytes and MFI of FoxP3 + or CD25 + lymphocytes (C) after 72 h culture of PBMC in a 37°C, 5% CO 2 environment with mitogens–ConA or PHA and MPA at different concentrations (1 μM, 10 μM, 100 μM) or without MPA (solvent control– 0.1% DMSO) (n = 7). Mean ± SEM. *p
    Figure Legend Snippet: The percentage of CD4 + CD25 + (A), CD4 + CD25 + FoxP3 + (B) lymphocytes and MFI of FoxP3 + or CD25 + lymphocytes (C) after 72 h culture of PBMC in a 37°C, 5% CO 2 environment with mitogens–ConA or PHA and MPA at different concentrations (1 μM, 10 μM, 100 μM) or without MPA (solvent control– 0.1% DMSO) (n = 7). Mean ± SEM. *p

    Techniques Used:

    The percentage and MFI of PCNA + lymphocytes (n = 7) (A) and the proliferation index of CFSE-labeled lymphocytes (n = 8) (B) after 72 h culture of PBMC in a 37°C, 5% CO 2 environment with mitogens–ConA or PHA and MPA at different concentrations (1 μM, 10 μM, 100 μM) or without MPA (solvent control– 0.1% DMSO) Mean ± SEM. ***p
    Figure Legend Snippet: The percentage and MFI of PCNA + lymphocytes (n = 7) (A) and the proliferation index of CFSE-labeled lymphocytes (n = 8) (B) after 72 h culture of PBMC in a 37°C, 5% CO 2 environment with mitogens–ConA or PHA and MPA at different concentrations (1 μM, 10 μM, 100 μM) or without MPA (solvent control– 0.1% DMSO) Mean ± SEM. ***p

    Techniques Used: Labeling

    The percentage and MFI of CD3 + (A) and CD8 + (B) lymphocytes after 72 h culture of PBMC in a 37°C, 5% CO 2 environment with mitogens–ConA or PHA and MPA at different concentrations (1 μM, 10 μM, 100 μM) or without MPA (solvent control– 0.1% DMSO) (n = 7). Mean ± SEM. *p
    Figure Legend Snippet: The percentage and MFI of CD3 + (A) and CD8 + (B) lymphocytes after 72 h culture of PBMC in a 37°C, 5% CO 2 environment with mitogens–ConA or PHA and MPA at different concentrations (1 μM, 10 μM, 100 μM) or without MPA (solvent control– 0.1% DMSO) (n = 7). Mean ± SEM. *p

    Techniques Used:

    60) Product Images from "Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2"

    Article Title: Anti-Leukemic Properties of Histamine in Monocytic Leukemia: The Role of NOX2

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2018.00218

    Targeting intracellular reactive oxygen species (ROS) promotes leukemic cell differentiation. (A,B) Measurement of ROS production by chemiluminescence following fMLF stimulation in the presence or absence of histamine dihydrochloride. (A) A representative curve showing the respiratory burst by dimethyl sulfoxide (DMSO)-differentiated wild-type (WT) or NOX2 -KO PLB-985. (B) ROS production by undifferentiated and DMSO-differentiated WT or NOX2 -KO PLB-985 cells. (C–E) Fluorescent measurement of intracellular ROS in WT and NOX2 -KO PLB-985 using flow cytometry: (C) cytoplasmic ROS, and (D,E) mitochondrial ROS. Antimycin A was used to trigger mitochondrial ROS production. (F) WT and NOX2 -KO PLB-985 cells were cultured in the presence or absence of luminol or isoluminol for 5 days and the percentage of CD11b + cells was determined by flow cytometry. Two-tailed paired or unpaired t -tests, one-way ANOVA; * p
    Figure Legend Snippet: Targeting intracellular reactive oxygen species (ROS) promotes leukemic cell differentiation. (A,B) Measurement of ROS production by chemiluminescence following fMLF stimulation in the presence or absence of histamine dihydrochloride. (A) A representative curve showing the respiratory burst by dimethyl sulfoxide (DMSO)-differentiated wild-type (WT) or NOX2 -KO PLB-985. (B) ROS production by undifferentiated and DMSO-differentiated WT or NOX2 -KO PLB-985 cells. (C–E) Fluorescent measurement of intracellular ROS in WT and NOX2 -KO PLB-985 using flow cytometry: (C) cytoplasmic ROS, and (D,E) mitochondrial ROS. Antimycin A was used to trigger mitochondrial ROS production. (F) WT and NOX2 -KO PLB-985 cells were cultured in the presence or absence of luminol or isoluminol for 5 days and the percentage of CD11b + cells was determined by flow cytometry. Two-tailed paired or unpaired t -tests, one-way ANOVA; * p

    Techniques Used: Cell Differentiation, Flow Cytometry, Cytometry, Cell Culture, Two Tailed Test

    Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p
    Figure Legend Snippet: Histamine dihydrochloride (HDC)-induced differentiation of leukemic cells is NOX2-dependent. (A) FACS-plots showing NOX2 and H 2 R expression on wild-type (WT) and NOX2 -KO PLB-985 cells. Expression of CD11b (B,C) , FPR1 (D) , and FPR2 (E) on WT and NOX2 -KO PLB-985 cells cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) as determined by flow cytometry. (F) FACS-plot showing NOX2 and H 2 R expression by OCI-AML3 cells. Expression of CD11b (G) , CD14 (H) , FPR1 (I) , and FPR2 (J) on OCI-AML3 cells cultured in the presence or absence of HDC or DMSO. Abbreviations: MFI, median fluorescence intensity. ANOVA; * p

    Techniques Used: FACS, Expressing, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

    Histamine dihydrochloride (HDC) selectively regulates gene expression in NOX2 + leukemic cells and exerts anti-leukemic efficacy in vivo . (A–C) Wild-type (WT) and NOX2-KO PLB-985 cells were cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) for 48 h. RNA was extracted and microarray analysis performed. (A) Heat map showing genes significantly ( p
    Figure Legend Snippet: Histamine dihydrochloride (HDC) selectively regulates gene expression in NOX2 + leukemic cells and exerts anti-leukemic efficacy in vivo . (A–C) Wild-type (WT) and NOX2-KO PLB-985 cells were cultured in the presence or absence of HDC or dimethyl sulfoxide (DMSO) for 48 h. RNA was extracted and microarray analysis performed. (A) Heat map showing genes significantly ( p

    Techniques Used: Expressing, In Vivo, Cell Culture, Microarray

    61) Product Images from "Potential therapeutic effects of N-butylidenephthalide from Radix Angelica Sinensis (Danggui) in human bladder cancer cells"

    Article Title: Potential therapeutic effects of N-butylidenephthalide from Radix Angelica Sinensis (Danggui) in human bladder cancer cells

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-017-2034-3

    Effects of BP on the viability of human bladder cancer cells. a Human bladder cancer cells (5637, BFTC, T24 and TCCSUP) were treated with 0.2% DMSO as vehicle control or 60 μg/ml BP for 24 h, were shown; Scale bar: 50 μm. b Human bladder cancer cells were treated with various concentration of BP (12.5 to 100 μg/ml) for 24 (■) and 48 h (■), respectively, and the survival rate was evaluated with MTT assay. Data are presented as means ± S.D. obtained from three different experiments. ** p
    Figure Legend Snippet: Effects of BP on the viability of human bladder cancer cells. a Human bladder cancer cells (5637, BFTC, T24 and TCCSUP) were treated with 0.2% DMSO as vehicle control or 60 μg/ml BP for 24 h, were shown; Scale bar: 50 μm. b Human bladder cancer cells were treated with various concentration of BP (12.5 to 100 μg/ml) for 24 (■) and 48 h (■), respectively, and the survival rate was evaluated with MTT assay. Data are presented as means ± S.D. obtained from three different experiments. ** p

    Techniques Used: Concentration Assay, MTT Assay

    BP induced mitochondrial-mediated apoptosis in human bladder cancer cells. a Human bladder cancer cells were treated with 0.2% DMSO (control) or 60 μg/ml BP for 72 h and then stained with the TUNEL assay. TUNEL positive cells (green fluorescence) were indicated by arrows. Scale bar: 50 μm. b Human bladder cancer cells were treated with 60 μg/ml BP for 0 to 72 h, and western blot analysis were performed for cleaved caspase-9 and -3. β-actin was used as an internal control. c MTT assay of human bladder cancer cells pretreated with caspase 3 inhibitor Z-DEVD-fmk (20 μM) for 1 h and then treated in the presence or absence of 60 μg/ml BP for 48 h. The values are the mean ± S.D. from three independent experiments, * p
    Figure Legend Snippet: BP induced mitochondrial-mediated apoptosis in human bladder cancer cells. a Human bladder cancer cells were treated with 0.2% DMSO (control) or 60 μg/ml BP for 72 h and then stained with the TUNEL assay. TUNEL positive cells (green fluorescence) were indicated by arrows. Scale bar: 50 μm. b Human bladder cancer cells were treated with 60 μg/ml BP for 0 to 72 h, and western blot analysis were performed for cleaved caspase-9 and -3. β-actin was used as an internal control. c MTT assay of human bladder cancer cells pretreated with caspase 3 inhibitor Z-DEVD-fmk (20 μM) for 1 h and then treated in the presence or absence of 60 μg/ml BP for 48 h. The values are the mean ± S.D. from three independent experiments, * p

    Techniques Used: Staining, TUNEL Assay, Fluorescence, Western Blot, MTT Assay

    62) Product Images from "Structure-based virtual screening and characterization of a novel IL-6 antagonistic compound from synthetic compound database"

    Article Title: Structure-based virtual screening and characterization of a novel IL-6 antagonistic compound from synthetic compound database

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S118457

    Compound 1 induced XG-7 cell apoptosis. Notes: ( A ) Morphological analysis of compound 1-treated XG-7 cells stained with Giemsa. NEG: NEG control (XG-7 cells with hIL-6), DMSO: 0.1% DMSO control and POS: POS control (XG-7 cells without hIL-6) and XG-7 cells treated for 72 hours with 0.3 μM and 3 μM of compound 1, respectively. The apoptosis cells were marked out with black arrows. Magnification: ×600. ( B ) Compound 1 induced DNA fragmentation in XG-7 cells. XG-7 cells were treated with different concentrations of compound 1 for 72 hours in the presence or absence of hIL-6. DNA was isolated and examined with 2% agarose gel electrophoresis. Marker: DL-2000 ladders. Abbreviations: NEG, negative; DMSO, dimethyl sulfoxide; POS, positive.
    Figure Legend Snippet: Compound 1 induced XG-7 cell apoptosis. Notes: ( A ) Morphological analysis of compound 1-treated XG-7 cells stained with Giemsa. NEG: NEG control (XG-7 cells with hIL-6), DMSO: 0.1% DMSO control and POS: POS control (XG-7 cells without hIL-6) and XG-7 cells treated for 72 hours with 0.3 μM and 3 μM of compound 1, respectively. The apoptosis cells were marked out with black arrows. Magnification: ×600. ( B ) Compound 1 induced DNA fragmentation in XG-7 cells. XG-7 cells were treated with different concentrations of compound 1 for 72 hours in the presence or absence of hIL-6. DNA was isolated and examined with 2% agarose gel electrophoresis. Marker: DL-2000 ladders. Abbreviations: NEG, negative; DMSO, dimethyl sulfoxide; POS, positive.

