human neuroblastoma cells sh sy5y  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH human neuroblastoma cells sh sy5y
    ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating <t>SH-SY5Y</t> cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.
    Human Neuroblastoma Cells Sh Sy5y, supplied by CLS Cell Lines Service GmbH, 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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    1) Product Images from "A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes"

    Article Title: A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0217269

    ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating SH-SY5Y cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.
    Figure Legend Snippet: ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating SH-SY5Y cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.

    Techniques Used: Modification, Methylation, Expressing

    ( a ) The tri-methylation of H3 K27 in SH-SY5Y cells was significantly decreased by propofol [25 μg/ml], while TNFα [10 ng/ml] did not show any changes after 24 h. ( b ) ChIP assays showed that the promoter regions of ACHE , BCHE and CHRNA7 bind to tri-methylated H3 K27. For the negative control (1) rabbit IgG was used instead of the specific tri met H3 K27 antibody (2), and the positive control contained an antibody against RNA polymerase and primers for GAPDH. ( c ) Propofol increased the expression of the histone de-acetylating HDAC1 approximately 1.5-fold. ( d ) The expression of KDM2A was increased more than 3-fold after 4h, and ( e ) the expression of DMNT3B was slightly increased over the first 4 hours. Error bars depict 2 x SE.
    Figure Legend Snippet: ( a ) The tri-methylation of H3 K27 in SH-SY5Y cells was significantly decreased by propofol [25 μg/ml], while TNFα [10 ng/ml] did not show any changes after 24 h. ( b ) ChIP assays showed that the promoter regions of ACHE , BCHE and CHRNA7 bind to tri-methylated H3 K27. For the negative control (1) rabbit IgG was used instead of the specific tri met H3 K27 antibody (2), and the positive control contained an antibody against RNA polymerase and primers for GAPDH. ( c ) Propofol increased the expression of the histone de-acetylating HDAC1 approximately 1.5-fold. ( d ) The expression of KDM2A was increased more than 3-fold after 4h, and ( e ) the expression of DMNT3B was slightly increased over the first 4 hours. Error bars depict 2 x SE.

    Techniques Used: Methylation, Negative Control, Positive Control, Expressing

    sh sy5y human neuroblastoma cell line  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH sh sy5y human neuroblastoma cell line
    Effect of the benzofuran-2-ones 6 – 9 on the viability of undifferentiated <t>SH-SY5Y</t> cells. Trypan blue exclusion assay showing the time course (0–72 h) of the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells treated with increasing concentrations (0–100 μM) of the benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.
    Sh Sy5y Human Neuroblastoma Cell Line, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration"

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    Journal: Life

    doi: 10.3390/life14040422

    Effect of the benzofuran-2-ones 6 – 9 on the viability of undifferentiated SH-SY5Y cells. Trypan blue exclusion assay showing the time course (0–72 h) of the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells treated with increasing concentrations (0–100 μM) of the benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.
    Figure Legend Snippet: Effect of the benzofuran-2-ones 6 – 9 on the viability of undifferentiated SH-SY5Y cells. Trypan blue exclusion assay showing the time course (0–72 h) of the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells treated with increasing concentrations (0–100 μM) of the benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.

    Techniques Used: Trypan Blue Exclusion Assay

    Effect of catechol stress on intracellular ROS production, HO-1 expression, proliferation, and viability of undifferentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Magnification: 10×. Scale bar: 100 μM. ( B ) Western blot analysis showing HO-1 expression in undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( C ) The graph shows the time course (0–72 h) of the proliferation (expressed as number of cells) of undifferentiated SH-SY5Y cells exposed or not (Ctrl, black line) to 10 μM catechol (blue line). Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). ( D ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. ( E ) Fluorescence analysis showing the cytoskeleton (Phalloidin-FITC, in green), nuclei (DAPI, blue) and the merged channels (Merge) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. Magnification: 40×. Scale bar: 20 μM.
    Figure Legend Snippet: Effect of catechol stress on intracellular ROS production, HO-1 expression, proliferation, and viability of undifferentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Magnification: 10×. Scale bar: 100 μM. ( B ) Western blot analysis showing HO-1 expression in undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( C ) The graph shows the time course (0–72 h) of the proliferation (expressed as number of cells) of undifferentiated SH-SY5Y cells exposed or not (Ctrl, black line) to 10 μM catechol (blue line). Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). ( D ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. ( E ) Fluorescence analysis showing the cytoskeleton (Phalloidin-FITC, in green), nuclei (DAPI, blue) and the merged channels (Merge) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. Magnification: 40×. Scale bar: 20 μM.

    Techniques Used: Expressing, Fluorescence, Staining, Western Blot, Trypan Blue Exclusion Assay

    Heme oxygenase-1 localization after catechol stress in undifferentiated SH-SY5Y cells. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in undifferentiated SH-SY5Y cells treated with 10 μM catechol for 6 h. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.
    Figure Legend Snippet: Heme oxygenase-1 localization after catechol stress in undifferentiated SH-SY5Y cells. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in undifferentiated SH-SY5Y cells treated with 10 μM catechol for 6 h. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.

    Techniques Used: Immunofluorescence

    Effects of catechol stress on the expression of HO-1 and viability of differentiated and undifferentiated SH-SY5Y cells. ( A ) Schematic representation of SH-SY5Y cell differentiation protocol. The image was created with BioRender.com. ( B ) Microphotographs showing the time course (0–6 days) of SH-SY5Y cells differentiation by phorbol 12-myristate, 13 acetate (PMA). Black arrows indicate outgrowing neurites. Upper images: 10× magnification. Bottom images represent the magnification (20×) of the areas delimited by black squares in the upper images. ( C ) Real-time PCR analysis showing the relative mRNA expression levels of the neuronal differentiation marker βIII-tubulin in SH-SY5Y cells after 6 days of PMA differentiation (differentiated) compared to undifferentiated cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( D ) Real-time PCR analysis showing the relative mRNA expression levels of HO-1 in differentiated and undifferentiated SH-SY5Y cells exposed or not (Ctrl) to 10 μM catechol for 6 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( E ) Western blot analysis showing the presence and expression of HO-1 protein isoforms in differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 6 h. ( F ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 24 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001).
    Figure Legend Snippet: Effects of catechol stress on the expression of HO-1 and viability of differentiated and undifferentiated SH-SY5Y cells. ( A ) Schematic representation of SH-SY5Y cell differentiation protocol. The image was created with BioRender.com. ( B ) Microphotographs showing the time course (0–6 days) of SH-SY5Y cells differentiation by phorbol 12-myristate, 13 acetate (PMA). Black arrows indicate outgrowing neurites. Upper images: 10× magnification. Bottom images represent the magnification (20×) of the areas delimited by black squares in the upper images. ( C ) Real-time PCR analysis showing the relative mRNA expression levels of the neuronal differentiation marker βIII-tubulin in SH-SY5Y cells after 6 days of PMA differentiation (differentiated) compared to undifferentiated cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( D ) Real-time PCR analysis showing the relative mRNA expression levels of HO-1 in differentiated and undifferentiated SH-SY5Y cells exposed or not (Ctrl) to 10 μM catechol for 6 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( E ) Western blot analysis showing the presence and expression of HO-1 protein isoforms in differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 6 h. ( F ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 24 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001).

    Techniques Used: Expressing, Cell Differentiation, Real-time Polymerase Chain Reaction, Marker, Western Blot, Trypan Blue Exclusion Assay

    Effects of the benzofuran-2-ones 6 – 9 on catechol-induced intracellular ROS production in differentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX, 10 μM) was included as a reference antioxidant. Magnification: 10×. Scale bar: 100 μM. ( B ) Cytofluorimetric analysis of dichlorodihydrofluorescein diacetate (DCF-DA) staining showing intracellular ROS levels, expressed as Mean Fluorescence Intensity (MFI), of differentiated SH-SY5Y cells exposed or not for 6 h to 10 μM catechol (in green) with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 , in blue). Trolox (TRX, 10 μM, in blue) was included as a reference antioxidant.
    Figure Legend Snippet: Effects of the benzofuran-2-ones 6 – 9 on catechol-induced intracellular ROS production in differentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX, 10 μM) was included as a reference antioxidant. Magnification: 10×. Scale bar: 100 μM. ( B ) Cytofluorimetric analysis of dichlorodihydrofluorescein diacetate (DCF-DA) staining showing intracellular ROS levels, expressed as Mean Fluorescence Intensity (MFI), of differentiated SH-SY5Y cells exposed or not for 6 h to 10 μM catechol (in green) with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 , in blue). Trolox (TRX, 10 μM, in blue) was included as a reference antioxidant.

    Techniques Used: Fluorescence, Staining

    Effects of the compound 9 on nuclear fragmentation and HO-1 localization in differentiated SH-SY5Y cells exposed to catechol. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in differentiated SH-SY5Y cells treated or not (ctrl) with 10 μM catechol for 6 h in presence or not of 10 μM compound 9 . White asterisks indicate damaged nuclei. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.
    Figure Legend Snippet: Effects of the compound 9 on nuclear fragmentation and HO-1 localization in differentiated SH-SY5Y cells exposed to catechol. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in differentiated SH-SY5Y cells treated or not (ctrl) with 10 μM catechol for 6 h in presence or not of 10 μM compound 9 . White asterisks indicate damaged nuclei. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.

