β galactosidase determination β gal assays Search Results


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  • 94
    Thermo Fisher β gal assay kit
    R4-uAb-mediated proteolysis of <t>β-gal</t> in mammalian cells. a , immunoblots of soluble ( top panels ) or insoluble ( bottom panel ) fractions prepared from HEK293T cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per
    β Gal Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β galactosidase determination β gal assays
    R4-uAb-mediated proteolysis of <t>β-gal</t> in mammalian cells. a , immunoblots of soluble ( top panels ) or insoluble ( bottom panel ) fractions prepared from HEK293T cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per
    β Galactosidase Determination β Gal Assays, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β galactosidase β gal staining kit
    TBI selectively increases p16 Ink4a expression and <t>SA-β-gal</t> activity in LKS + cells. (A) Quantification of p16 Ink4a mRNA expression in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice. Top panel: Amplification profiles of representative real-time RT-PCR assays. Bottom panel: A minimal estimate of the fold increase in p16 Ink4a mRNA expression is presented as the mean ± SE of 3 amplification assays. (B) Quantification of p16 Ink4a -expressing cells in LKS - and LKS + populations by flow cytometry. The expression of p16 Ink4a in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice was determined by flow cytometry as described in “Materials and methods.” Top panel: Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS - and LKS + cell gates are shown. The numbers marked in the plots are the percentage of p16 Ink4a -positive cells. Bottom panel: The percentage of p16 Ink4a-positive cells is presented as the mean ± SE (n = 3). (C) Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS + subpopulation from irradiated (day 14 after TBI) and control Ink4a/Arf KO or wild-type mice. (D) Representative immunofluorescent (IF) staining of p16 Ink4a in sorted LKS - and LKS + cells from irradiated and control mice. (E) Representative SA-β-gal staining in sorted LKS - and LKS + cells from irradiated and control mice. * P
    β Galactosidase β Gal Staining Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioVision senescence associated β galactosidase activity determination senescence associated β galactosidase sa βgal activity
    TBI selectively increases p16 Ink4a expression and <t>SA-β-gal</t> activity in LKS + cells. (A) Quantification of p16 Ink4a mRNA expression in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice. Top panel: Amplification profiles of representative real-time RT-PCR assays. Bottom panel: A minimal estimate of the fold increase in p16 Ink4a mRNA expression is presented as the mean ± SE of 3 amplification assays. (B) Quantification of p16 Ink4a -expressing cells in LKS - and LKS + populations by flow cytometry. The expression of p16 Ink4a in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice was determined by flow cytometry as described in “Materials and methods.” Top panel: Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS - and LKS + cell gates are shown. The numbers marked in the plots are the percentage of p16 Ink4a -positive cells. Bottom panel: The percentage of p16 Ink4a-positive cells is presented as the mean ± SE (n = 3). (C) Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS + subpopulation from irradiated (day 14 after TBI) and control Ink4a/Arf KO or wild-type mice. (D) Representative immunofluorescent (IF) staining of p16 Ink4a in sorted LKS - and LKS + cells from irradiated and control mice. (E) Representative SA-β-gal staining in sorted LKS - and LKS + cells from irradiated and control mice. * P
    Senescence Associated β Galactosidase Activity Determination Senescence Associated β Galactosidase Sa βgal Activity, supplied by BioVision, used in various techniques. Bioz Stars score: 96/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher β galactosidase β gal filter lift assay
    Interaction of nibrin subfragments with full-length Mre11 in the yeast two-hybrid system. Full-length nibrin and subfragments as indicated in the diagram were expressed as fusion proteins in pAS2-1 and individually tested for interaction with full-length Mre11 expressed in pACT2. Numbers flanking individual fragments indicate amino acid positions. <t>β-Gal</t> activity was measured by liquid assay as described in Materials and Methods. For each combination, three or more independent colonies were tested in duplicate for β-Gal production. The results are normalized to the β-Gal activity of the pAS2-1 vector alone. Growth was assessed by observing the viability of cotransformants on SD medium lacking uracil, lysine, tryptophan, leucine, and histidine and supplemented with 30 mM 3-amino-1,2,4-triazole.
    β Galactosidase β Gal Filter Lift Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa β gal kit
    Interaction of nibrin subfragments with full-length Mre11 in the yeast two-hybrid system. Full-length nibrin and subfragments as indicated in the diagram were expressed as fusion proteins in pAS2-1 and individually tested for interaction with full-length Mre11 expressed in pACT2. Numbers flanking individual fragments indicate amino acid positions. <t>β-Gal</t> activity was measured by liquid assay as described in Materials and Methods. For each combination, three or more independent colonies were tested in duplicate for β-Gal production. The results are normalized to the β-Gal activity of the pAS2-1 vector alone. Growth was assessed by observing the viability of cotransformants on SD medium lacking uracil, lysine, tryptophan, leucine, and histidine and supplemented with 30 mM 3-amino-1,2,4-triazole.