    Techniques Used: Staining, Isolation, Agarose Gel Electrophoresis, Marker

    The detection of cytotoxic effect of compound 1 with MTT assay. Notes: L929 cells ( A ) and SP2/0 cells ( B ) were treated with different concentrations of compound 1 for 72 hours. Average results from a triplex experiment are shown together with the mean ± SD values. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; NEG, negative; DMSO, dimethyl sulfoxide; ns, not significant.
    Figure Legend Snippet: The detection of cytotoxic effect of compound 1 with MTT assay. Notes: L929 cells ( A ) and SP2/0 cells ( B ) were treated with different concentrations of compound 1 for 72 hours. Average results from a triplex experiment are shown together with the mean ± SD values. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; NEG, negative; DMSO, dimethyl sulfoxide; ns, not significant.

    Techniques Used: MTT Assay, Standard Deviation

    The inhibition effect of compounds on XG-7 cells by cell proliferation assays. Notes: ( A – C ) show results of MTT, 3 H-TdR and ATPlite assay, respectively. ( D ) The inhibition ratio of compound 1 by three different assays. NEG: NEG control (XG-7 cells with 2 ng/mL hIL-6), DMSO: DMSO control (XG-7 cells with hIL-6 and 0.1% DMSO) and POS: POS control (XG-7 cells without hIL-6). Represented data are mean ± SD (n=3). * P
    Figure Legend Snippet: The inhibition effect of compounds on XG-7 cells by cell proliferation assays. Notes: ( A – C ) show results of MTT, 3 H-TdR and ATPlite assay, respectively. ( D ) The inhibition ratio of compound 1 by three different assays. NEG: NEG control (XG-7 cells with 2 ng/mL hIL-6), DMSO: DMSO control (XG-7 cells with hIL-6 and 0.1% DMSO) and POS: POS control (XG-7 cells without hIL-6). Represented data are mean ± SD (n=3). * P

    Techniques Used: Inhibition, MTT Assay

    63) Product Images from "Specific Inhibition of HIV Infection by the Action of Spironolactone in T Cells"

    Article Title: Specific Inhibition of HIV Infection by the Action of Spironolactone in T Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01722-16

    Spironolactone induces degradation of XPB, but not of other general transcription components, without affecting cell viability or proliferation. (A) TFIIH protein levels in Jurkat T cells cultured in the presence of DMSO, SP, or EPL. (Left) Representative Western blot. (Right) Quantification of the results of four independent experiments. (B) XPB, XPD, GAPDH, and Rad52 mRNA levels in Jurkat T cells treated with DMSO, SP, or EPL. rq, relative quantity; NF, normalization factor. (C) Viability of Jurkat T cells using the MTT test. The cells were treated with DMSO or SP for up to 3 days. (D) Viability of primary CD4 + T cells using the MTT test. The cells were treated with DMSO or SP. Unless otherwise indicated, SP or EPL was added to the culture medium at a concentration of 10 μM for 48 h. The error bars represent SEM.
    Figure Legend Snippet: Spironolactone induces degradation of XPB, but not of other general transcription components, without affecting cell viability or proliferation. (A) TFIIH protein levels in Jurkat T cells cultured in the presence of DMSO, SP, or EPL. (Left) Representative Western blot. (Right) Quantification of the results of four independent experiments. (B) XPB, XPD, GAPDH, and Rad52 mRNA levels in Jurkat T cells treated with DMSO, SP, or EPL. rq, relative quantity; NF, normalization factor. (C) Viability of Jurkat T cells using the MTT test. The cells were treated with DMSO or SP for up to 3 days. (D) Viability of primary CD4 + T cells using the MTT test. The cells were treated with DMSO or SP. Unless otherwise indicated, SP or EPL was added to the culture medium at a concentration of 10 μM for 48 h. The error bars represent SEM.

    Techniques Used: Cell Culture, Western Blot, MTT Assay, Concentration Assay

    Spironolactone inhibits HIV-1 and HIV-2 infection of Jurkat T cells. (A) HIV-1 NL4.3 infection of Jurkat T cells treated or not with SP. The number of infected cells was measured by Gag labeling followed by flow cytometry analysis. P = 0.005. (B) HIV-1 NL4.3 infection of Jurkat T cells treated or not with EPL. P = 0.0027. (C) Jurkat T cell infection with HIV-1 NL4.3 at an MOI of 0.05. SP was added to the culture medium at the concentrations indicated. The number of infected cells was measured by Gag labeling followed by flow cytometry analysis, and the IC 50 was determined. (D) HIV-2 GL-AN infection of Jurkat T cells treated or not with SP. P = 0.005. (E) Kinetics of HIV-1 infection in the presence or absence of SP for 12 days. Jurkat T cells were infected with HIV-1 NL4.3 and cultured with DMSO or SP; the medium was changed every 3 days. For some cells at 3 dpi, SP was replaced by RPMI medium without further addition of the drug. For some cells at 6 dpi, DMSO was replaced by SP. The results are the means of four independent experiments performed in duplicate (A and D) and of two independent experiments performed in triplicate (B, C, and E). (A, B, and C) The values were normalized, taking as 100% the value obtained for one replicate of control cells treated with DMSO. Unless otherwise indicated, SP or EPL was added to the culture medium at a concentration of 10 μM for 48 h. The error bars represent SEM. **, P
    Figure Legend Snippet: Spironolactone inhibits HIV-1 and HIV-2 infection of Jurkat T cells. (A) HIV-1 NL4.3 infection of Jurkat T cells treated or not with SP. The number of infected cells was measured by Gag labeling followed by flow cytometry analysis. P = 0.005. (B) HIV-1 NL4.3 infection of Jurkat T cells treated or not with EPL. P = 0.0027. (C) Jurkat T cell infection with HIV-1 NL4.3 at an MOI of 0.05. SP was added to the culture medium at the concentrations indicated. The number of infected cells was measured by Gag labeling followed by flow cytometry analysis, and the IC 50 was determined. (D) HIV-2 GL-AN infection of Jurkat T cells treated or not with SP. P = 0.005. (E) Kinetics of HIV-1 infection in the presence or absence of SP for 12 days. Jurkat T cells were infected with HIV-1 NL4.3 and cultured with DMSO or SP; the medium was changed every 3 days. For some cells at 3 dpi, SP was replaced by RPMI medium without further addition of the drug. For some cells at 6 dpi, DMSO was replaced by SP. The results are the means of four independent experiments performed in duplicate (A and D) and of two independent experiments performed in triplicate (B, C, and E). (A, B, and C) The values were normalized, taking as 100% the value obtained for one replicate of control cells treated with DMSO. Unless otherwise indicated, SP or EPL was added to the culture medium at a concentration of 10 μM for 48 h. The error bars represent SEM. **, P

    Techniques Used: Infection, Labeling, Flow Cytometry, Cytometry, Cell Culture, Concentration Assay

    Spironolactone, but not eplerenone, inhibits HIV-1 transduction. (A) XPB protein levels in Jurkat T cells treated with DMSO, SP, or EPL. (B) Jurkat T cell transduction with the single-cycle HIV-1-CMV-GFP. Cells were cultured with or without SP. The number of GFP + cells was analyzed by flow cytometry. P = 0.005. (C) Jurkat T cell transduction with the pseudotyped single-cycle HIV-1-Luc (HIV-1-VSV-G-NL4.3-luciferase-Δenv-Δnef). Cells were cultured or not with SP. P = 0.0004. (D) XPB protein level in Jurkat T cells after HIV-1-Luc transduction and cell culture with DMSO, SP, or EPL. X/G represents the ratio of XPB to GAPDH protein levels. (E) Jurkat T cell transduction with the single-cycle HIV-1 Luc. Cells were treated or not with EPL. P = 0.1977. ns, no statistically significant difference. (F) Jurkat T cell transduction with two different amounts of the single-cycle HIV-1-Luc. Cells were cultured with increasing concentrations of SP, and the IC 50 was determined. (G) Jurkat cell transduction with the single-cycle HIV-2 ROD9 (HIV-2-Rod9-VSV-G-GFP-Δenv). Cells were treated or not with SP. The results are the means of three independent experiments performed in duplicate. P = 0.0004. (H) Jurkat cell transduction with the single-cycle HIV-2 ROD9, treated or not with EPL. The results are the means of three independent experiments performed in duplicate. P = 0.1977. The values were normalized, taking as 100% the value obtained for one replicate of control cells treated with DMSO. The error bars represent standard errors of the mean (SEM). Unless otherwise indicated, SP or EPL was added to the culture medium at a concentration of 10 μM for 48 h. **, P
    Figure Legend Snippet: Spironolactone, but not eplerenone, inhibits HIV-1 transduction. (A) XPB protein levels in Jurkat T cells treated with DMSO, SP, or EPL. (B) Jurkat T cell transduction with the single-cycle HIV-1-CMV-GFP. Cells were cultured with or without SP. The number of GFP + cells was analyzed by flow cytometry. P = 0.005. (C) Jurkat T cell transduction with the pseudotyped single-cycle HIV-1-Luc (HIV-1-VSV-G-NL4.3-luciferase-Δenv-Δnef). Cells were cultured or not with SP. P = 0.0004. (D) XPB protein level in Jurkat T cells after HIV-1-Luc transduction and cell culture with DMSO, SP, or EPL. X/G represents the ratio of XPB to GAPDH protein levels. (E) Jurkat T cell transduction with the single-cycle HIV-1 Luc. Cells were treated or not with EPL. P = 0.1977. ns, no statistically significant difference. (F) Jurkat T cell transduction with two different amounts of the single-cycle HIV-1-Luc. Cells were cultured with increasing concentrations of SP, and the IC 50 was determined. (G) Jurkat cell transduction with the single-cycle HIV-2 ROD9 (HIV-2-Rod9-VSV-G-GFP-Δenv). Cells were treated or not with SP. The results are the means of three independent experiments performed in duplicate. P = 0.0004. (H) Jurkat cell transduction with the single-cycle HIV-2 ROD9, treated or not with EPL. The results are the means of three independent experiments performed in duplicate. P = 0.1977. The values were normalized, taking as 100% the value obtained for one replicate of control cells treated with DMSO. The error bars represent standard errors of the mean (SEM). Unless otherwise indicated, SP or EPL was added to the culture medium at a concentration of 10 μM for 48 h. **, P

    Techniques Used: Transduction, Cell Culture, Flow Cytometry, Cytometry, Luciferase, Concentration Assay

    64) Product Images from "Changes of Sexual Behaviors in Rapamycin-injected Cichlid Fish Astatotilapia burtoni Males"

    Article Title: Changes of Sexual Behaviors in Rapamycin-injected Cichlid Fish Astatotilapia burtoni Males

    Journal: Development & Reproduction

    doi: 10.12717/DR.2016.20.3.267

    Representative testes of cichlid dominant males. The testes of rapamycin- or DMSO-injected fish were fixed and examined by routine hematoxylin and eosin staining method. Scale bars indicate 500 μm. Sg, spermatogonia; Sc, spermatocyte; sd, spermatid.
    Figure Legend Snippet: Representative testes of cichlid dominant males. The testes of rapamycin- or DMSO-injected fish were fixed and examined by routine hematoxylin and eosin staining method. Scale bars indicate 500 μm. Sg, spermatogonia; Sc, spermatocyte; sd, spermatid.