    Techniques Used: Immunofluorescence

    Effects of the benzofuran-2-ones 6 – 9 on HO-1 expression and viability of differentiated SH-SY5Y cells exposed to catechol stress. ( A ) Real-time PCR analysis showing the time course (0–6 h) of induction of HO-1 mRNA expression under catechol stress in differentiated SH-SY5Y cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( B ) Western blot analysis showing the expression of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol or to 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. ( C ) Western blot analysis showing the expression levels of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. Results are expressed as the mean ± SD of three independent experiments. ( D ) Real-time PCR analysis showing HO-1 mRNA levels in differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol and to 10 μM TRX or 9 in presence or not of 10 μM catechol. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( E ) Cytofluorimetric analysis of propidium iodide (PI) staining showing necrotic differentiated SH-SY5Y cells (expressed as percentage) exposed or not (Ctrl) for 24 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.
    Figure Legend Snippet: Effects of the benzofuran-2-ones 6 – 9 on HO-1 expression and viability of differentiated SH-SY5Y cells exposed to catechol stress. ( A ) Real-time PCR analysis showing the time course (0–6 h) of induction of HO-1 mRNA expression under catechol stress in differentiated SH-SY5Y cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( B ) Western blot analysis showing the expression of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol or to 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. ( C ) Western blot analysis showing the expression levels of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. Results are expressed as the mean ± SD of three independent experiments. ( D ) Real-time PCR analysis showing HO-1 mRNA levels in differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol and to 10 μM TRX or 9 in presence or not of 10 μM catechol. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( E ) Cytofluorimetric analysis of propidium iodide (PI) staining showing necrotic differentiated SH-SY5Y cells (expressed as percentage) exposed or not (Ctrl) for 24 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining

    sh sy5y human neuroblastoma cell line  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH sh sy5y human neuroblastoma cell line
    Sh Sy5y Human Neuroblastoma Cell Line, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sh sy5y human neuroblastoma cell line - by Bioz Stars, 2024-05
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    human neuroblastoma cells sh sy5y  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH human neuroblastoma cells sh sy5y
    Activity and mRNA expression of AChE and BChE after incubation with midazolam A) the intracellular cholinesterase activity increased 24 h after midazolam exposure (n = 4; p = 0.01) and was measured by fluorometric assay B) ACHE mRNA quantified by qPCR expression was increased 24 hours after midazolam exposure in <t>SH-SY5Y</t> cells (n = 3; p<0.01) C) BCHE mRNA quantified by qPCR expression was increased 24 hours after midazolam exposure in SH-SY5Y cells (n = 3; p = 0.03). Data are presented as mean ± standard deviation. The reported p-value refers to the Dunnett’s post-hoc test, comparing the underlying columns at the ends of each bar.
    Human Neuroblastoma Cells Sh Sy5y, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human neuroblastoma cells sh sy5y/product/CLS Cell Lines Service GmbH
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human neuroblastoma cells sh sy5y - by Bioz Stars, 2024-05
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    1) Product Images from "Midazolam impacts acetyl—And butyrylcholinesterase genes: An epigenetic explanation for postoperative delirium?"

    Article Title: Midazolam impacts acetyl—And butyrylcholinesterase genes: An epigenetic explanation for postoperative delirium?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0271119

    Activity and mRNA expression of AChE and BChE after incubation with midazolam A) the intracellular cholinesterase activity increased 24 h after midazolam exposure (n = 4; p = 0.01) and was measured by fluorometric assay B) ACHE mRNA quantified by qPCR expression was increased 24 hours after midazolam exposure in SH-SY5Y cells (n = 3; p<0.01) C) BCHE mRNA quantified by qPCR expression was increased 24 hours after midazolam exposure in SH-SY5Y cells (n = 3; p = 0.03). Data are presented as mean ± standard deviation. The reported p-value refers to the Dunnett’s post-hoc test, comparing the underlying columns at the ends of each bar.
    Figure Legend Snippet: Activity and mRNA expression of AChE and BChE after incubation with midazolam A) the intracellular cholinesterase activity increased 24 h after midazolam exposure (n = 4; p = 0.01) and was measured by fluorometric assay B) ACHE mRNA quantified by qPCR expression was increased 24 hours after midazolam exposure in SH-SY5Y cells (n = 3; p<0.01) C) BCHE mRNA quantified by qPCR expression was increased 24 hours after midazolam exposure in SH-SY5Y cells (n = 3; p = 0.03). Data are presented as mean ± standard deviation. The reported p-value refers to the Dunnett’s post-hoc test, comparing the underlying columns at the ends of each bar.

    Techniques Used: Activity Assay, Expressing, Incubation, Standard Deviation

    Methylation of BCHE in neuronal SH-SY5Y and U343 cells after midazolam incubation A) BCHE intron 2 methylation reduced after midazolam (50 μg/ml) exposure of SH-SY5Y cells (n = 3; p = 0.01) analyzed by methylation specific PCR. B) ELISA showed that histone H3 lysine 4 di-methylation (H3K4me2) decreased in U343 cells after incubation with 250 ng/ml midazolam (n = 3; p = 0.02) C) Chip-Assay confirmed binding of BCHE promoter region (90 bp) to H3K4me2; a 100 bp DNA Ladder was utilized; lanes 1, 7 show incubation with H3K27 antibody; lanes 2 and 6 show incubation with H3K4 antibody; lane 4 shows negative control without antibody and lane 5 shows positive control with RNA-polymerase II antibody (two experiments out of three are shown; n = 3). D) ACHE -571-/-670 promoter methylation was not affected by midazolam (50 μg/ml) exposure of SH-SY5Y cells (n = 3; p = n.s.) analyzed by methylation specific PCR. Data are presented as mean ± standard deviation.
    Figure Legend Snippet: Methylation of BCHE in neuronal SH-SY5Y and U343 cells after midazolam incubation A) BCHE intron 2 methylation reduced after midazolam (50 μg/ml) exposure of SH-SY5Y cells (n = 3; p = 0.01) analyzed by methylation specific PCR. B) ELISA showed that histone H3 lysine 4 di-methylation (H3K4me2) decreased in U343 cells after incubation with 250 ng/ml midazolam (n = 3; p = 0.02) C) Chip-Assay confirmed binding of BCHE promoter region (90 bp) to H3K4me2; a 100 bp DNA Ladder was utilized; lanes 1, 7 show incubation with H3K27 antibody; lanes 2 and 6 show incubation with H3K4 antibody; lane 4 shows negative control without antibody and lane 5 shows positive control with RNA-polymerase II antibody (two experiments out of three are shown; n = 3). D) ACHE -571-/-670 promoter methylation was not affected by midazolam (50 μg/ml) exposure of SH-SY5Y cells (n = 3; p = n.s.) analyzed by methylation specific PCR. Data are presented as mean ± standard deviation.

    Techniques Used: Methylation, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Positive Control, Standard Deviation

    Increased expression of lysine specific demethylase ( KDM1A ) in different cells after midazolam [50 μg/ml] exposure for 24 hours analyzed by qPCR. KDM1A expression increased in U343 (n = 3; A, in peripheral blood mononuclear cells (PBMCs) (n = 8; B) and in SH-SY5Y (n = 3; C). Flumazenil did not reduce KDM1A expression (n = 6, D). Data are presented as mean ± standard deviation. The reported p-value refers to the Dunnett’s post-hoc test, comparing the underlying columns at the ends of each bar.
    Figure Legend Snippet: Increased expression of lysine specific demethylase ( KDM1A ) in different cells after midazolam [50 μg/ml] exposure for 24 hours analyzed by qPCR. KDM1A expression increased in U343 (n = 3; A, in peripheral blood mononuclear cells (PBMCs) (n = 8; B) and in SH-SY5Y (n = 3; C). Flumazenil did not reduce KDM1A expression (n = 6, D). Data are presented as mean ± standard deviation. The reported p-value refers to the Dunnett’s post-hoc test, comparing the underlying columns at the ends of each bar.

    Techniques Used: Expressing, Standard Deviation

    human neuroblastoma cell lines sh sy5y  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH human neuroblastoma cell lines sh sy5y
    Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) <t>SH-SY5Y</t> and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.
    Human Neuroblastoma Cell Lines Sh Sy5y, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis"

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23073849

    Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.
    Figure Legend Snippet: Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.