    Techniques Used: Injection, Fluorescence In Situ Hybridization, Staining

    Changes in chasing behavior of cichlid dominant males. Behavior was recorded by a digital video camera at each morning (08:00 AM) with light onset, counted for 20 min later, and expressed as relative percentages compared to those of non-injected animals (–10 h). After 10 h of the first day recording, intraventricular injection was done with rapamycin (20 μg/μL) or same volume of DMSO (0 h) and fish was further maintained up to +26 h. Data are plotted as mean ± SEM (n=3).
    Figure Legend Snippet: Changes in chasing behavior of cichlid dominant males. Behavior was recorded by a digital video camera at each morning (08:00 AM) with light onset, counted for 20 min later, and expressed as relative percentages compared to those of non-injected animals (–10 h). After 10 h of the first day recording, intraventricular injection was done with rapamycin (20 μg/μL) or same volume of DMSO (0 h) and fish was further maintained up to +26 h. Data are plotted as mean ± SEM (n=3).

    Techniques Used: Injection, Fluorescence In Situ Hybridization

    Representative GnRH1-immunoreactive neurons of cichlid dominant male brains. Rapamycin- or DMSO-injected fish were sacrificed and the brains were applied to an immunohistochemical examination. GnRH1 neurons in the preoptic areas of brains with were detected with a polyclonal guinea pig antibody for cichlid GnRH1 and detailed procedures are described in Materials and Methods. Scale bars indicate 10 μm.
    Figure Legend Snippet: Representative GnRH1-immunoreactive neurons of cichlid dominant male brains. Rapamycin- or DMSO-injected fish were sacrificed and the brains were applied to an immunohistochemical examination. GnRH1 neurons in the preoptic areas of brains with were detected with a polyclonal guinea pig antibody for cichlid GnRH1 and detailed procedures are described in Materials and Methods. Scale bars indicate 10 μm.

    Techniques Used: Injection, Fluorescence In Situ Hybridization, Immunohistochemistry

    65) Product Images from "An Untargeted Metabolomics Approach to Investigate the Metabolic Modulations of HepG2 Cells Exposed to Low Doses of Bisphenol A and 17β-Estradiol"

    Article Title: An Untargeted Metabolomics Approach to Investigate the Metabolic Modulations of HepG2 Cells Exposed to Low Doses of Bisphenol A and 17β-Estradiol

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2018.00571

    Two-dimensional PLS-DA scores plot of HepG2 cell extracts integrated 1 H-NMR spectra for E2 exposure. Each dot or star represents an observation projected onto the first (horizontal axis) and the second (vertical axis) PLS-DA latent variables. E2 doses are shown in different colors: DMSO (green; N = 17), E2 10 −9 M (light orange; N = 12), E2 10 −12 M (dark orange; N = 12), E2 10 −15 M (dark red; N = 12) (R 2 Y = 81.6% and Q 2 = 0.635).
    Figure Legend Snippet: Two-dimensional PLS-DA scores plot of HepG2 cell extracts integrated 1 H-NMR spectra for E2 exposure. Each dot or star represents an observation projected onto the first (horizontal axis) and the second (vertical axis) PLS-DA latent variables. E2 doses are shown in different colors: DMSO (green; N = 17), E2 10 −9 M (light orange; N = 12), E2 10 −12 M (dark orange; N = 12), E2 10 −15 M (dark red; N = 12) (R 2 Y = 81.6% and Q 2 = 0.635).

    Techniques Used: Nuclear Magnetic Resonance

    Two-dimensional PLS-DA score plot of HepG2 cell extracts integrated 1 H-NMR spectra for BPA exposure. Each star represents an observation projected onto the first (horizontal axis) and the second (vertical axis) PLS-DA latent variables. BPA doses are shown in different colors: DMSO (green; N = 17), BPA 10 −6 (light orange; N = 12), BPA 10 −9 (dark orange; N = 12), BPA 10 −12 (dark red; N = 12) (R 2 Y = 62.2% and Q 2 = 0.561).
    Figure Legend Snippet: Two-dimensional PLS-DA score plot of HepG2 cell extracts integrated 1 H-NMR spectra for BPA exposure. Each star represents an observation projected onto the first (horizontal axis) and the second (vertical axis) PLS-DA latent variables. BPA doses are shown in different colors: DMSO (green; N = 17), BPA 10 −6 (light orange; N = 12), BPA 10 −9 (dark orange; N = 12), BPA 10 −12 (dark red; N = 12) (R 2 Y = 62.2% and Q 2 = 0.561).

    Techniques Used: Nuclear Magnetic Resonance

    Two-dimensional PLS-DA scores plot (Axis 1 and 2) of HepG2 cell extracts integrated 1 H-NMR spectra for BPA and E2 exposure. Each dot or star represents an observation projected onto the first (horizontal axis) and the second (vertical axis) PLS-DA latent variables. Different symbols are used for BPA (4-point star) and E2 (circle) exposure. Doses are shown as follow: DMSO: green ( N = 17), BPA 10 −6 M: light orange ( N = 11), BPA 10 −9 M: dark orange; ( N = 12), BPA 10 −12 M: dark red ( N = 12), E2 10 −9 M: light orange ( N = 11), E2 10 −12 M: dark orange ( N = 12), E2 10 −15 M: dark red ( N = 12); (R 2 Y = 59.5% and Q 2 = 0.508).
    Figure Legend Snippet: Two-dimensional PLS-DA scores plot (Axis 1 and 2) of HepG2 cell extracts integrated 1 H-NMR spectra for BPA and E2 exposure. Each dot or star represents an observation projected onto the first (horizontal axis) and the second (vertical axis) PLS-DA latent variables. Different symbols are used for BPA (4-point star) and E2 (circle) exposure. Doses are shown as follow: DMSO: green ( N = 17), BPA 10 −6 M: light orange ( N = 11), BPA 10 −9 M: dark orange; ( N = 12), BPA 10 −12 M: dark red ( N = 12), E2 10 −9 M: light orange ( N = 11), E2 10 −12 M: dark orange ( N = 12), E2 10 −15 M: dark red ( N = 12); (R 2 Y = 59.5% and Q 2 = 0.508).

    Techniques Used: Nuclear Magnetic Resonance

    66) Product Images from "TGF-? Signaling Regulates Pancreatic ?-Cell Proliferation through Control of Cell Cycle Regulator p27 Expression"

    Article Title: TGF-? Signaling Regulates Pancreatic ?-Cell Proliferation through Control of Cell Cycle Regulator p27 Expression

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.12035

    Effects of TGF-β signaling on nuclear localization of p27 (green). To stimulate TGF-β signaling, HIT-T15 cells were transfected with pcDNA.3.1.mRFP1 (red), which served as a transfectant indicator, and either empty vector, pcDNA3.1 ( A ) or a constitutively active form of TRβ-I, ALK5* ( B ). ( C ) Nuclear p27 integrated density (A. U., arbitrary unit) in ( A ) and ( B ). To inhibit TGF-β signaling, HIT-T15 cells were treated with DMSO ( D ) or 2 µM SB-431542 ( E ). ( F ) Nuclear p27 integrated density (A. U.) in ( D ) and ( E ). Nuclei were detected with TO-PRO-3 iodide (blue) ( A, B ) or PI (red) ( D, E ). The data are mean±SD (error bars) of a representative experiment. * p
    Figure Legend Snippet: Effects of TGF-β signaling on nuclear localization of p27 (green). To stimulate TGF-β signaling, HIT-T15 cells were transfected with pcDNA.3.1.mRFP1 (red), which served as a transfectant indicator, and either empty vector, pcDNA3.1 ( A ) or a constitutively active form of TRβ-I, ALK5* ( B ). ( C ) Nuclear p27 integrated density (A. U., arbitrary unit) in ( A ) and ( B ). To inhibit TGF-β signaling, HIT-T15 cells were treated with DMSO ( D ) or 2 µM SB-431542 ( E ). ( F ) Nuclear p27 integrated density (A. U.) in ( D ) and ( E ). Nuclei were detected with TO-PRO-3 iodide (blue) ( A, B ) or PI (red) ( D, E ). The data are mean±SD (error bars) of a representative experiment. * p

    Techniques Used: Transfection, Plasmid Preparation

    Effects of TGF-β signaling on cell proliferation of HIT-T15 cells. ( A ) HIT-T15 cells were cultured in 10% FBS RPMI 1640 medium with or without 5 ng/ml TGF-β1 for 1 day, 2 days, 3 days and 4 days (6 independent repeats of experiments). ( B ) HIT-T15 cells were cultured in 10% FBS RPMI 1640 medium containing DMSO or 2 µM SB-431542 for 1 day, 2 days, 3 days and 4 days. Six repeats of the experiment were done independently. The data are mean±SD (error bars) of a representative experiment. * p
    Figure Legend Snippet: Effects of TGF-β signaling on cell proliferation of HIT-T15 cells. ( A ) HIT-T15 cells were cultured in 10% FBS RPMI 1640 medium with or without 5 ng/ml TGF-β1 for 1 day, 2 days, 3 days and 4 days (6 independent repeats of experiments). ( B ) HIT-T15 cells were cultured in 10% FBS RPMI 1640 medium containing DMSO or 2 µM SB-431542 for 1 day, 2 days, 3 days and 4 days. Six repeats of the experiment were done independently. The data are mean±SD (error bars) of a representative experiment. * p

    Techniques Used: Cell Culture

    Effects of TGF-β signaling on expression of p27. (A) HIT-T15 cells were treated with or without 5 ng/ml TGF-β1, and/or with DMSO or 2 µM SB-431542, for 2 days. Western blotting of the whole cell extracts for p27, and GAPDH, which was used as a control. (B) Results are displayed as the relative protein level of p27. Six repeats of the Western blotting were done independently. The data are mean±SD (error bars) of a representative experiment. * p
    Figure Legend Snippet: Effects of TGF-β signaling on expression of p27. (A) HIT-T15 cells were treated with or without 5 ng/ml TGF-β1, and/or with DMSO or 2 µM SB-431542, for 2 days. Western blotting of the whole cell extracts for p27, and GAPDH, which was used as a control. (B) Results are displayed as the relative protein level of p27. Six repeats of the Western blotting were done independently. The data are mean±SD (error bars) of a representative experiment. * p

    Techniques Used: Expressing, Western Blot

    67) Product Images from "3-Methylcholanthrene Induces Differential Recruitment of Aryl Hydrocarbon Receptor to Human Promoters"

    Article Title: 3-Methylcholanthrene Induces Differential Recruitment of Aryl Hydrocarbon Receptor to Human Promoters

    Journal: Toxicological sciences : an official journal of the Society of Toxicology

    doi: 10.1093/toxsci/kfq096

    Repeat dosing with 3MC but not TCDD results in changes in mRNA expression levels of AHR target genes. T-47D cells were treated with DMSO, 3MC (1μM), or TCDD (10nM) for 24 h only or were re-dosed with each of the ligands for an additional 6 h.
    Figure Legend Snippet: Repeat dosing with 3MC but not TCDD results in changes in mRNA expression levels of AHR target genes. T-47D cells were treated with DMSO, 3MC (1μM), or TCDD (10nM) for 24 h only or were re-dosed with each of the ligands for an additional 6 h.

    Techniques Used: Expressing

    3MC-induced recruitment of AHR to regions identified by ChIP-chip. Quantification of AHR binding was determined as fold induction above IgG DMSO and is expressed as the mean of three independent replicates. Regions were chosen to cover a range of enrichment
    Figure Legend Snippet: 3MC-induced recruitment of AHR to regions identified by ChIP-chip. Quantification of AHR binding was determined as fold induction above IgG DMSO and is expressed as the mean of three independent replicates. Regions were chosen to cover a range of enrichment

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay

    3MC induces gene-specific changes in histone H3 acetylation and methylation. T-47D cells were treated with 1μM 3MC or DMSO for 1 and 4 h. ChIP assays were performed with specific antibodies against IgG, H3, H3K4Me2, and H3K9Ac. Immunoprecipitated
    Figure Legend Snippet: 3MC induces gene-specific changes in histone H3 acetylation and methylation. T-47D cells were treated with 1μM 3MC or DMSO for 1 and 4 h. ChIP assays were performed with specific antibodies against IgG, H3, H3K4Me2, and H3K9Ac. Immunoprecipitated

    Techniques Used: Methylation, Chromatin Immunoprecipitation, Immunoprecipitation

    68) Product Images from "Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD"

    Article Title: Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0144864

    Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in StHdh Q7/7 and StHdh Q111/111 cells at 0 mins and 10 mins of 100ng/ml EGF stimulation, either with or without a prior 2 hour incubation with 500nM AKT inhibitor VIII or 1μM MEK 1/2 inhibitor. In cases where inhibitors were not used, cells were incubated with the equivalent volume of DMSO for the same amount of time prior to treatment and processing. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. Grey asterisks indicate genotypic differences. N = 3 replications *p
    Figure Legend Snippet: Mean absorbance (Abs) read at 450nm following a sandwich ELISA protocol for the detection of phosphorylated A. AKT1 and B. MEK1 in StHdh Q7/7 and StHdh Q111/111 cells at 0 mins and 10 mins of 100ng/ml EGF stimulation, either with or without a prior 2 hour incubation with 500nM AKT inhibitor VIII or 1μM MEK 1/2 inhibitor. In cases where inhibitors were not used, cells were incubated with the equivalent volume of DMSO for the same amount of time prior to treatment and processing. Error bars = ±SEM. Black asterisks denote a significant difference from 0 mins + DMSO. Grey asterisks indicate genotypic differences. N = 3 replications *p

    Techniques Used: Sandwich ELISA, Incubation

    Relative quantitation (RQ) values representing gene expression fold change of Egr1 , Arc and Ngfib in StHdh Q7/7 and StHdh Q111/111 cells following inhibition with either 500nM AKT inhibitor VIII, 1μM MEK 1/2 inhibitor or the equivalent volume of DMSO, followed by 0 or 2 hours of 100ng/ml EGF stimulation. Statistical analysis was conducted on ΔC t values. Error bars = ± SEM. Asterisks denote a significant difference from 0 + DMSO, hashes indicate a significant difference from EGF + DMSO. Data representative of two experiments. N = 6.*/# p
    Figure Legend Snippet: Relative quantitation (RQ) values representing gene expression fold change of Egr1 , Arc and Ngfib in StHdh Q7/7 and StHdh Q111/111 cells following inhibition with either 500nM AKT inhibitor VIII, 1μM MEK 1/2 inhibitor or the equivalent volume of DMSO, followed by 0 or 2 hours of 100ng/ml EGF stimulation. Statistical analysis was conducted on ΔC t values. Error bars = ± SEM. Asterisks denote a significant difference from 0 + DMSO, hashes indicate a significant difference from EGF + DMSO. Data representative of two experiments. N = 6.*/# p

    Techniques Used: Quantitation Assay, Expressing, Inhibition

    A. StHdh Q7/7 , B. StHdh Q7/111 and C. StHdh Q111/111 cells treated with either AKT inhibitor VIII, MEK 1/2 inhibitor, or the equivalent volume of DMSO for 2 hours prior to 0, 5, 15 and 30 mins stimulation with 100ng/ml EGF, then probed with amino-terminal huntingtin antibody Mab2166. Scale bar = 20μm. D-E. Quantification of mean pixel intensity (MPI) from images represented in A-C for the D. Nuclear/Cytoplasmic (N/C) ratio and E. Nuclear/Perinuclear (N/P) ratio. Error bars = SEM. Light grey bars and asterisks signify statistically significant differences between DMSO conditions. Black asterisks and hashes indicate statistically significant differences between DMSO vs AKT inhibitor conditions and DMSO vs MEK inhibitor conditions, respectively. Data representative of three experiments. n = 85–135. */# p
    Figure Legend Snippet: A. StHdh Q7/7 , B. StHdh Q7/111 and C. StHdh Q111/111 cells treated with either AKT inhibitor VIII, MEK 1/2 inhibitor, or the equivalent volume of DMSO for 2 hours prior to 0, 5, 15 and 30 mins stimulation with 100ng/ml EGF, then probed with amino-terminal huntingtin antibody Mab2166. Scale bar = 20μm. D-E. Quantification of mean pixel intensity (MPI) from images represented in A-C for the D. Nuclear/Cytoplasmic (N/C) ratio and E. Nuclear/Perinuclear (N/P) ratio. Error bars = SEM. Light grey bars and asterisks signify statistically significant differences between DMSO conditions. Black asterisks and hashes indicate statistically significant differences between DMSO vs AKT inhibitor conditions and DMSO vs MEK inhibitor conditions, respectively. Data representative of three experiments. n = 85–135. */# p

    Techniques Used:

    69) Product Images from "Anti-Inflammatory Effects of Licorice and Roasted Licorice Extracts on TPA-Induced Acute Inflammation and Collagen-Induced Arthritis in Mice"

    Article Title: Anti-Inflammatory Effects of Licorice and Roasted Licorice Extracts on TPA-Induced Acute Inflammation and Collagen-Induced Arthritis in Mice

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2010/709378

    Effects of LE and rLE on the serum TNF- α and IL-1 β levels, and pro-inflammatory immune response of spleen cell in CIA mice. CIA mice with the clinical signs of arthritis were orally administered the vehicle (PBS containing 1% DMSO), LE (10 mg/kg) or rLE (10 mg/kg) once daily from day 25 to day 45. Control mice were neither immunized with CII nor treated with extracts. (a) The serum levels of TNF- α and IL-1 β were measured by ELISA assay. (b) Single-cell suspensions from spleens were obtained from control and all CIA mice. The proliferation of spleen cells stimulated with 50 μ g/ml denatured CII for 72 hours was assessed using a BrdU cell proliferation ELISA kit (left), and TNF- α and IL-1 β levels were measured in conditioned media of spleen cells stimulated with 50 μ g/ml denatured CII or 5 μ g/ml LPS for 48 hours, using each specific ELISA kit (middle and right). Data is expressed as mean ± SE of 5 mice per group. # P
    Figure Legend Snippet: Effects of LE and rLE on the serum TNF- α and IL-1 β levels, and pro-inflammatory immune response of spleen cell in CIA mice. CIA mice with the clinical signs of arthritis were orally administered the vehicle (PBS containing 1% DMSO), LE (10 mg/kg) or rLE (10 mg/kg) once daily from day 25 to day 45. Control mice were neither immunized with CII nor treated with extracts. (a) The serum levels of TNF- α and IL-1 β were measured by ELISA assay. (b) Single-cell suspensions from spleens were obtained from control and all CIA mice. The proliferation of spleen cells stimulated with 50 μ g/ml denatured CII for 72 hours was assessed using a BrdU cell proliferation ELISA kit (left), and TNF- α and IL-1 β levels were measured in conditioned media of spleen cells stimulated with 50 μ g/ml denatured CII or 5 μ g/ml LPS for 48 hours, using each specific ELISA kit (middle and right). Data is expressed as mean ± SE of 5 mice per group. # P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effects of LE and rLE on TPA-induced mouse ear edema. The right ear of each ICR mouse was topically treated with LE and rLE (1.0 and 2.0 mg/ear) in 50 μ l vehicle (DMSO-acetone: 15–85, v/v) 30 minutes prior to the application of 5 nmol TPA in 50 μ l vehicle. The left ears were treated with vehicle alone. Four hours later, edema was measured as the increase in the weight of the right ear punch over that of the left. Data are expressed as means ± SE of 5 mice per group. * P
    Figure Legend Snippet: Effects of LE and rLE on TPA-induced mouse ear edema. The right ear of each ICR mouse was topically treated with LE and rLE (1.0 and 2.0 mg/ear) in 50 μ l vehicle (DMSO-acetone: 15–85, v/v) 30 minutes prior to the application of 5 nmol TPA in 50 μ l vehicle. The left ears were treated with vehicle alone. Four hours later, edema was measured as the increase in the weight of the right ear punch over that of the left. Data are expressed as means ± SE of 5 mice per group. * P

    Techniques Used: Mouse Assay

    70) Product Images from "Raised Activity of L-Type Calcium Channels Renders Neurons Prone to Form Paroxysmal Depolarization Shifts"

    Article Title: Raised Activity of L-Type Calcium Channels Renders Neurons Prone to Form Paroxysmal Depolarization Shifts

    Journal: Neuromolecular Medicine

    doi: 10.1007/s12017-013-8234-1

    LTCCs can both enhance and reduce low-Mg 2+ -induced seizure-like activity. Seizure-like activity was induced repeatedly for 2-min with 5-min intervals, and DMSO, BayK or isradipine was co-administered in the low-Mg 2+ saline. To account for differences in the delay till the onset, 90 s of SLA out of 120-s superfusion time were evaluated by determining the area between the trace and a baseline set at the membrane potential just prior to the change of the superfusion saline (dashed line in the original traces below). a – c The graphs show the results for all neurons ( a , n = 14) and after separation of the data according to whether an increase ( b , n = 7) or a decrease in the area ( c , n = 7) was determined for SLA in BayK. Data are shown normalized to control areas (DMSO). Statistical difference between BayK and isradipine data was tested using Wilcoxon matched-pairs signed rank test (n.s. not significant; * P
    Figure Legend Snippet: LTCCs can both enhance and reduce low-Mg 2+ -induced seizure-like activity. Seizure-like activity was induced repeatedly for 2-min with 5-min intervals, and DMSO, BayK or isradipine was co-administered in the low-Mg 2+ saline. To account for differences in the delay till the onset, 90 s of SLA out of 120-s superfusion time were evaluated by determining the area between the trace and a baseline set at the membrane potential just prior to the change of the superfusion saline (dashed line in the original traces below). a – c The graphs show the results for all neurons ( a , n = 14) and after separation of the data according to whether an increase ( b , n = 7) or a decrease in the area ( c , n = 7) was determined for SLA in BayK. Data are shown normalized to control areas (DMSO). Statistical difference between BayK and isradipine data was tested using Wilcoxon matched-pairs signed rank test (n.s. not significant; * P