    Techniques Used: Expressing, Western Blot, Incubation

    Enhancement of cell surface p75NTR expression by VPA and entinostat. ( A ) The SH-SY5Y cells were incubated for 24 h with either vehicle, 1 mM VPA, or 1 µM entinostat. Thereafter, the cells were treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and the solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total cell extract (cell lysate) and precipitated proteins (surface protein) were analyzed for p75NTR by Western blot. The bar graphs report the changes in the cell surface p75NTR levels that were normalized normalized to pan-cadherin (cadherin), a plasma membrane marker. Values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test. ( B ) SH-SY5Y cells that were grown on glass coverslips were treated for 24 h with either vehicle ( a ) or 1 µM entinostat (entin) ( b , c ), fixed, and incubated overnight with ATTO-488-conjugated anti-p75NTR (extracellular) antibody. In ( c ) the cells were preincubated with a mouse monoclonal unconjugated antibody directed against the extracellular domain of the receptor before the addition of the labeled antibody. The images were analyzed for p75NTR expression (green color) by fluorescence microscopy. The nuclei were stained in blue with 4′-6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar = 25 µm. The values that are shown in the scatter plot are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test.
    Figure Legend Snippet: Enhancement of cell surface p75NTR expression by VPA and entinostat. ( A ) The SH-SY5Y cells were incubated for 24 h with either vehicle, 1 mM VPA, or 1 µM entinostat. Thereafter, the cells were treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and the solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total cell extract (cell lysate) and precipitated proteins (surface protein) were analyzed for p75NTR by Western blot. The bar graphs report the changes in the cell surface p75NTR levels that were normalized normalized to pan-cadherin (cadherin), a plasma membrane marker. Values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test. ( B ) SH-SY5Y cells that were grown on glass coverslips were treated for 24 h with either vehicle ( a ) or 1 µM entinostat (entin) ( b , c ), fixed, and incubated overnight with ATTO-488-conjugated anti-p75NTR (extracellular) antibody. In ( c ) the cells were preincubated with a mouse monoclonal unconjugated antibody directed against the extracellular domain of the receptor before the addition of the labeled antibody. The images were analyzed for p75NTR expression (green color) by fluorescence microscopy. The nuclei were stained in blue with 4′-6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar = 25 µm. The values that are shown in the scatter plot are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test.

    Techniques Used: Expressing, Incubation, Isolation, Western Blot, Membrane, Marker, Labeling, Fluorescence, Microscopy, Staining

    Exposure to p75IgG-Sap induces p75NTR internalization, intracellular saporin-S6 delivery, and citotoxicity in entinostat-treated SH-SY5Y neuroblastoma cells. ( A ) The cells were treated for 24 h with 1 µM entinostat and the cell lysates were analyzed for p75NTR immunoreactivity by Western blot using either a non-targeted preimmune antibody conjugated to saporin-S6 (IgG-Sap) (1:50) or p75IgG-Sap (1:50). The images are representative of three separate experiments. ( B ). The cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed for additional 24 h with either vehicle or p75IgG-Sap (30 nM). Biotinylated cell surface proteins were isolated and analyzed for p75NTR expression. The values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle; ### , p < 0.001 by ANOVA followed by by Tukey’s test. ( C ) The cells were treated with 1 µM entinostat for 24 h and then exposed to either p75IgG-Sap (30 nM) or IgG-Sap (30 nM) for additional 24 h. The cells were then analyzed for p75NTR (green color) and LAMP1 (red color) localization by immunofluorescence. ( D ) The cells were incubated for 24 h with either vehicle or entinostat (1 µM), exposed to p75IgG-Sap (30 nM) for additional 24 h, and then analyzed for saporin-S6 (green color) and LAMP1 (red color) expression by immunofluorescence. In ( C , D ) the cell nuclei were stained in blue with DAPI. Scale bar = 25 µm. Values that are reported in the scatter plots are the mean ± SD of three independent experiments # p < 0.05; ## p < 0.01 by Student’s t -test. ( E ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently exposed to the indicated concentrations of p75IgG-Sap for 24 h. Cell viability was assayed by propidium iodide (PI) staining (red color). The values are the mean ± SD of five independent experiments. * p < 0.05 vs. vehicle; # p < 0.05, ### p < 0.001 by ANOVA followed by Tukey’s test. ( F ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then exposed to either vehicle or 30 nM p75IgG-Sap. The images were obtained by phase-contrast microscopy and are representative of five separate experiments. ( G – J ) Cell viability was determined by a luminescence assay; ( G ) The cells were treated as indicated in ( F ); ( H , I ) The cells were treated with either vehicle or 1 µM entinostat and then incubated for 24 h with vehicle, 30 nM IgG-Sap, or 30 nM saporin-S6 (Sap); ( J ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then incubated with 30 nM p75IgG-Sap in the absence and in the presence of 100 nM p75IgG added 2 h before the immunotoxin. The values are expressed as percent of control (vehicle) the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle; ### p < 0.001, ns = not significant by ANOVA followed by Tukey’s test.
    Figure Legend Snippet: Exposure to p75IgG-Sap induces p75NTR internalization, intracellular saporin-S6 delivery, and citotoxicity in entinostat-treated SH-SY5Y neuroblastoma cells. ( A ) The cells were treated for 24 h with 1 µM entinostat and the cell lysates were analyzed for p75NTR immunoreactivity by Western blot using either a non-targeted preimmune antibody conjugated to saporin-S6 (IgG-Sap) (1:50) or p75IgG-Sap (1:50). The images are representative of three separate experiments. ( B ). The cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed for additional 24 h with either vehicle or p75IgG-Sap (30 nM). Biotinylated cell surface proteins were isolated and analyzed for p75NTR expression. The values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle; ### , p < 0.001 by ANOVA followed by by Tukey’s test. ( C ) The cells were treated with 1 µM entinostat for 24 h and then exposed to either p75IgG-Sap (30 nM) or IgG-Sap (30 nM) for additional 24 h. The cells were then analyzed for p75NTR (green color) and LAMP1 (red color) localization by immunofluorescence. ( D ) The cells were incubated for 24 h with either vehicle or entinostat (1 µM), exposed to p75IgG-Sap (30 nM) for additional 24 h, and then analyzed for saporin-S6 (green color) and LAMP1 (red color) expression by immunofluorescence. In ( C , D ) the cell nuclei were stained in blue with DAPI. Scale bar = 25 µm. Values that are reported in the scatter plots are the mean ± SD of three independent experiments # p < 0.05; ## p < 0.01 by Student’s t -test. ( E ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently exposed to the indicated concentrations of p75IgG-Sap for 24 h. Cell viability was assayed by propidium iodide (PI) staining (red color). The values are the mean ± SD of five independent experiments. * p < 0.05 vs. vehicle; # p < 0.05, ### p < 0.001 by ANOVA followed by Tukey’s test. ( F ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then exposed to either vehicle or 30 nM p75IgG-Sap. The images were obtained by phase-contrast microscopy and are representative of five separate experiments. ( G – J ) Cell viability was determined by a luminescence assay; ( G ) The cells were treated as indicated in ( F ); ( H , I ) The cells were treated with either vehicle or 1 µM entinostat and then incubated for 24 h with vehicle, 30 nM IgG-Sap, or 30 nM saporin-S6 (Sap); ( J ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then incubated with 30 nM p75IgG-Sap in the absence and in the presence of 100 nM p75IgG added 2 h before the immunotoxin. The values are expressed as percent of control (vehicle) the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle; ### p < 0.001, ns = not significant by ANOVA followed by Tukey’s test.

    Techniques Used: Western Blot, Incubation, Isolation, Expressing, Immunofluorescence, Staining, Microscopy, Luminescence Assay

    The immunotoxin p75IgG-Sap potentiates entinostat-induced apoptosis of human neuroblastoma cells. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed to either vehicle or 30 nM p75IgG-Sap for 24 h. The cell lysates were analyzed for cleaved and procaspase 3 levels by Western blot. ( B , D ) SH-SY5Y ( B ) and LAN-1 ( D ) cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently incubated with 30 nM p75IgG-Sap. The cells were analyzed for cleaved caspase 3 expression (green color) by immunofluorescence microscopy. The nuclei were stained in blue with DAPI. Scale bar = 50 µm. ( C , E ) SH-SY5Y and LAN-1 cells were treated as in ( B , D ) and the caspase 3/7 activity was determined by a luminescence assay. ( F , G ) SH-SY5Y and LAN-1 cells were pretreated for 24 h with either the vehicle or the indicated concentrations of entinostat and then exposed for additional 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for PARP cleavage. ( H ) SH-SY5Y cells were treated as in ( A ) and then analyzed for DNA fragmentation by using an in situ fluorimetric TUNEL assay. Scale bar = 50 µm. ( I ) SH-SY5Y cells were treated for 24 h with either the vehicle or 0.3 µM entinostat and then exposed for 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for survivin levels. Values are the mean ± SD of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle; ### p < 0.001 by ANOVA followed by Tukey’s test.
    Figure Legend Snippet: The immunotoxin p75IgG-Sap potentiates entinostat-induced apoptosis of human neuroblastoma cells. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed to either vehicle or 30 nM p75IgG-Sap for 24 h. The cell lysates were analyzed for cleaved and procaspase 3 levels by Western blot. ( B , D ) SH-SY5Y ( B ) and LAN-1 ( D ) cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently incubated with 30 nM p75IgG-Sap. The cells were analyzed for cleaved caspase 3 expression (green color) by immunofluorescence microscopy. The nuclei were stained in blue with DAPI. Scale bar = 50 µm. ( C , E ) SH-SY5Y and LAN-1 cells were treated as in ( B , D ) and the caspase 3/7 activity was determined by a luminescence assay. ( F , G ) SH-SY5Y and LAN-1 cells were pretreated for 24 h with either the vehicle or the indicated concentrations of entinostat and then exposed for additional 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for PARP cleavage. ( H ) SH-SY5Y cells were treated as in ( A ) and then analyzed for DNA fragmentation by using an in situ fluorimetric TUNEL assay. Scale bar = 50 µm. ( I ) SH-SY5Y cells were treated for 24 h with either the vehicle or 0.3 µM entinostat and then exposed for 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for survivin levels. Values are the mean ± SD of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle; ### p < 0.001 by ANOVA followed by Tukey’s test.