    Techniques Used: Activity Assay

    Levels of LTCC-mediated calcium currents in primary hippocampal neurons. a LTCC-mediated current components in total voltage-gated calcium currents were determined by applying ramp depolarizations (0.5 mV/ms) from −80 mV (=holding potential) to +50 mV and measurement of calcium current reduction upon a 90-s administration of 3 μM isradipine. The three traces depict the peak currents evoked under control conditions (DMSO), 3 μM isradipine and after washout of the dihydropyridine. b The reversible reduction was monitored by reading the peak of currents that were elicited every 10 s (e.g., sweeps 8–16 in the experiment shown). c Percentage of isradipine inhibited current with respect to total voltage-activated currents calculated from measurements as shown in a , b . Neurons were grouped according to the age of the cultures, as indicated on the x- axes. Neurons that had been kept in culture for at least 10 days but not longer than 2 weeks were allocated to the ≤14 days in vitro (DIV) group ( n = 16), neurons that had been maintained in culture for more than 4 weeks and maximally up to 5 weeks were allocated to the > 28 DIV group ( n = 19). n for the ≤21 DIV and ≤28 DIV was 17 and 15, respectively. Considerably variation of LTCC current density exists in all age groups, yet statistically groups do not significantly differ from each other. d Same data as in c. LTCC current density (pA/pF) was determined by relating of the dihydropyridine-sensitive current component to cell capacitance as a measure of cell surface. To highlight the intrinsic variation, data in c and d are shown as box-plots with min to max whiskers
    Figure Legend Snippet: Levels of LTCC-mediated calcium currents in primary hippocampal neurons. a LTCC-mediated current components in total voltage-gated calcium currents were determined by applying ramp depolarizations (0.5 mV/ms) from −80 mV (=holding potential) to +50 mV and measurement of calcium current reduction upon a 90-s administration of 3 μM isradipine. The three traces depict the peak currents evoked under control conditions (DMSO), 3 μM isradipine and after washout of the dihydropyridine. b The reversible reduction was monitored by reading the peak of currents that were elicited every 10 s (e.g., sweeps 8–16 in the experiment shown). c Percentage of isradipine inhibited current with respect to total voltage-activated currents calculated from measurements as shown in a , b . Neurons were grouped according to the age of the cultures, as indicated on the x- axes. Neurons that had been kept in culture for at least 10 days but not longer than 2 weeks were allocated to the ≤14 days in vitro (DIV) group ( n = 16), neurons that had been maintained in culture for more than 4 weeks and maximally up to 5 weeks were allocated to the > 28 DIV group ( n = 19). n for the ≤21 DIV and ≤28 DIV was 17 and 15, respectively. Considerably variation of LTCC current density exists in all age groups, yet statistically groups do not significantly differ from each other. d Same data as in c. LTCC current density (pA/pF) was determined by relating of the dihydropyridine-sensitive current component to cell capacitance as a measure of cell surface. To highlight the intrinsic variation, data in c and d are shown as box-plots with min to max whiskers

    Techniques Used: Mass Spectrometry, In Vitro

    Effect of LTCC activity on EPSPs-2. Pharmacological potentiation of LTCCs augments (short) super-threshold synaptic potentials (“spike events,” a ) and promotes the formation of depolarization shifts (see middle traces in a ), but at the same time leaves sub-threshold EPSPs (“small events,” b ) unaltered. Isradipine reverses the effect of BayK. Each graph shows an overlay of 5 arbitrarily chosen EPSPs recorded in DMSO ( dark blue traces ), BayK ( green traces ) and isradipine ( red traces ). c Overlay of representative traces from this experiment recorded under the three experimental conditions. d Statistical comparisons of small event and spike event data, respectively, from a total of 12 experiments identical to the one illustrated in a – c (see main text for details). n.s. indicates a lack of statistical significance, *** P value
    Figure Legend Snippet: Effect of LTCC activity on EPSPs-2. Pharmacological potentiation of LTCCs augments (short) super-threshold synaptic potentials (“spike events,” a ) and promotes the formation of depolarization shifts (see middle traces in a ), but at the same time leaves sub-threshold EPSPs (“small events,” b ) unaltered. Isradipine reverses the effect of BayK. Each graph shows an overlay of 5 arbitrarily chosen EPSPs recorded in DMSO ( dark blue traces ), BayK ( green traces ) and isradipine ( red traces ). c Overlay of representative traces from this experiment recorded under the three experimental conditions. d Statistical comparisons of small event and spike event data, respectively, from a total of 12 experiments identical to the one illustrated in a – c (see main text for details). n.s. indicates a lack of statistical significance, *** P value

    Techniques Used: Activity Assay

    71) Product Images from "Astilbin Inhibits the Activity of Sortase A from Streptococcus mutans"

    Article Title: Astilbin Inhibits the Activity of Sortase A from Streptococcus mutans

    Journal: Molecules

    doi: 10.3390/molecules24030465

    The minimum inhibitory concentration (MIC) of astilbin against S. mutans and the growth curves of S. mutans treated with astilbin. ( A ) The growth state of S. mutans in the presence of different concentrations of astilbin. “Neg” represents the negative control group and “Pos” represents the positive control group. The blank group contained only the brain heart infusion (BHI) broth and the tested S. mutans , the negative control group was treated with 1% dimethyl sulfoxide (DMSO), and the positive control group was treated with 20 μg/mL of chlorhexidine. ( B ) The growth curves of S. mutans treated with different concentrations of astilbin. The initial inoculum was approximately 1 × 10 6 colony-forming units/mL. The growth rates were measured by determining the optical density (OD) every two hours for one day.
    Figure Legend Snippet: The minimum inhibitory concentration (MIC) of astilbin against S. mutans and the growth curves of S. mutans treated with astilbin. ( A ) The growth state of S. mutans in the presence of different concentrations of astilbin. “Neg” represents the negative control group and “Pos” represents the positive control group. The blank group contained only the brain heart infusion (BHI) broth and the tested S. mutans , the negative control group was treated with 1% dimethyl sulfoxide (DMSO), and the positive control group was treated with 20 μg/mL of chlorhexidine. ( B ) The growth curves of S. mutans treated with different concentrations of astilbin. The initial inoculum was approximately 1 × 10 6 colony-forming units/mL. The growth rates were measured by determining the optical density (OD) every two hours for one day.

    Techniques Used: Concentration Assay, Negative Control, Positive Control

    Inhibition of S. mutans biofilm formation by astilbin. ( A ) Photograph of S. mutans biofilms grown in the wells of a 96-well plate. Cells were cultured in BHI media containing different concentrations of astilbin for 16 h and stained with 0.1% crystal violet. The group treated with 128 μg/mL of morin served as a positive control (Con). The DMSO treatment group served as a negative control. ( B ) Quantification of the biomass of S. mutans treated with astilbin. The data were obtained from three independent experiments. Significant differences between groups were accepted at ** p
    Figure Legend Snippet: Inhibition of S. mutans biofilm formation by astilbin. ( A ) Photograph of S. mutans biofilms grown in the wells of a 96-well plate. Cells were cultured in BHI media containing different concentrations of astilbin for 16 h and stained with 0.1% crystal violet. The group treated with 128 μg/mL of morin served as a positive control (Con). The DMSO treatment group served as a negative control. ( B ) Quantification of the biomass of S. mutans treated with astilbin. The data were obtained from three independent experiments. Significant differences between groups were accepted at ** p

    Techniques Used: Inhibition, Cell Culture, Staining, Positive Control, Negative Control

    72) Product Images from "Crosstalk between Hedgehog pathway and energy pathways in human adipose-derived stem cells: A deep sequencing analysis of polysome-associated RNA"

    Article Title: Crosstalk between Hedgehog pathway and energy pathways in human adipose-derived stem cells: A deep sequencing analysis of polysome-associated RNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26533-y

    Comparison between differentially expressed mRNAs in free and polysomal fractions. ( A ) Venn diagram shows the number of differentially represented mRNAs in the ribosome-free fraction (mRNA not associated with polysome) from cells treated with cyclopamine and purmorphamine compared to that from cells treated with DMSO. ( B ) Comparative enrichment analyzes of the mRNAs that increased over the control (DMSO). ( C ) Scheme representing the free and polysomal fraction mRNA and their respective biological processes. ( D , E ) Venn diagram shows the number of up-represented and down-represented mRNAs in ADSCs treated with cyclopamine ( D ) and purmorphamine ( E ).
    Figure Legend Snippet: Comparison between differentially expressed mRNAs in free and polysomal fractions. ( A ) Venn diagram shows the number of differentially represented mRNAs in the ribosome-free fraction (mRNA not associated with polysome) from cells treated with cyclopamine and purmorphamine compared to that from cells treated with DMSO. ( B ) Comparative enrichment analyzes of the mRNAs that increased over the control (DMSO). ( C ) Scheme representing the free and polysomal fraction mRNA and their respective biological processes. ( D , E ) Venn diagram shows the number of up-represented and down-represented mRNAs in ADSCs treated with cyclopamine ( D ) and purmorphamine ( E ).

    Techniques Used:

    The transcriptional factor GLI1 is located in the nucleus of ADSCs. ( A , B ) qRT-PCR analysis of the level of GLI1 and PTCH1 mRNA in ADSCs treated with purmorphamine and cyclopamine during 1, 3 and 5 days; ( A ) GLI1 mRNA ( B ) PTCH1 mRNA. GAPDH and POLR2A were used as an internal housekeeping gene control. (Biological replicates = 2–6, each pont represent of the average of the technical triplicate, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). ( C ) Indirect immunofluorescence staining of GLI1 (green) in ADSCs after 24 h of DMSO, purmorphamine, or cyclopamine treatment. Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm. ( D ) High-throughput imaging: GLI1+ staining intensity in the nucleus of ADSCs treated with DMSO, purmorphamine, and cyclopamine for 24 h. Object Number represents each cell that received a number according to the reading of the image. ( E – H ) Percentage of cells GLI1+ in to the nucleus and cytoplasm treated with DMSO (control), purmorphamine, and cyclopamine (n = 4–5). ( E ) Percentage of cells GLI1+ nuclei; ( F ) Percentage of cells GLI1+ Nuclei Low intensity; ( G ) Percentage of cells GLI1+ Nuclei High intensity; ( H ) Percentage of cells GLI1+ Cytoplasmic. There were no statistically significant differences between group means as determined by one-way ANOVA.
    Figure Legend Snippet: The transcriptional factor GLI1 is located in the nucleus of ADSCs. ( A , B ) qRT-PCR analysis of the level of GLI1 and PTCH1 mRNA in ADSCs treated with purmorphamine and cyclopamine during 1, 3 and 5 days; ( A ) GLI1 mRNA ( B ) PTCH1 mRNA. GAPDH and POLR2A were used as an internal housekeeping gene control. (Biological replicates = 2–6, each pont represent of the average of the technical triplicate, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). ( C ) Indirect immunofluorescence staining of GLI1 (green) in ADSCs after 24 h of DMSO, purmorphamine, or cyclopamine treatment. Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm. ( D ) High-throughput imaging: GLI1+ staining intensity in the nucleus of ADSCs treated with DMSO, purmorphamine, and cyclopamine for 24 h. Object Number represents each cell that received a number according to the reading of the image. ( E – H ) Percentage of cells GLI1+ in to the nucleus and cytoplasm treated with DMSO (control), purmorphamine, and cyclopamine (n = 4–5). ( E ) Percentage of cells GLI1+ nuclei; ( F ) Percentage of cells GLI1+ Nuclei Low intensity; ( G ) Percentage of cells GLI1+ Nuclei High intensity; ( H ) Percentage of cells GLI1+ Cytoplasmic. There were no statistically significant differences between group means as determined by one-way ANOVA.

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, High Throughput Screening Assay, Imaging

    Different mRNAs associated with polysomes from ADSCs treated with cyclopamine and purmorphamine. ( A ) Polysomal profile of ADSCs treated with DMSO, cyclopamine, and purmorphamine. ( B ) Venn diagram shows the number of mRNAs associated with the polysomes in cells treated with cyclopamine and purmorphamine compared to that in cells treated with DMSO. ( C ), ( D ) Venn diagram shows the mRNAs in to Energy pathways term in each condition ( C ). (F , G ) Gene ontology (Panther analysis) shows the biological processes involving the mRNAs associated with the polysomes in cells treated with ( F ) cyclopamine and ( G ) purmorphamine. ( H , I ) Representation of gene networks associated with lipid metabolism (Ingenuity Pathway Analysis) for both treatment: Cells treated with cyclopamine ( H ) and purmorphamine ( I ). Green: down-represented in polysomes. Red: up-represented in polysomes.
    Figure Legend Snippet: Different mRNAs associated with polysomes from ADSCs treated with cyclopamine and purmorphamine. ( A ) Polysomal profile of ADSCs treated with DMSO, cyclopamine, and purmorphamine. ( B ) Venn diagram shows the number of mRNAs associated with the polysomes in cells treated with cyclopamine and purmorphamine compared to that in cells treated with DMSO. ( C ), ( D ) Venn diagram shows the mRNAs in to Energy pathways term in each condition ( C ). (F , G ) Gene ontology (Panther analysis) shows the biological processes involving the mRNAs associated with the polysomes in cells treated with ( F ) cyclopamine and ( G ) purmorphamine. ( H , I ) Representation of gene networks associated with lipid metabolism (Ingenuity Pathway Analysis) for both treatment: Cells treated with cyclopamine ( H ) and purmorphamine ( I ). Green: down-represented in polysomes. Red: up-represented in polysomes.

    Techniques Used:

    73) Product Images from "Regulation of Protein Tyrosine Kinase Signaling by Substrate Degradation during Brain Development"

    Article Title: Regulation of Protein Tyrosine Kinase Signaling by Substrate Degradation during Brain Development

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.24.9293-9302.2003

    Dab1 is polyubiquitinated and degraded via the proteasome pathway in response to the presence of Reelin. (A) Mouse cortical neuron cultures were treated for 5 h with mock (lane 1) or Reelin-containing (lanes 2 to 6) supernatant in the presence or absence of DMSO (lane 3), MG132 ([10 μM]) (lane 4), epoxomicin (Epoxo [10 μM]) (lane 5), or chloroquine (Chloro [10 μM]) (lane 6). Total lysates were subjected to SDS-PAGE and Western blot analysis using anti-Dab1 antibodies to detect total Dab1 (Dab1 WB), an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated Dab1 [pDab1(Tyr) WB], or an anti-Abl antibody (Abl WB) as a loading control. (B) Neuron cultures were treated for 1 h with mock (lane 1) or Reelin-containing (lane 2) supernatant in the presence of MG132 (10 μM) to inhibit degradation of polyubiquitinated proteins by the proteasome. Neuron lysates were then subjected to immunoprecipitation with anti-Dab1 antibodies (Dab1 IP), and the immunoprecipitates were analyzed by SDS-7% PAGE and Western blotting with anti-Dab1 antibodies (Dab1 WB). The respective positions of unmodified Dab1 (Dab1) and high-molecular-mass Dab1 species (arrow) are indicated. (C) Neuron cultures were treated as described for panel B except that MG132 was added after 0 min (lanes 1 and 2) or 15 min (lanes 3 and 4) of mock (lanes 1 and 3) or Reelin (lanes 2 and 4) treatment. Ubiquitinated proteins were immunoprecipitated from neuron lysates with the FK2 antibody (FK2 IP) and analyzed by SDS-7% PAGE and Western blotting with anti-Dab1 antibodies (Dab1 WB). The position of polyubiquitinated Dab1 (Ubn-Dab1) is indicated.
    Figure Legend Snippet: Dab1 is polyubiquitinated and degraded via the proteasome pathway in response to the presence of Reelin. (A) Mouse cortical neuron cultures were treated for 5 h with mock (lane 1) or Reelin-containing (lanes 2 to 6) supernatant in the presence or absence of DMSO (lane 3), MG132 ([10 μM]) (lane 4), epoxomicin (Epoxo [10 μM]) (lane 5), or chloroquine (Chloro [10 μM]) (lane 6). Total lysates were subjected to SDS-PAGE and Western blot analysis using anti-Dab1 antibodies to detect total Dab1 (Dab1 WB), an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated Dab1 [pDab1(Tyr) WB], or an anti-Abl antibody (Abl WB) as a loading control. (B) Neuron cultures were treated for 1 h with mock (lane 1) or Reelin-containing (lane 2) supernatant in the presence of MG132 (10 μM) to inhibit degradation of polyubiquitinated proteins by the proteasome. Neuron lysates were then subjected to immunoprecipitation with anti-Dab1 antibodies (Dab1 IP), and the immunoprecipitates were analyzed by SDS-7% PAGE and Western blotting with anti-Dab1 antibodies (Dab1 WB). The respective positions of unmodified Dab1 (Dab1) and high-molecular-mass Dab1 species (arrow) are indicated. (C) Neuron cultures were treated as described for panel B except that MG132 was added after 0 min (lanes 1 and 2) or 15 min (lanes 3 and 4) of mock (lanes 1 and 3) or Reelin (lanes 2 and 4) treatment. Ubiquitinated proteins were immunoprecipitated from neuron lysates with the FK2 antibody (FK2 IP) and analyzed by SDS-7% PAGE and Western blotting with anti-Dab1 antibodies (Dab1 WB). The position of polyubiquitinated Dab1 (Ubn-Dab1) is indicated.

    Techniques Used: SDS Page, Western Blot, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    Reelin stimulation induces degradation of Dab1 in primary cortical neurons. (A) Mouse cortical neuron cultures were left untreated (lanes 1 and 13) or treated with Reelin-containing (lanes 2 and 3, 5 to 7, and 9 to 12) or mock (lanes 4 and 8) supernatant for the indicated times. Total lysates were subjected to SDS-7.5% PAGE and Western blot analysis using an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated Dab1 [pDab1(Tyr) WB], anti-Dab1 antibodies to detect total Dab1 (Dab1 WB), anti-β catenin antibody (β catenin WB), or anti-Fyn antibodies (Fyn WB). (B) Quantification of the data presented in panel A was used to determine relative levels of tyrosine-phosphorylated Dab1 [pDab1(Tyr)] and total Dab1 after Reelin stimulation for various times. In both cases, levels were normalized to those in untreated neuron cultures (set at 1.0). (C) Neuron cultures were treated with 20 μg of cycloheximide/ml (CHX; lanes 1 to 4) and either Reelin-containing (lane 5) or mock (lane 6) supernatant for the indicated times. Total lysates were subjected to SDS-8% PAGE and Western blot analysis using anti-Dab1 antibodies (Dab1 WB) or an anti-Abl antibody (Abl WB) as a loading control. (D) Neuron cultures were treated for 5 h with mock (lanes 1, 3, and 5) or Reelin-containing (lanes 2, 4, and 6) supernatant, washed, and returned to normal growth medium for the indicated times. Total lysates were subjected to SDS-PAGE and Western blot analysis using anti-Dab1 antibodies to detect total Dab1 (Dab1 WB) or an anti-Abl antibody (Abl WB) as a loading control. (E) Dab1 can be resolved into two forms, Dab1 p80L and p80U. Lysates were prepared from neuron cultures treated for 15 min with Reelin-containing or mock supernatant as indicated. A portion was analyzed directly by Western blotting of serial diluted samples: 50 (lanes 1 and 2), 25 (lane 3), 12.5 (lane 4), and 6.25 (lane 5) μg. A second portion was immunoprecipitated (IP) with an anti-phosphotyrosine antibody (4G10 IP), and the immunoprecipitates were analyzed in parallel with the total lysates by SDS-9% PAGE (see Materials and Methods for details) and Western blotting with an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated Dab1 [pDab1(Tyr) WB] or anti-Dab1 antibodies (Dab1 WB). The respective positions of Dab1 p80 of slower electrophoretic mobility (upper band [p80U]) and Dab1 p80 of faster mobility (lower band [p80L]) are indicated. Note that only Dab1 p80U is tyrosine phosphorylated and that Dab1 p80U represents approximately two-thirds of total Dab1 after 15 min of Reelin stimulation. The quantity of Reelin-induced tyrosine-phosphorylated Dab1 in the 4G10 IP is equivalent to that present in 25 μg of total lysate, while the quantity of Dab1 p80U in the 4G10 IP is equivalent to two-thirds of that present in 6.25 μg of total lysate; therefore, after 15 min of Reelin stimulation, tyrosine-phosphorylated Dab1 represents ∼15% (6.25/25 × 2/3 = 1/6 [∼15%]) of total Dab1 equivalent to ∼25% (6.25/25 = 1/4 [25%]) of Dab1 p80U. (F) Brain lysates prepared from E16.5 embryos were subjected to immunoprecipitation with anti-Dab1 antibodies (Dab1 IP). The immunoprecipitate was treated with λ protein phosphatase (λ-PPase) (lane 1) or left untreated (lane 2) before analysis using SDS-9% PAGE and Western blotting with anti-Dab1 antibodies (Dab1 WB) was performed. (G) Neuron cultures were treated for 1 h with 0.4 μM (lanes 2 and 5), 2 μM (lanes 3 and 6), or 10 μM (lanes 4 and 7) calyculin A (lanes 2 to 4), okadaic acid (lanes 5 to 7), or DMSO (lane 1). Total lysates were subjected to SDS-9% PAGE and Western blot analysis using anti-Dab1 antibodies (Dab1 WB).
    Figure Legend Snippet: Reelin stimulation induces degradation of Dab1 in primary cortical neurons. (A) Mouse cortical neuron cultures were left untreated (lanes 1 and 13) or treated with Reelin-containing (lanes 2 and 3, 5 to 7, and 9 to 12) or mock (lanes 4 and 8) supernatant for the indicated times. Total lysates were subjected to SDS-7.5% PAGE and Western blot analysis using an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated Dab1 [pDab1(Tyr) WB], anti-Dab1 antibodies to detect total Dab1 (Dab1 WB), anti-β catenin antibody (β catenin WB), or anti-Fyn antibodies (Fyn WB). (B) Quantification of the data presented in panel A was used to determine relative levels of tyrosine-phosphorylated Dab1 [pDab1(Tyr)] and total Dab1 after Reelin stimulation for various times. In both cases, levels were normalized to those in untreated neuron cultures (set at 1.0). (C) Neuron cultures were treated with 20 μg of cycloheximide/ml (CHX; lanes 1 to 4) and either Reelin-containing (lane 5) or mock (lane 6) supernatant for the indicated times. Total lysates were subjected to SDS-8% PAGE and Western blot analysis using anti-Dab1 antibodies (Dab1 WB) or an anti-Abl antibody (Abl WB) as a loading control. (D) Neuron cultures were treated for 5 h with mock (lanes 1, 3, and 5) or Reelin-containing (lanes 2, 4, and 6) supernatant, washed, and returned to normal growth medium for the indicated times. Total lysates were subjected to SDS-PAGE and Western blot analysis using anti-Dab1 antibodies to detect total Dab1 (Dab1 WB) or an anti-Abl antibody (Abl WB) as a loading control. (E) Dab1 can be resolved into two forms, Dab1 p80L and p80U. Lysates were prepared from neuron cultures treated for 15 min with Reelin-containing or mock supernatant as indicated. A portion was analyzed directly by Western blotting of serial diluted samples: 50 (lanes 1 and 2), 25 (lane 3), 12.5 (lane 4), and 6.25 (lane 5) μg. A second portion was immunoprecipitated (IP) with an anti-phosphotyrosine antibody (4G10 IP), and the immunoprecipitates were analyzed in parallel with the total lysates by SDS-9% PAGE (see Materials and Methods for details) and Western blotting with an anti-phosphotyrosine antibody to detect tyrosine-phosphorylated Dab1 [pDab1(Tyr) WB] or anti-Dab1 antibodies (Dab1 WB). The respective positions of Dab1 p80 of slower electrophoretic mobility (upper band [p80U]) and Dab1 p80 of faster mobility (lower band [p80L]) are indicated. Note that only Dab1 p80U is tyrosine phosphorylated and that Dab1 p80U represents approximately two-thirds of total Dab1 after 15 min of Reelin stimulation. The quantity of Reelin-induced tyrosine-phosphorylated Dab1 in the 4G10 IP is equivalent to that present in 25 μg of total lysate, while the quantity of Dab1 p80U in the 4G10 IP is equivalent to two-thirds of that present in 6.25 μg of total lysate; therefore, after 15 min of Reelin stimulation, tyrosine-phosphorylated Dab1 represents ∼15% (6.25/25 × 2/3 = 1/6 [∼15%]) of total Dab1 equivalent to ∼25% (6.25/25 = 1/4 [25%]) of Dab1 p80U. (F) Brain lysates prepared from E16.5 embryos were subjected to immunoprecipitation with anti-Dab1 antibodies (Dab1 IP). The immunoprecipitate was treated with λ protein phosphatase (λ-PPase) (lane 1) or left untreated (lane 2) before analysis using SDS-9% PAGE and Western blotting with anti-Dab1 antibodies (Dab1 WB) was performed. (G) Neuron cultures were treated for 1 h with 0.4 μM (lanes 2 and 5), 2 μM (lanes 3 and 6), or 10 μM (lanes 4 and 7) calyculin A (lanes 2 to 4), okadaic acid (lanes 5 to 7), or DMSO (lane 1). Total lysates were subjected to SDS-9% PAGE and Western blot analysis using anti-Dab1 antibodies (Dab1 WB).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page, Immunoprecipitation