    Techniques Used: Western Blot, Incubation, Expressing, Immunofluorescence, Microscopy, Staining, Activity Assay, Luminescence Assay, In Situ, TUNEL Assay

    Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.
    Figure Legend Snippet: Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.

    Techniques Used: Light Microscopy, Incubation, Western Blot

    The combination treatment with p75IgG-Sap enhances entinostat cytotoxicity in neuroblastoma multicell spheroids. ( A ) A total of 24 h after cell seeding, SH-SY5Y multicell spheroids (mcs) were preincubated for 24 h with either vehicle or 1 µM entinostat (entin). Thereafter (time 0), the spheroids were incubated for the indicated time periods with either vehicle, 30 nM p75IgG-Sap, 1 µM entinostat, or the combination of entinostat plus p75IgG-Sap. Digital images were acquired by light microscopy at the indicated time points. The mcs size of each experimental group is reported as percent of the control (vehicle at time 0). Scale bar = 200 µm. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. entinostat alone by ANOVA followed by Bonferroni’s test. ( B ) SH-SY5Y spheroids that were grown for six days were pretreated for 48 h with either vehicle or 1 µM entinostat and the exposed for 72 h to either the vehicle or 30 nM p75IgG-Sap. At the end of the incubation, digital images were acquired by light microscopy. The mcs size of each experimental group is reported as percent of the control (vehicle + vehicle). Scale bar = 200 µm. ( C ) SH-SY5Y spheroids that were grown for six days and treated as in ( B ) were analyzed for cleaved PARP by Western blot. Values are the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle. ## p < 0.01, ### p < 0.001 by ANOVA followed by Tukey’s test.
    Figure Legend Snippet: The combination treatment with p75IgG-Sap enhances entinostat cytotoxicity in neuroblastoma multicell spheroids. ( A ) A total of 24 h after cell seeding, SH-SY5Y multicell spheroids (mcs) were preincubated for 24 h with either vehicle or 1 µM entinostat (entin). Thereafter (time 0), the spheroids were incubated for the indicated time periods with either vehicle, 30 nM p75IgG-Sap, 1 µM entinostat, or the combination of entinostat plus p75IgG-Sap. Digital images were acquired by light microscopy at the indicated time points. The mcs size of each experimental group is reported as percent of the control (vehicle at time 0). Scale bar = 200 µm. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. entinostat alone by ANOVA followed by Bonferroni’s test. ( B ) SH-SY5Y spheroids that were grown for six days were pretreated for 48 h with either vehicle or 1 µM entinostat and the exposed for 72 h to either the vehicle or 30 nM p75IgG-Sap. At the end of the incubation, digital images were acquired by light microscopy. The mcs size of each experimental group is reported as percent of the control (vehicle + vehicle). Scale bar = 200 µm. ( C ) SH-SY5Y spheroids that were grown for six days and treated as in ( B ) were analyzed for cleaved PARP by Western blot. Values are the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle. ## p < 0.01, ### p < 0.001 by ANOVA followed by Tukey’s test.

    Techniques Used: Incubation, Light Microscopy, Western Blot

    Expression of p75NTR and histone H3 acetylation in neuroblastoma tumor xenografts and different organs of entinostat-treated mice. ( A , C – F ) Athymic nude mice bearing xenograft of SH-SY5Y cells were treated daily for 10 days with either the vehicle or entinostat (20 mg/kg) by oral gavage. The mice were sacrificed, the tumor xenografts and the indicated organs were resected, and the tissue extracts were analyzed for p75NTR levels by Western blot. ( B ) The levels of acetylated histone H3 were determined in neuroblastoma tumor xenografts of vehicle- and entinostat-treated mice by Western blot. Each lane was loaded with a sample that was obtained from an individual animal (in ( A , B ): four animals that were treated with vehicle and five animals that were treated with entinostat; in ( C – F ), three animals that were treated with either vehicle or entinostat). Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD of three independent Western blots. * p < 0.05, *** p < 0.001, ns = not significant by Student’s t test.
    Figure Legend Snippet: Expression of p75NTR and histone H3 acetylation in neuroblastoma tumor xenografts and different organs of entinostat-treated mice. ( A , C – F ) Athymic nude mice bearing xenograft of SH-SY5Y cells were treated daily for 10 days with either the vehicle or entinostat (20 mg/kg) by oral gavage. The mice were sacrificed, the tumor xenografts and the indicated organs were resected, and the tissue extracts were analyzed for p75NTR levels by Western blot. ( B ) The levels of acetylated histone H3 were determined in neuroblastoma tumor xenografts of vehicle- and entinostat-treated mice by Western blot. Each lane was loaded with a sample that was obtained from an individual animal (in ( A , B ): four animals that were treated with vehicle and five animals that were treated with entinostat; in ( C – F ), three animals that were treated with either vehicle or entinostat). Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD of three independent Western blots. * p < 0.05, *** p < 0.001, ns = not significant by Student’s t test.

    Techniques Used: Expressing, Western Blot

    Cytotoxic effects of p75IgG-Sap in entinostat-treated neuroblastoma tumor xenografts. ( A ) Nude mice bearing xenografts of SH-SY5Y cells were treated for 10 days with either the vehicle or entinostat as indicated in , and then injected twice with either the vehicle or p75IgG-Sap (5.0 µg) into two distal sites of the tumor. The scatter plot indicates the values (mean ± SD) of the tumor volumes that were measured 48 h after the last intratumoral injection. ( B ) Representative images of tumor xenografts that were acquired following resection from each experimental group. ( C ) Tumor xenografts of mice that were treated as indicated in ( A ) were analyzed for PARP cleavage and survivin expression by Western blot. Each lane was loaded with a sample th acquired at was obtained from an individual animal. Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD. ( D ) Tumor xenograft fragments were incubated for 96 h in complete growth medium. Thereafter, the medium was changed and the cell aggregates were analyzed by light microscopy to examine the growth and adhesion to the substrate. The scatter plot indicates the percent of tumor adhesion (mean ± SD) for each experimental group. * p < 0.05, ** p < 0.01 vs. vehicle; ## p < 0.01 by ANOVA followed by Tukey’s test.
    Figure Legend Snippet: Cytotoxic effects of p75IgG-Sap in entinostat-treated neuroblastoma tumor xenografts. ( A ) Nude mice bearing xenografts of SH-SY5Y cells were treated for 10 days with either the vehicle or entinostat as indicated in , and then injected twice with either the vehicle or p75IgG-Sap (5.0 µg) into two distal sites of the tumor. The scatter plot indicates the values (mean ± SD) of the tumor volumes that were measured 48 h after the last intratumoral injection. ( B ) Representative images of tumor xenografts that were acquired following resection from each experimental group. ( C ) Tumor xenografts of mice that were treated as indicated in ( A ) were analyzed for PARP cleavage and survivin expression by Western blot. Each lane was loaded with a sample th acquired at was obtained from an individual animal. Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD. ( D ) Tumor xenograft fragments were incubated for 96 h in complete growth medium. Thereafter, the medium was changed and the cell aggregates were analyzed by light microscopy to examine the growth and adhesion to the substrate. The scatter plot indicates the percent of tumor adhesion (mean ± SD) for each experimental group. * p < 0.05, ** p < 0.01 vs. vehicle; ## p < 0.01 by ANOVA followed by Tukey’s test.

    Techniques Used: Injection, Expressing, Western Blot, Incubation, Light Microscopy

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    Structured Review

    CLS Cell Lines Service GmbH human neuroblastoma cells sh sy5y
    ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating <t>SH-SY5Y</t> cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.
    Human Neuroblastoma Cells Sh Sy5y, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human neuroblastoma cells sh sy5y/product/CLS Cell Lines Service GmbH
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes"

    Article Title: A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0217269

    ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating SH-SY5Y cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.
    Figure Legend Snippet: ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating SH-SY5Y cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.

    Techniques Used: Modification, Methylation, Expressing

    ( a ) The tri-methylation of H3 K27 in SH-SY5Y cells was significantly decreased by propofol [25 μg/ml], while TNFα [10 ng/ml] did not show any changes after 24 h. ( b ) ChIP assays showed that the promoter regions of ACHE , BCHE and CHRNA7 bind to tri-methylated H3 K27. For the negative control (1) rabbit IgG was used instead of the specific tri met H3 K27 antibody (2), and the positive control contained an antibody against RNA polymerase and primers for GAPDH. ( c ) Propofol increased the expression of the histone de-acetylating HDAC1 approximately 1.5-fold. ( d ) The expression of KDM2A was increased more than 3-fold after 4h, and ( e ) the expression of DMNT3B was slightly increased over the first 4 hours. Error bars depict 2 x SE.
    Figure Legend Snippet: ( a ) The tri-methylation of H3 K27 in SH-SY5Y cells was significantly decreased by propofol [25 μg/ml], while TNFα [10 ng/ml] did not show any changes after 24 h. ( b ) ChIP assays showed that the promoter regions of ACHE , BCHE and CHRNA7 bind to tri-methylated H3 K27. For the negative control (1) rabbit IgG was used instead of the specific tri met H3 K27 antibody (2), and the positive control contained an antibody against RNA polymerase and primers for GAPDH. ( c ) Propofol increased the expression of the histone de-acetylating HDAC1 approximately 1.5-fold. ( d ) The expression of KDM2A was increased more than 3-fold after 4h, and ( e ) the expression of DMNT3B was slightly increased over the first 4 hours. Error bars depict 2 x SE.

    Techniques Used: Methylation, Negative Control, Positive Control, Expressing

    human neuroblastoma sh sy5y cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH human neuroblastoma sh sy5y cells
    Human Neuroblastoma Sh Sy5y Cells, supplied by CLS Cell Lines Service GmbH, 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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    CLS Cell Lines Service GmbH human neuroblastoma cells sh sy5y
    ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating <t>SH-SY5Y</t> cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.
    Human Neuroblastoma Cells Sh Sy5y, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human neuroblastoma cells sh sy5y/product/CLS Cell Lines Service GmbH
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    CLS Cell Lines Service GmbH sh sy5y human neuroblastoma cell line
    Effect of the benzofuran-2-ones 6 – 9 on the viability of undifferentiated <t>SH-SY5Y</t> cells. Trypan blue exclusion assay showing the time course (0–72 h) of the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells treated with increasing concentrations (0–100 μM) of the benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.
    Sh Sy5y Human Neuroblastoma Cell Line, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sh sy5y human neuroblastoma cell line/product/CLS Cell Lines Service GmbH
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    CLS Cell Lines Service GmbH human neuroblastoma cell lines sh sy5y
    Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) <t>SH-SY5Y</t> and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.
    Human Neuroblastoma Cell Lines Sh Sy5y, supplied by CLS Cell Lines Service GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLS Cell Lines Service GmbH human neuroblastoma sh sy5y cells
    Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) <t>SH-SY5Y</t> and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.
    Human Neuroblastoma Sh Sy5y Cells, supplied by CLS Cell Lines Service GmbH, 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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    ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating SH-SY5Y cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.

    Journal: PLoS ONE

    Article Title: A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes

    doi: 10.1371/journal.pone.0217269

    Figure Lengend Snippet: ( a ) We could not detect any significantly modified methylation in the promoter of the ACHE gene (area -1703/-1559) and BCHE gene (intron 2), apart from a decreased methylation in the promoter of the CHRNA7 gene (area -1185/-1010; p = 0.011) after stimulating SH-SY5Y cells with 25 μg/ml propofol for 2 h. Methylation of CHRNA7 recovered after 4h to almost unstimulated levels. ( b ) The artificial de-methylation of the epigenome of SH-SY5Y cells using 5-aza-2’ deoxycytidine (ADC, 50 μM) resulted in a visible decrease of overall methylation as measured in the three promoter regions. ( c ) This de-methylation reduced the expression of CHRNA7 mRNA (p = 0.034) but did not affect ACHE or BCHE significantly. ( d ) Propofol did not change the expression of proinflammatory (TNFα and IL-6) and anti-inflammatory cytokines (IL-10). Error bars depict 2 x SE.

    Article Snippet: The human neuroblastoma cells SH-SY5Y (origin: Cell Lines Service, CLS, Eppelheim Germany, SH-SY5Y item number: 300154) were cultured at 37°C and 5% CO 2 in Dulbecco’s modified Eagle medium (DMEM; Gibco, Darmstadt, Germany) with 10% foetal calf serum (FCS; Gibco, Darmstadt, Germany) and 1% penicillin/streptomycin (Penstrep; Gibco, Darmstadt, Germany).

    Techniques: Modification, Methylation, Expressing

    ( a ) The tri-methylation of H3 K27 in SH-SY5Y cells was significantly decreased by propofol [25 μg/ml], while TNFα [10 ng/ml] did not show any changes after 24 h. ( b ) ChIP assays showed that the promoter regions of ACHE , BCHE and CHRNA7 bind to tri-methylated H3 K27. For the negative control (1) rabbit IgG was used instead of the specific tri met H3 K27 antibody (2), and the positive control contained an antibody against RNA polymerase and primers for GAPDH. ( c ) Propofol increased the expression of the histone de-acetylating HDAC1 approximately 1.5-fold. ( d ) The expression of KDM2A was increased more than 3-fold after 4h, and ( e ) the expression of DMNT3B was slightly increased over the first 4 hours. Error bars depict 2 x SE.

    Journal: PLoS ONE

    Article Title: A novel understanding of postoperative complications: In vitro study of the impact of propofol on epigenetic modifications in cholinergic genes

    doi: 10.1371/journal.pone.0217269

    Figure Lengend Snippet: ( a ) The tri-methylation of H3 K27 in SH-SY5Y cells was significantly decreased by propofol [25 μg/ml], while TNFα [10 ng/ml] did not show any changes after 24 h. ( b ) ChIP assays showed that the promoter regions of ACHE , BCHE and CHRNA7 bind to tri-methylated H3 K27. For the negative control (1) rabbit IgG was used instead of the specific tri met H3 K27 antibody (2), and the positive control contained an antibody against RNA polymerase and primers for GAPDH. ( c ) Propofol increased the expression of the histone de-acetylating HDAC1 approximately 1.5-fold. ( d ) The expression of KDM2A was increased more than 3-fold after 4h, and ( e ) the expression of DMNT3B was slightly increased over the first 4 hours. Error bars depict 2 x SE.

    Article Snippet: The human neuroblastoma cells SH-SY5Y (origin: Cell Lines Service, CLS, Eppelheim Germany, SH-SY5Y item number: 300154) were cultured at 37°C and 5% CO 2 in Dulbecco’s modified Eagle medium (DMEM; Gibco, Darmstadt, Germany) with 10% foetal calf serum (FCS; Gibco, Darmstadt, Germany) and 1% penicillin/streptomycin (Penstrep; Gibco, Darmstadt, Germany).

    Techniques: Methylation, Negative Control, Positive Control, Expressing

    Effect of the benzofuran-2-ones 6 – 9 on the viability of undifferentiated SH-SY5Y cells. Trypan blue exclusion assay showing the time course (0–72 h) of the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells treated with increasing concentrations (0–100 μM) of the benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Effect of the benzofuran-2-ones 6 – 9 on the viability of undifferentiated SH-SY5Y cells. Trypan blue exclusion assay showing the time course (0–72 h) of the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells treated with increasing concentrations (0–100 μM) of the benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Trypan Blue Exclusion Assay

    Effect of catechol stress on intracellular ROS production, HO-1 expression, proliferation, and viability of undifferentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Magnification: 10×. Scale bar: 100 μM. ( B ) Western blot analysis showing HO-1 expression in undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( C ) The graph shows the time course (0–72 h) of the proliferation (expressed as number of cells) of undifferentiated SH-SY5Y cells exposed or not (Ctrl, black line) to 10 μM catechol (blue line). Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). ( D ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. ( E ) Fluorescence analysis showing the cytoskeleton (Phalloidin-FITC, in green), nuclei (DAPI, blue) and the merged channels (Merge) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. Magnification: 40×. Scale bar: 20 μM.

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Effect of catechol stress on intracellular ROS production, HO-1 expression, proliferation, and viability of undifferentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Magnification: 10×. Scale bar: 100 μM. ( B ) Western blot analysis showing HO-1 expression in undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol compared to 250 μM H 2 O 2 . Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( C ) The graph shows the time course (0–72 h) of the proliferation (expressed as number of cells) of undifferentiated SH-SY5Y cells exposed or not (Ctrl, black line) to 10 μM catechol (blue line). Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (*: p < 0.05; **: p < 0.01; ***: p < 0.001). ( D ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. ( E ) Fluorescence analysis showing the cytoskeleton (Phalloidin-FITC, in green), nuclei (DAPI, blue) and the merged channels (Merge) of undifferentiated SH-SY5Y cells exposed or not (Ctrl) for 72 h to 10 μM catechol. Magnification: 40×. Scale bar: 20 μM.