    74) Product Images from "Piperlongumine Inhibits Akt Phosphorylation to Reverse Resistance to Cisplatin in Human Non-Small Cell Lung Cancer Cells via ROS Regulation"

    Article Title: Piperlongumine Inhibits Akt Phosphorylation to Reverse Resistance to Cisplatin in Human Non-Small Cell Lung Cancer Cells via ROS Regulation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2019.01178

    Intracellular ROS generation induced by PL was blocked by NAC. (A) PL downregulates the network of Akt signaling to reverse resistance of A549/Cis cells. A549/Cis cells were treated with DMSO or 5 μM PL for 24 h. The effects on Akt, Akt (Ser473), FoxO3a, FoxO3a (Ser318/321), Nrf2, P-gp, p53, BAD, BAD (Ser75), BAD (Ser99), and Bcl-xL protein expression were evaluated by western blot analysis. (B) relative protein expression levels were quantified using ImageJ. Phospho-protein levels were normalized to corresponding protein and the other protein levels were normalized to GAPDH. Data are expressed as the mean ± SD of three independent experiments. (C , D , E) intracellular ROS generation induced by increasing doses of PL was stained with 10 μM DCFH-DA and blocked by pre-incubated with 3 mM NAC for 2 h before exposure to PL. Intracellular ROS generation was measured by flow cytometry (C , D) or fluorescence microscope (E) . (F , G) DCF-DA mean fluorescence density was quantified using ImageJ. Data are expressed as the mean ± SD of three independent experiments. Bars = SD. * P
    Figure Legend Snippet: Intracellular ROS generation induced by PL was blocked by NAC. (A) PL downregulates the network of Akt signaling to reverse resistance of A549/Cis cells. A549/Cis cells were treated with DMSO or 5 μM PL for 24 h. The effects on Akt, Akt (Ser473), FoxO3a, FoxO3a (Ser318/321), Nrf2, P-gp, p53, BAD, BAD (Ser75), BAD (Ser99), and Bcl-xL protein expression were evaluated by western blot analysis. (B) relative protein expression levels were quantified using ImageJ. Phospho-protein levels were normalized to corresponding protein and the other protein levels were normalized to GAPDH. Data are expressed as the mean ± SD of three independent experiments. (C , D , E) intracellular ROS generation induced by increasing doses of PL was stained with 10 μM DCFH-DA and blocked by pre-incubated with 3 mM NAC for 2 h before exposure to PL. Intracellular ROS generation was measured by flow cytometry (C , D) or fluorescence microscope (E) . (F , G) DCF-DA mean fluorescence density was quantified using ImageJ. Data are expressed as the mean ± SD of three independent experiments. Bars = SD. * P

    Techniques Used: Expressing, Western Blot, Staining, Incubation, Flow Cytometry, Cytometry, Fluorescence, Microscopy

    NAC can block the resistance reversal effect of PL in A549/Cis cells. (A) NAC antagonized the effect of PL combined with cisplatin on inducing apoptosis in A549/Cis cells. A549/Cis cells were treated with DMSO, 5 μM PL, 37.5 μg/ml cisplatin, 5 μM PL and 37.5 μg/ml cisplatin combined, 5 μM PL and 3mM NAC combined or 5 μM PL plus 37.5 μg/ml cisplatin, and 3mM NAC combined for 24 h. (B) the percentage of cell apoptosis was determined by Annexin–V/PI staining and flow cytometry in three independent experiments, and graphed by GraphPad Prism 5. (C) A549/Cis cells were treated with PL plus cisplatin alone or in combination with NAC at 24 h and cell viability was measured by an MTT assay. (D) A549/Cis cells were treated with increased dose of cisplatin alone, combined with PL or combined with PL and NAC, and the cell proliferation was measured by EdU assay and, (E) the fluorescence density was quantified using ImageJ. (F) A549/Cis cells were treated with DMSO, 3mM NAC, 5 μM PL alone, or in combination with NAC for 24 h. The effects on Akt and Akt (Ser-473) protein expression were evaluated by western blot analysis. (G) relative protein expression levels were quantified using ImageJ and normalized to Akt. Data are expressed as the mean ± SD of three independent experiments. Bars = SD. *** P
    Figure Legend Snippet: NAC can block the resistance reversal effect of PL in A549/Cis cells. (A) NAC antagonized the effect of PL combined with cisplatin on inducing apoptosis in A549/Cis cells. A549/Cis cells were treated with DMSO, 5 μM PL, 37.5 μg/ml cisplatin, 5 μM PL and 37.5 μg/ml cisplatin combined, 5 μM PL and 3mM NAC combined or 5 μM PL plus 37.5 μg/ml cisplatin, and 3mM NAC combined for 24 h. (B) the percentage of cell apoptosis was determined by Annexin–V/PI staining and flow cytometry in three independent experiments, and graphed by GraphPad Prism 5. (C) A549/Cis cells were treated with PL plus cisplatin alone or in combination with NAC at 24 h and cell viability was measured by an MTT assay. (D) A549/Cis cells were treated with increased dose of cisplatin alone, combined with PL or combined with PL and NAC, and the cell proliferation was measured by EdU assay and, (E) the fluorescence density was quantified using ImageJ. (F) A549/Cis cells were treated with DMSO, 3mM NAC, 5 μM PL alone, or in combination with NAC for 24 h. The effects on Akt and Akt (Ser-473) protein expression were evaluated by western blot analysis. (G) relative protein expression levels were quantified using ImageJ and normalized to Akt. Data are expressed as the mean ± SD of three independent experiments. Bars = SD. *** P

    Techniques Used: Blocking Assay, Staining, Flow Cytometry, Cytometry, MTT Assay, EdU Assay, Fluorescence, Expressing, Western Blot

    Piperlongumine (PL) enhances the chemosensitivity of A549/Cis cells to cisplatin. A549 and A549/Cis cells were treated with various concentrations of cisplatin at 24 h (A) or 48 h (B) , or treated with various concentrations of PL at 24 h (C) . (D) A549/Cis cells were treated with cisplatin alone or in combination with PL at 24 h and the cell viability was measured by an MTT assay. DMSO was used as the negative control. Five replicates were made for each concentration of the drugs. The results are expressed as the mean ± SD (n = 3) of three independent experiments.
    Figure Legend Snippet: Piperlongumine (PL) enhances the chemosensitivity of A549/Cis cells to cisplatin. A549 and A549/Cis cells were treated with various concentrations of cisplatin at 24 h (A) or 48 h (B) , or treated with various concentrations of PL at 24 h (C) . (D) A549/Cis cells were treated with cisplatin alone or in combination with PL at 24 h and the cell viability was measured by an MTT assay. DMSO was used as the negative control. Five replicates were made for each concentration of the drugs. The results are expressed as the mean ± SD (n = 3) of three independent experiments.

    Techniques Used: MTT Assay, Negative Control, Concentration Assay

    75) Product Images from "Inhibition of extracellular signal-regulated kinase 1/2 signaling has beneficial effects on skeletal muscle in a mouse model of Emery-Dreifuss muscular dystrophy caused by lamin A/C gene mutation"

    Article Title: Inhibition of extracellular signal-regulated kinase 1/2 signaling has beneficial effects on skeletal muscle in a mouse model of Emery-Dreifuss muscular dystrophy caused by lamin A/C gene mutation

    Journal: Skeletal Muscle

    doi: 10.1186/2044-5040-3-17

    Selumetinib inhibits ERK1/2 phosphorylation in skeletal muscles from Lmna H222P/H222P mice. Representative immunoblots using antibodies against phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2) to probe proteins extracts from quadriceps, diaphragm, and tibialis anterior from 20-week-old male Lmna H222P/H222P mice treated with selumetinib or DMSO for 4 weeks. The immunoblot shown is representative of three separately performed experiments.
    Figure Legend Snippet: Selumetinib inhibits ERK1/2 phosphorylation in skeletal muscles from Lmna H222P/H222P mice. Representative immunoblots using antibodies against phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2) to probe proteins extracts from quadriceps, diaphragm, and tibialis anterior from 20-week-old male Lmna H222P/H222P mice treated with selumetinib or DMSO for 4 weeks. The immunoblot shown is representative of three separately performed experiments.