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Expressing, Fluorescence, Staining, Western Blot, Trypan Blue Exclusion Assay

    Heme oxygenase-1 localization after catechol stress in undifferentiated SH-SY5Y cells. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in undifferentiated SH-SY5Y cells treated with 10 μM catechol for 6 h. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Heme oxygenase-1 localization after catechol stress in undifferentiated SH-SY5Y cells. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in undifferentiated SH-SY5Y cells treated with 10 μM catechol for 6 h. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Immunofluorescence

    Effects of catechol stress on the expression of HO-1 and viability of differentiated and undifferentiated SH-SY5Y cells. ( A ) Schematic representation of SH-SY5Y cell differentiation protocol. The image was created with BioRender.com. ( B ) Microphotographs showing the time course (0–6 days) of SH-SY5Y cells differentiation by phorbol 12-myristate, 13 acetate (PMA). Black arrows indicate outgrowing neurites. Upper images: 10× magnification. Bottom images represent the magnification (20×) of the areas delimited by black squares in the upper images. ( C ) Real-time PCR analysis showing the relative mRNA expression levels of the neuronal differentiation marker βIII-tubulin in SH-SY5Y cells after 6 days of PMA differentiation (differentiated) compared to undifferentiated cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( D ) Real-time PCR analysis showing the relative mRNA expression levels of HO-1 in differentiated and undifferentiated SH-SY5Y cells exposed or not (Ctrl) to 10 μM catechol for 6 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( E ) Western blot analysis showing the presence and expression of HO-1 protein isoforms in differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 6 h. ( F ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 24 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001).

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Effects of catechol stress on the expression of HO-1 and viability of differentiated and undifferentiated SH-SY5Y cells. ( A ) Schematic representation of SH-SY5Y cell differentiation protocol. The image was created with BioRender.com. ( B ) Microphotographs showing the time course (0–6 days) of SH-SY5Y cells differentiation by phorbol 12-myristate, 13 acetate (PMA). Black arrows indicate outgrowing neurites. Upper images: 10× magnification. Bottom images represent the magnification (20×) of the areas delimited by black squares in the upper images. ( C ) Real-time PCR analysis showing the relative mRNA expression levels of the neuronal differentiation marker βIII-tubulin in SH-SY5Y cells after 6 days of PMA differentiation (differentiated) compared to undifferentiated cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( D ) Real-time PCR analysis showing the relative mRNA expression levels of HO-1 in differentiated and undifferentiated SH-SY5Y cells exposed or not (Ctrl) to 10 μM catechol for 6 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001). ( E ) Western blot analysis showing the presence and expression of HO-1 protein isoforms in differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 6 h. ( F ) Trypan blue exclusion assay showing the viability (expressed as percentage of control) of differentiated and undifferentiated SH-SY5Y cells exposed or not to 10 μM catechol for 24 h. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by Student’s t -test (***: p < 0.001).

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Expressing, Cell Differentiation, Real-time Polymerase Chain Reaction, Marker, Western Blot, Trypan Blue Exclusion Assay

    Effects of the benzofuran-2-ones 6 – 9 on catechol-induced intracellular ROS production in differentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX, 10 μM) was included as a reference antioxidant. Magnification: 10×. Scale bar: 100 μM. ( B ) Cytofluorimetric analysis of dichlorodihydrofluorescein diacetate (DCF-DA) staining showing intracellular ROS levels, expressed as Mean Fluorescence Intensity (MFI), of differentiated SH-SY5Y cells exposed or not for 6 h to 10 μM catechol (in green) with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 , in blue). Trolox (TRX, 10 μM, in blue) was included as a reference antioxidant.

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Effects of the benzofuran-2-ones 6 – 9 on catechol-induced intracellular ROS production in differentiated SH-SY5Y cells. ( A ) Representative fluorescence microphotographs showing intracellular ROS (in green) by dichlorodihydrofluorescein diacetate (DCF-DA) staining of differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX, 10 μM) was included as a reference antioxidant. Magnification: 10×. Scale bar: 100 μM. ( B ) Cytofluorimetric analysis of dichlorodihydrofluorescein diacetate (DCF-DA) staining showing intracellular ROS levels, expressed as Mean Fluorescence Intensity (MFI), of differentiated SH-SY5Y cells exposed or not for 6 h to 10 μM catechol (in green) with or without 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 , in blue). Trolox (TRX, 10 μM, in blue) was included as a reference antioxidant.

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Fluorescence, Staining

    Effects of the compound 9 on nuclear fragmentation and HO-1 localization in differentiated SH-SY5Y cells exposed to catechol. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in differentiated SH-SY5Y cells treated or not (ctrl) with 10 μM catechol for 6 h in presence or not of 10 μM compound 9 . White asterisks indicate damaged nuclei. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Effects of the compound 9 on nuclear fragmentation and HO-1 localization in differentiated SH-SY5Y cells exposed to catechol. Immunofluorescence analysis showing nuclei (DAPI, blue), HO-1 protein (red) and the merged channels in differentiated SH-SY5Y cells treated or not (ctrl) with 10 μM catechol for 6 h in presence or not of 10 μM compound 9 . White asterisks indicate damaged nuclei. White arrows indicate perinuclear localization of HO-1. Magnification: 40×. Scale bar: 20 μM.

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Immunofluorescence

    Effects of the benzofuran-2-ones 6 – 9 on HO-1 expression and viability of differentiated SH-SY5Y cells exposed to catechol stress. ( A ) Real-time PCR analysis showing the time course (0–6 h) of induction of HO-1 mRNA expression under catechol stress in differentiated SH-SY5Y cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( B ) Western blot analysis showing the expression of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol or to 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. ( C ) Western blot analysis showing the expression levels of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. Results are expressed as the mean ± SD of three independent experiments. ( D ) Real-time PCR analysis showing HO-1 mRNA levels in differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol and to 10 μM TRX or 9 in presence or not of 10 μM catechol. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( E ) Cytofluorimetric analysis of propidium iodide (PI) staining showing necrotic differentiated SH-SY5Y cells (expressed as percentage) exposed or not (Ctrl) for 24 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.

    Journal: Life

    Article Title: In Vitro Evaluation of the Antioxidant Capacity of 3,3-Disubstituted-3H-benzofuran-2-one Derivatives in a Cellular Model of Neurodegeneration

    doi: 10.3390/life14040422

    Figure Lengend Snippet: Effects of the benzofuran-2-ones 6 – 9 on HO-1 expression and viability of differentiated SH-SY5Y cells exposed to catechol stress. ( A ) Real-time PCR analysis showing the time course (0–6 h) of induction of HO-1 mRNA expression under catechol stress in differentiated SH-SY5Y cells. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( B ) Western blot analysis showing the expression of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol or to 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. ( C ) Western blot analysis showing the expression levels of HO-1 protein in differentiated SH-SY5Y exposed or not (Ctrl) for 6 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant. Results are expressed as the mean ± SD of three independent experiments. ( D ) Real-time PCR analysis showing HO-1 mRNA levels in differentiated SH-SY5Y cells exposed or not (Ctrl) for 6 h to 10 μM catechol and to 10 μM TRX or 9 in presence or not of 10 μM catechol. Results are expressed as the mean ± SD of three independent experiments. Statistical significance was assessed by one-way ANOVA (***: p < 0.001). ( E ) Cytofluorimetric analysis of propidium iodide (PI) staining showing necrotic differentiated SH-SY5Y cells (expressed as percentage) exposed or not (Ctrl) for 24 h to 10 μM catechol in presence or not of 10 μM benzofuran-2-ones ( 6 , 7 , 8 and 9 ). Trolox (TRX) was included as a reference antioxidant.

    Article Snippet: The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining

    Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: Induction of p75NTR expression in human neuroblastoma cell lines by VPA and entinostat. ( A ) The cells were exposed for 24 h to either vehicle, 1 mM VPA, or 1 µM entinostat (entin) and then analyzed for p75NTR protein levels by Western blot. The values are the mean ± SD of four independent experiments. The position of molecular mass standards is reported in the right side of the blots. *** p < 0.001 vs. control (vehicle) by Student’s t test. ( B ) SH-SY5Y and LAN-1 cells were treated as indicated in ( A ) and then analyzed for CASZ1 protein levels. The values are the mean ± SD of four (SH-SY5Y) and three (LAN-1) experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (vehicle). # p < 0.05, ## p < 0.01 vs. control (vehicle) by ANOVA followed by Tukey’s test. ( C ) SH-SY5Y and Kelly cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat. The cell lysates were analyzed for p75NTR protein expression. Values are the mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. control (vehicle) by Student’s t test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Expressing, Western Blot, Incubation