    Techniques Used: Mouse Assay, Western Blot

    Selumetinib from 16 to 20 weeks of age improves skeletal muscle pathology and function in Lmna H222P/H222P mice. ( A ) Expression of Myh3 in Lmna H222P/H222P mice measured using real-time quantitative RT-PCR. White bars show relative RNA expression levels in skeletal muscles of DMSO-treated (white bars) and selumetinib-treated (black bars) mice. Values are means ± SEM for n = 5 mice per group; the real time RT-PCR was performed in triplicate with the different RNA sample; * P
    Figure Legend Snippet: Selumetinib from 16 to 20 weeks of age improves skeletal muscle pathology and function in Lmna H222P/H222P mice. ( A ) Expression of Myh3 in Lmna H222P/H222P mice measured using real-time quantitative RT-PCR. White bars show relative RNA expression levels in skeletal muscles of DMSO-treated (white bars) and selumetinib-treated (black bars) mice. Values are means ± SEM for n = 5 mice per group; the real time RT-PCR was performed in triplicate with the different RNA sample; * P

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, RNA Expression

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    Article Snippet: Dimethyl sulfoxide (DMSO), Rifampicin (RIF), 3,3′-Diindolylmethane (DIM), Ketoconazole (KET), Valspodar (PSC-833) and Rhodamine 123 (R123) were purchased from Sigma–Aldrich. .. The pcDNA3, pcDNA3-hPXR, FLAG-pcDNA3, FLAG-pcDNA3-hPXR, pEF-rPXR WT, pEF-rPXR F305L, pGL3-CYP3A4-luc and pGL3-CMV-Renilla luciferase plasmids were previously described ( ; ; ; , ). pcDNA3-mPXR plasmid was provided by Dr. Steven Kliewer (University of Texas Southwestern Medical Center).

    Article Title: Unlike for Human Monocytes after LPS Activation, Release of TNF-? by THP-1 Cells Is Produced by a TACE Catalytically Different from Constitutive TACE
    Article Snippet: Dimethyl Sulfoxide (DMSO), Hank's balanced salt solution (HBSS), Histopaque-1077, Lipopolisaccharide (LPS) from Salmonella abortus equi, RPMI 1640, SigmaFast OPD (O-phenylenediamine dihydrochloride), Thimerosal (Mercury-[(o-carboxyphenyl)thio]ethyl sodium salt), Tween 20 and Zinc chloride ZnCl2 were obtained through Sigma-Aldrich (Saint-Quentin Fallavier, France). .. Dimethyl Sulfoxide (DMSO), Hank's balanced salt solution (HBSS), Histopaque-1077, Lipopolisaccharide (LPS) from Salmonella abortus equi, RPMI 1640, SigmaFast OPD (O-phenylenediamine dihydrochloride), Thimerosal (Mercury-[(o-carboxyphenyl)thio]ethyl sodium salt), Tween 20 and Zinc chloride ZnCl2 were obtained through Sigma-Aldrich (Saint-Quentin Fallavier, France).

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  • 90
    Millipore bq 123
    Bq 123, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bq 123/product/Millipore
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    90
    Millipore sphingosine 1 phosphate
    Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous <t>sphingosine-1-phosphate</t> (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. Histogram represents quantitative analysis of Ki67 positive BM-MSC cell nuclei expressed as percentage of the total nuclei number. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were collected and counted as reported in Section 2 . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p
    Sphingosine 1 Phosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pepstatin a
    Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous <t>sphingosine-1-phosphate</t> (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. Histogram represents quantitative analysis of Ki67 positive BM-MSC cell nuclei expressed as percentage of the total nuclei number. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were collected and counted as reported in Section 2 . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p
    Pepstatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. Histogram represents quantitative analysis of Ki67 positive BM-MSC cell nuclei expressed as percentage of the total nuclei number. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were collected and counted as reported in Section 2 . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p

    Journal: Stem Cells International

    Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

    doi: 10.1155/2018/5034679

    Figure Lengend Snippet: Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. Histogram represents quantitative analysis of Ki67 positive BM-MSC cell nuclei expressed as percentage of the total nuclei number. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were collected and counted as reported in Section 2 . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p

    Article Snippet: The cells were plated at low- (3–5,000 cells cm−2 ) and high- (15–20,000 cells cm−2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

    Techniques: Incubation, Immunofluorescence, Expressing, Marker, Fluorescence, Cell Counting, Western Blot, SDS Page

    MMP-2 expression and activity. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P) or 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative immunofluorescence confocal images of fixed cells on glass coverslips immunostained with antibodies against MMP-2 (green). Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. (b) Densitometric analysis of the intensity of the MMP-2 fluorescence signal performed on digitized images. (c) Zymography. A representative gelatin zymography of MMP-2 from conditioned media obtained from BM-MSCs incubated in absence (vehicle) or in presence of W146 or SEW2871 for 48 h. Densitometry scanning from at least three separate experiments was performed and data, expressed as relative OD values (a.u.) to those of control group (vehicle) set to 100, are reported in the histogram. Data are mean ± S.E.M. Significance of difference in (b) (one-way ANOVA and Newman-Keuls multiple comparison tests), ∗ p

    Journal: Stem Cells International

    Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

    doi: 10.1155/2018/5034679

    Figure Lengend Snippet: MMP-2 expression and activity. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P) or 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative immunofluorescence confocal images of fixed cells on glass coverslips immunostained with antibodies against MMP-2 (green). Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. (b) Densitometric analysis of the intensity of the MMP-2 fluorescence signal performed on digitized images. (c) Zymography. A representative gelatin zymography of MMP-2 from conditioned media obtained from BM-MSCs incubated in absence (vehicle) or in presence of W146 or SEW2871 for 48 h. Densitometry scanning from at least three separate experiments was performed and data, expressed as relative OD values (a.u.) to those of control group (vehicle) set to 100, are reported in the histogram. Data are mean ± S.E.M. Significance of difference in (b) (one-way ANOVA and Newman-Keuls multiple comparison tests), ∗ p

    Article Snippet: The cells were plated at low- (3–5,000 cells cm−2 ) and high- (15–20,000 cells cm−2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

    Techniques: Expressing, Activity Assay, Cell Culture, Immunofluorescence, Fluorescence, Zymography, Incubation

    Cytoskeleton organization and cortactin expression. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of the following compounds: 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, and 2 μ M S1PR1 receptor agonist, SEW2871 and/or MMP-2/9 inhibitor, SB-3CT (5 μ M or 10 μ M). (a–h) Representative immunofluorescence confocal images of cells cultured on glass coverslips in the indicated experimental conditions, fixed and stained with Alexa 568-phalloidin to visualize actin filaments (red) and immunostained with antibodies against cortactin (green). Scale bar 50 μ m. Arrows indicate filopodia and arrowheads indicate lamellipodia (L). (A–D) Magnifications of the indicated squared regions of interest showing the red and green fluorescence signals separately and together. Yellow-orange colour indicates colocalization between the two fluorescence signals. Scale bar 12 μ m. The images are representative of at least three independent experiments with similar results. (i) Densitometric analysis of the intensity of the cortactin fluorescence signal performed on digitized images. Data are mean ± S.E.M. Significance of differences (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p

    Journal: Stem Cells International

    Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

    doi: 10.1155/2018/5034679

    Figure Lengend Snippet: Cytoskeleton organization and cortactin expression. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of the following compounds: 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, and 2 μ M S1PR1 receptor agonist, SEW2871 and/or MMP-2/9 inhibitor, SB-3CT (5 μ M or 10 μ M). (a–h) Representative immunofluorescence confocal images of cells cultured on glass coverslips in the indicated experimental conditions, fixed and stained with Alexa 568-phalloidin to visualize actin filaments (red) and immunostained with antibodies against cortactin (green). Scale bar 50 μ m. Arrows indicate filopodia and arrowheads indicate lamellipodia (L). (A–D) Magnifications of the indicated squared regions of interest showing the red and green fluorescence signals separately and together. Yellow-orange colour indicates colocalization between the two fluorescence signals. Scale bar 12 μ m. The images are representative of at least three independent experiments with similar results. (i) Densitometric analysis of the intensity of the cortactin fluorescence signal performed on digitized images. Data are mean ± S.E.M. Significance of differences (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p

    Article Snippet: The cells were plated at low- (3–5,000 cells cm−2 ) and high- (15–20,000 cells cm−2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

    Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Fluorescence

    S1P receptor subtype expression and SphK/S1PR axis role in cell gelatinolytic activity. (a and c) Expression of S1P receptors by reverse transcription (RT) and real-time PCR analysis. mRNA were determined by RT of total RNA (1 μ g) obtained from BM-MSCs at low- (L-) and high- (H-) density culture and 2 μ l of cDNA (for S1PR1, S1PR2, and S1PR3 detection) or 4 μ l of cDNA (for S1PR4 and S1PR5 detection) were amplified as described in Section 2 . Representative agarose gels of amplified DNA are shown. GAPDH amplification was used for data normalization. (b and d) Quantification of mRNA expression by real-time PCR analysis. Data are reported as mean ± S.E.M. of the ratio between the fold of variation of S1P receptor expression obtained from high- and low-density BM-MSCs culture. (e and f) BM-MSCs seeded onto fluorescein-labeled gelatin substrate- (DQ gelatin-) coated plastic culture plates (e) or glass coverslips (f) were cultured for 48 h in absence (vehicle) or in presence of the following compounds: 5 μ M sphingosine kinase inhibitor (iSK), 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, and 2 μ M S1PR1 receptor agonist, SEW2871. (e) Spectrophotometrical quantification of the DQ gelatin fluorescence intensity revealed after proteolytic digestion of the gelatin by MMP gelatinases. (f) Representative superimposed DIC (grey) and fluorescent confocal microscopy images (green; gelatin fluorescence intensity) of fixed cells. Scale bar 30 μ m. Histogram shows the densitometric analysis of the intensity of the gelatin fluorescence signals performed on digitized images. Data reported as mean ± S.E.M. are representative of at least three independent experiments with similar results. Significance of differences in (b) and (d) (Student's t -test), ∗ p

    Journal: Stem Cells International

    Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

    doi: 10.1155/2018/5034679

    Figure Lengend Snippet: S1P receptor subtype expression and SphK/S1PR axis role in cell gelatinolytic activity. (a and c) Expression of S1P receptors by reverse transcription (RT) and real-time PCR analysis. mRNA were determined by RT of total RNA (1 μ g) obtained from BM-MSCs at low- (L-) and high- (H-) density culture and 2 μ l of cDNA (for S1PR1, S1PR2, and S1PR3 detection) or 4 μ l of cDNA (for S1PR4 and S1PR5 detection) were amplified as described in Section 2 . Representative agarose gels of amplified DNA are shown. GAPDH amplification was used for data normalization. (b and d) Quantification of mRNA expression by real-time PCR analysis. Data are reported as mean ± S.E.M. of the ratio between the fold of variation of S1P receptor expression obtained from high- and low-density BM-MSCs culture. (e and f) BM-MSCs seeded onto fluorescein-labeled gelatin substrate- (DQ gelatin-) coated plastic culture plates (e) or glass coverslips (f) were cultured for 48 h in absence (vehicle) or in presence of the following compounds: 5 μ M sphingosine kinase inhibitor (iSK), 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, and 2 μ M S1PR1 receptor agonist, SEW2871. (e) Spectrophotometrical quantification of the DQ gelatin fluorescence intensity revealed after proteolytic digestion of the gelatin by MMP gelatinases. (f) Representative superimposed DIC (grey) and fluorescent confocal microscopy images (green; gelatin fluorescence intensity) of fixed cells. Scale bar 30 μ m. Histogram shows the densitometric analysis of the intensity of the gelatin fluorescence signals performed on digitized images. Data reported as mean ± S.E.M. are representative of at least three independent experiments with similar results. Significance of differences in (b) and (d) (Student's t -test), ∗ p

    Article Snippet: The cells were plated at low- (3–5,000 cells cm−2 ) and high- (15–20,000 cells cm−2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

    Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Amplification, Labeling, Cell Culture, Fluorescence, Confocal Microscopy