    Enhancement of cell surface p75NTR expression by VPA and entinostat. ( A ) The SH-SY5Y cells were incubated for 24 h with either vehicle, 1 mM VPA, or 1 µM entinostat. Thereafter, the cells were treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and the solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total cell extract (cell lysate) and precipitated proteins (surface protein) were analyzed for p75NTR by Western blot. The bar graphs report the changes in the cell surface p75NTR levels that were normalized normalized to pan-cadherin (cadherin), a plasma membrane marker. Values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test. ( B ) SH-SY5Y cells that were grown on glass coverslips were treated for 24 h with either vehicle ( a ) or 1 µM entinostat (entin) ( b , c ), fixed, and incubated overnight with ATTO-488-conjugated anti-p75NTR (extracellular) antibody. In ( c ) the cells were preincubated with a mouse monoclonal unconjugated antibody directed against the extracellular domain of the receptor before the addition of the labeled antibody. The images were analyzed for p75NTR expression (green color) by fluorescence microscopy. The nuclei were stained in blue with 4′-6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar = 25 µm. The values that are shown in the scatter plot are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: Enhancement of cell surface p75NTR expression by VPA and entinostat. ( A ) The SH-SY5Y cells were incubated for 24 h with either vehicle, 1 mM VPA, or 1 µM entinostat. Thereafter, the cells were treated with the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido) hexanoate and the solubilized proteins were isolated by precipitation with streptavidin-conjugated agarose beads. The total cell extract (cell lysate) and precipitated proteins (surface protein) were analyzed for p75NTR by Western blot. The bar graphs report the changes in the cell surface p75NTR levels that were normalized normalized to pan-cadherin (cadherin), a plasma membrane marker. Values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test. ( B ) SH-SY5Y cells that were grown on glass coverslips were treated for 24 h with either vehicle ( a ) or 1 µM entinostat (entin) ( b , c ), fixed, and incubated overnight with ATTO-488-conjugated anti-p75NTR (extracellular) antibody. In ( c ) the cells were preincubated with a mouse monoclonal unconjugated antibody directed against the extracellular domain of the receptor before the addition of the labeled antibody. The images were analyzed for p75NTR expression (green color) by fluorescence microscopy. The nuclei were stained in blue with 4′-6-diamidino-2phenylindole dihydrochloride (DAPI). Scale bar = 25 µm. The values that are shown in the scatter plot are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle by Student’s t -test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Expressing, Incubation, Isolation, Western Blot, Membrane, Marker, Labeling, Fluorescence, Microscopy, Staining

    Exposure to p75IgG-Sap induces p75NTR internalization, intracellular saporin-S6 delivery, and citotoxicity in entinostat-treated SH-SY5Y neuroblastoma cells. ( A ) The cells were treated for 24 h with 1 µM entinostat and the cell lysates were analyzed for p75NTR immunoreactivity by Western blot using either a non-targeted preimmune antibody conjugated to saporin-S6 (IgG-Sap) (1:50) or p75IgG-Sap (1:50). The images are representative of three separate experiments. ( B ). The cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed for additional 24 h with either vehicle or p75IgG-Sap (30 nM). Biotinylated cell surface proteins were isolated and analyzed for p75NTR expression. The values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle; ### , p < 0.001 by ANOVA followed by by Tukey’s test. ( C ) The cells were treated with 1 µM entinostat for 24 h and then exposed to either p75IgG-Sap (30 nM) or IgG-Sap (30 nM) for additional 24 h. The cells were then analyzed for p75NTR (green color) and LAMP1 (red color) localization by immunofluorescence. ( D ) The cells were incubated for 24 h with either vehicle or entinostat (1 µM), exposed to p75IgG-Sap (30 nM) for additional 24 h, and then analyzed for saporin-S6 (green color) and LAMP1 (red color) expression by immunofluorescence. In ( C , D ) the cell nuclei were stained in blue with DAPI. Scale bar = 25 µm. Values that are reported in the scatter plots are the mean ± SD of three independent experiments # p < 0.05; ## p < 0.01 by Student’s t -test. ( E ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently exposed to the indicated concentrations of p75IgG-Sap for 24 h. Cell viability was assayed by propidium iodide (PI) staining (red color). The values are the mean ± SD of five independent experiments. * p < 0.05 vs. vehicle; # p < 0.05, ### p < 0.001 by ANOVA followed by Tukey’s test. ( F ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then exposed to either vehicle or 30 nM p75IgG-Sap. The images were obtained by phase-contrast microscopy and are representative of five separate experiments. ( G – J ) Cell viability was determined by a luminescence assay; ( G ) The cells were treated as indicated in ( F ); ( H , I ) The cells were treated with either vehicle or 1 µM entinostat and then incubated for 24 h with vehicle, 30 nM IgG-Sap, or 30 nM saporin-S6 (Sap); ( J ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then incubated with 30 nM p75IgG-Sap in the absence and in the presence of 100 nM p75IgG added 2 h before the immunotoxin. The values are expressed as percent of control (vehicle) the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle; ### p < 0.001, ns = not significant by ANOVA followed by Tukey’s test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: Exposure to p75IgG-Sap induces p75NTR internalization, intracellular saporin-S6 delivery, and citotoxicity in entinostat-treated SH-SY5Y neuroblastoma cells. ( A ) The cells were treated for 24 h with 1 µM entinostat and the cell lysates were analyzed for p75NTR immunoreactivity by Western blot using either a non-targeted preimmune antibody conjugated to saporin-S6 (IgG-Sap) (1:50) or p75IgG-Sap (1:50). The images are representative of three separate experiments. ( B ). The cells were incubated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed for additional 24 h with either vehicle or p75IgG-Sap (30 nM). Biotinylated cell surface proteins were isolated and analyzed for p75NTR expression. The values are the mean ± SD of three independent experiments. *** p < 0.001 vs. vehicle; ### , p < 0.001 by ANOVA followed by by Tukey’s test. ( C ) The cells were treated with 1 µM entinostat for 24 h and then exposed to either p75IgG-Sap (30 nM) or IgG-Sap (30 nM) for additional 24 h. The cells were then analyzed for p75NTR (green color) and LAMP1 (red color) localization by immunofluorescence. ( D ) The cells were incubated for 24 h with either vehicle or entinostat (1 µM), exposed to p75IgG-Sap (30 nM) for additional 24 h, and then analyzed for saporin-S6 (green color) and LAMP1 (red color) expression by immunofluorescence. In ( C , D ) the cell nuclei were stained in blue with DAPI. Scale bar = 25 µm. Values that are reported in the scatter plots are the mean ± SD of three independent experiments # p < 0.05; ## p < 0.01 by Student’s t -test. ( E ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently exposed to the indicated concentrations of p75IgG-Sap for 24 h. Cell viability was assayed by propidium iodide (PI) staining (red color). The values are the mean ± SD of five independent experiments. * p < 0.05 vs. vehicle; # p < 0.05, ### p < 0.001 by ANOVA followed by Tukey’s test. ( F ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then exposed to either vehicle or 30 nM p75IgG-Sap. The images were obtained by phase-contrast microscopy and are representative of five separate experiments. ( G – J ) Cell viability was determined by a luminescence assay; ( G ) The cells were treated as indicated in ( F ); ( H , I ) The cells were treated with either vehicle or 1 µM entinostat and then incubated for 24 h with vehicle, 30 nM IgG-Sap, or 30 nM saporin-S6 (Sap); ( J ) The cells were treated for 24 h with either vehicle or 1 µM entinostat and then incubated with 30 nM p75IgG-Sap in the absence and in the presence of 100 nM p75IgG added 2 h before the immunotoxin. The values are expressed as percent of control (vehicle) the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle; ### p < 0.001, ns = not significant by ANOVA followed by Tukey’s test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Western Blot, Incubation, Isolation, Expressing, Immunofluorescence, Staining, Microscopy, Luminescence Assay

    The immunotoxin p75IgG-Sap potentiates entinostat-induced apoptosis of human neuroblastoma cells. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed to either vehicle or 30 nM p75IgG-Sap for 24 h. The cell lysates were analyzed for cleaved and procaspase 3 levels by Western blot. ( B , D ) SH-SY5Y ( B ) and LAN-1 ( D ) cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently incubated with 30 nM p75IgG-Sap. The cells were analyzed for cleaved caspase 3 expression (green color) by immunofluorescence microscopy. The nuclei were stained in blue with DAPI. Scale bar = 50 µm. ( C , E ) SH-SY5Y and LAN-1 cells were treated as in ( B , D ) and the caspase 3/7 activity was determined by a luminescence assay. ( F , G ) SH-SY5Y and LAN-1 cells were pretreated for 24 h with either the vehicle or the indicated concentrations of entinostat and then exposed for additional 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for PARP cleavage. ( H ) SH-SY5Y cells were treated as in ( A ) and then analyzed for DNA fragmentation by using an in situ fluorimetric TUNEL assay. Scale bar = 50 µm. ( I ) SH-SY5Y cells were treated for 24 h with either the vehicle or 0.3 µM entinostat and then exposed for 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for survivin levels. Values are the mean ± SD of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle; ### p < 0.001 by ANOVA followed by Tukey’s test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: The immunotoxin p75IgG-Sap potentiates entinostat-induced apoptosis of human neuroblastoma cells. ( A ) SH-SY5Y cells were treated for 24 h with either vehicle or the indicated concentrations of entinostat (entin) and then exposed to either vehicle or 30 nM p75IgG-Sap for 24 h. The cell lysates were analyzed for cleaved and procaspase 3 levels by Western blot. ( B , D ) SH-SY5Y ( B ) and LAN-1 ( D ) cells were treated for 24 h with either vehicle or 1 µM entinostat and subsequently incubated with 30 nM p75IgG-Sap. The cells were analyzed for cleaved caspase 3 expression (green color) by immunofluorescence microscopy. The nuclei were stained in blue with DAPI. Scale bar = 50 µm. ( C , E ) SH-SY5Y and LAN-1 cells were treated as in ( B , D ) and the caspase 3/7 activity was determined by a luminescence assay. ( F , G ) SH-SY5Y and LAN-1 cells were pretreated for 24 h with either the vehicle or the indicated concentrations of entinostat and then exposed for additional 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for PARP cleavage. ( H ) SH-SY5Y cells were treated as in ( A ) and then analyzed for DNA fragmentation by using an in situ fluorimetric TUNEL assay. Scale bar = 50 µm. ( I ) SH-SY5Y cells were treated for 24 h with either the vehicle or 0.3 µM entinostat and then exposed for 24 h to either the vehicle or 30 nM p75IgG-Sap. The cell lysates were analyzed for survivin levels. Values are the mean ± SD of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle; ### p < 0.001 by ANOVA followed by Tukey’s test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Western Blot, Incubation, Expressing, Immunofluorescence, Microscopy, Staining, Activity Assay, Luminescence Assay, In Situ, TUNEL Assay

    Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: Upregulation of p75NTR and induction of apoptosis by entinostat in neuroblastoma spheroids. ( A , D ) Light microscopy images of IMR-32 and SH-SY5Y multicell spheroids (mcs) that were incubated for 72 h with either vehicle or 1 µM entinostat. The scatter plots report the values of mcs sizes expressed as percent of control (vehicle). Scale bar = 200 µm. ( B , C , E , F ) The spheroids were treated as indicated in ( A , D ) and then analyzed for p75NTR levels and PARP cleavage by Western blot. The values are the mean ± SD of four individual experiments. ** p < 0.01, *** p < 0.001 vs. vehicle by Student’s t -test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Light Microscopy, Incubation, Western Blot

    The combination treatment with p75IgG-Sap enhances entinostat cytotoxicity in neuroblastoma multicell spheroids. ( A ) A total of 24 h after cell seeding, SH-SY5Y multicell spheroids (mcs) were preincubated for 24 h with either vehicle or 1 µM entinostat (entin). Thereafter (time 0), the spheroids were incubated for the indicated time periods with either vehicle, 30 nM p75IgG-Sap, 1 µM entinostat, or the combination of entinostat plus p75IgG-Sap. Digital images were acquired by light microscopy at the indicated time points. The mcs size of each experimental group is reported as percent of the control (vehicle at time 0). Scale bar = 200 µm. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. entinostat alone by ANOVA followed by Bonferroni’s test. ( B ) SH-SY5Y spheroids that were grown for six days were pretreated for 48 h with either vehicle or 1 µM entinostat and the exposed for 72 h to either the vehicle or 30 nM p75IgG-Sap. At the end of the incubation, digital images were acquired by light microscopy. The mcs size of each experimental group is reported as percent of the control (vehicle + vehicle). Scale bar = 200 µm. ( C ) SH-SY5Y spheroids that were grown for six days and treated as in ( B ) were analyzed for cleaved PARP by Western blot. Values are the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle. ## p < 0.01, ### p < 0.001 by ANOVA followed by Tukey’s test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: The combination treatment with p75IgG-Sap enhances entinostat cytotoxicity in neuroblastoma multicell spheroids. ( A ) A total of 24 h after cell seeding, SH-SY5Y multicell spheroids (mcs) were preincubated for 24 h with either vehicle or 1 µM entinostat (entin). Thereafter (time 0), the spheroids were incubated for the indicated time periods with either vehicle, 30 nM p75IgG-Sap, 1 µM entinostat, or the combination of entinostat plus p75IgG-Sap. Digital images were acquired by light microscopy at the indicated time points. The mcs size of each experimental group is reported as percent of the control (vehicle at time 0). Scale bar = 200 µm. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. entinostat alone by ANOVA followed by Bonferroni’s test. ( B ) SH-SY5Y spheroids that were grown for six days were pretreated for 48 h with either vehicle or 1 µM entinostat and the exposed for 72 h to either the vehicle or 30 nM p75IgG-Sap. At the end of the incubation, digital images were acquired by light microscopy. The mcs size of each experimental group is reported as percent of the control (vehicle + vehicle). Scale bar = 200 µm. ( C ) SH-SY5Y spheroids that were grown for six days and treated as in ( B ) were analyzed for cleaved PARP by Western blot. Values are the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle. ## p < 0.01, ### p < 0.001 by ANOVA followed by Tukey’s test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Incubation, Light Microscopy, Western Blot

    Expression of p75NTR and histone H3 acetylation in neuroblastoma tumor xenografts and different organs of entinostat-treated mice. ( A , C – F ) Athymic nude mice bearing xenograft of SH-SY5Y cells were treated daily for 10 days with either the vehicle or entinostat (20 mg/kg) by oral gavage. The mice were sacrificed, the tumor xenografts and the indicated organs were resected, and the tissue extracts were analyzed for p75NTR levels by Western blot. ( B ) The levels of acetylated histone H3 were determined in neuroblastoma tumor xenografts of vehicle- and entinostat-treated mice by Western blot. Each lane was loaded with a sample that was obtained from an individual animal (in ( A , B ): four animals that were treated with vehicle and five animals that were treated with entinostat; in ( C – F ), three animals that were treated with either vehicle or entinostat). Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD of three independent Western blots. * p < 0.05, *** p < 0.001, ns = not significant by Student’s t test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: Expression of p75NTR and histone H3 acetylation in neuroblastoma tumor xenografts and different organs of entinostat-treated mice. ( A , C – F ) Athymic nude mice bearing xenograft of SH-SY5Y cells were treated daily for 10 days with either the vehicle or entinostat (20 mg/kg) by oral gavage. The mice were sacrificed, the tumor xenografts and the indicated organs were resected, and the tissue extracts were analyzed for p75NTR levels by Western blot. ( B ) The levels of acetylated histone H3 were determined in neuroblastoma tumor xenografts of vehicle- and entinostat-treated mice by Western blot. Each lane was loaded with a sample that was obtained from an individual animal (in ( A , B ): four animals that were treated with vehicle and five animals that were treated with entinostat; in ( C – F ), three animals that were treated with either vehicle or entinostat). Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD of three independent Western blots. * p < 0.05, *** p < 0.001, ns = not significant by Student’s t test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Expressing, Western Blot

    Cytotoxic effects of p75IgG-Sap in entinostat-treated neuroblastoma tumor xenografts. ( A ) Nude mice bearing xenografts of SH-SY5Y cells were treated for 10 days with either the vehicle or entinostat as indicated in , and then injected twice with either the vehicle or p75IgG-Sap (5.0 µg) into two distal sites of the tumor. The scatter plot indicates the values (mean ± SD) of the tumor volumes that were measured 48 h after the last intratumoral injection. ( B ) Representative images of tumor xenografts that were acquired following resection from each experimental group. ( C ) Tumor xenografts of mice that were treated as indicated in ( A ) were analyzed for PARP cleavage and survivin expression by Western blot. Each lane was loaded with a sample th acquired at was obtained from an individual animal. Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD. ( D ) Tumor xenograft fragments were incubated for 96 h in complete growth medium. Thereafter, the medium was changed and the cell aggregates were analyzed by light microscopy to examine the growth and adhesion to the substrate. The scatter plot indicates the percent of tumor adhesion (mean ± SD) for each experimental group. * p < 0.05, ** p < 0.01 vs. vehicle; ## p < 0.01 by ANOVA followed by Tukey’s test.

    Journal: International Journal of Molecular Sciences

    Article Title: Upregulation of p75NTR by Histone Deacetylase Inhibitors Sensitizes Human Neuroblastoma Cells to Targeted Immunotoxin-Induced Apoptosis

    doi: 10.3390/ijms23073849

    Figure Lengend Snippet: Cytotoxic effects of p75IgG-Sap in entinostat-treated neuroblastoma tumor xenografts. ( A ) Nude mice bearing xenografts of SH-SY5Y cells were treated for 10 days with either the vehicle or entinostat as indicated in , and then injected twice with either the vehicle or p75IgG-Sap (5.0 µg) into two distal sites of the tumor. The scatter plot indicates the values (mean ± SD) of the tumor volumes that were measured 48 h after the last intratumoral injection. ( B ) Representative images of tumor xenografts that were acquired following resection from each experimental group. ( C ) Tumor xenografts of mice that were treated as indicated in ( A ) were analyzed for PARP cleavage and survivin expression by Western blot. Each lane was loaded with a sample th acquired at was obtained from an individual animal. Scatter plots indicate the absolute values of densitometric ratios and are the mean ± SD. ( D ) Tumor xenograft fragments were incubated for 96 h in complete growth medium. Thereafter, the medium was changed and the cell aggregates were analyzed by light microscopy to examine the growth and adhesion to the substrate. The scatter plot indicates the percent of tumor adhesion (mean ± SD) for each experimental group. * p < 0.05, ** p < 0.01 vs. vehicle; ## p < 0.01 by ANOVA followed by Tukey’s test.

    Article Snippet: Human neuroblastoma cell lines SH-SY5Y, LAN-1, BE(2)-C, and IMR 32 were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK), whereas the cell lines Kelly and NB-1 were from CLS Cell Lines Service GmbH (Eppelheim, Germany) and JCRB Cell Bank (Japan), respectively.

    Techniques: Injection, Expressing, Western Blot, Incubation, Light Microscopy