    β Gal Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β galactosidase
    Kallistatin ( KS ) deficiency in mouse lung endothelial cells increases senescence, oxidative stress and inflammation. Endothelial marker CD 31 expression in mouse endothelial cells determined by PCR (A) (n = 3/group). Representative images of immunostaining of CD 31 (green), with nuclei (Hoechst 33342, blue) and their merged images are shown (B) (n = 3/group). Identification of mouse kallistatin depletion (del) by target null gene DNA electrophoresis (C) (n = 3/group). Representative immunoblots of mouse KS protein (D) (n = 3/group), and mouse KS mRNA levels (E) (n = 6/group). Quantitative analysis of SA <t>‐β‐gal</t> activity (F), p16 INK 4a and PAI ‐1 mRNA levels (G, H), superoxide formation (I), NADPH oxidase activity (J), VCAM ‐1, ICAM ‐1 and IL ‐6 mRNA levels (K‐M) in mouse endothelial cells with or without H 2 O 2 treatment (n = 6/group). Values are expressed as mean ± SEM. * P
    β Galactosidase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β gal
    Kallistatin ( KS ) deficiency in mouse lung endothelial cells increases senescence, oxidative stress and inflammation. Endothelial marker CD 31 expression in mouse endothelial cells determined by PCR (A) (n = 3/group). Representative images of immunostaining of CD 31 (green), with nuclei (Hoechst 33342, blue) and their merged images are shown (B) (n = 3/group). Identification of mouse kallistatin depletion (del) by target null gene DNA electrophoresis (C) (n = 3/group). Representative immunoblots of mouse KS protein (D) (n = 3/group), and mouse KS mRNA levels (E) (n = 6/group). Quantitative analysis of SA <t>‐β‐gal</t> activity (F), p16 INK 4a and PAI ‐1 mRNA levels (G, H), superoxide formation (I), NADPH oxidase activity (J), VCAM ‐1, ICAM ‐1 and IL ‐6 mRNA levels (K‐M) in mouse endothelial cells with or without H 2 O 2 treatment (n = 6/group). Values are expressed as mean ± SEM. * P
    β Gal, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 639 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc sa β gal kit
    Kallistatin ( KS ) deficiency in mouse lung endothelial cells increases senescence, oxidative stress and inflammation. Endothelial marker CD 31 expression in mouse endothelial cells determined by PCR (A) (n = 3/group). Representative images of immunostaining of CD 31 (green), with nuclei (Hoechst 33342, blue) and their merged images are shown (B) (n = 3/group). Identification of mouse kallistatin depletion (del) by target null gene DNA electrophoresis (C) (n = 3/group). Representative immunoblots of mouse KS protein (D) (n = 3/group), and mouse KS mRNA levels (E) (n = 6/group). Quantitative analysis of SA <t>‐β‐gal</t> activity (F), p16 INK 4a and PAI ‐1 mRNA levels (G, H), superoxide formation (I), NADPH oxidase activity (J), VCAM ‐1, ICAM ‐1 and IL ‐6 mRNA levels (K‐M) in mouse endothelial cells with or without H 2 O 2 treatment (n = 6/group). Values are expressed as mean ± SEM. * P
    Sa β Gal Kit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim β gal elisa
    Kallistatin ( KS ) deficiency in mouse lung endothelial cells increases senescence, oxidative stress and inflammation. Endothelial marker CD 31 expression in mouse endothelial cells determined by PCR (A) (n = 3/group). Representative images of immunostaining of CD 31 (green), with nuclei (Hoechst 33342, blue) and their merged images are shown (B) (n = 3/group). Identification of mouse kallistatin depletion (del) by target null gene DNA electrophoresis (C) (n = 3/group). Representative immunoblots of mouse KS protein (D) (n = 3/group), and mouse KS mRNA levels (E) (n = 6/group). Quantitative analysis of SA <t>‐β‐gal</t> activity (F), p16 INK 4a and PAI ‐1 mRNA levels (G, H), superoxide formation (I), NADPH oxidase activity (J), VCAM ‐1, ICAM ‐1 and IL ‐6 mRNA levels (K‐M) in mouse endothelial cells with or without H 2 O 2 treatment (n = 6/group). Values are expressed as mean ± SEM. * P
    β Gal Elisa, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime sa β gal kit
    SIP1 inhibition attenuates GADD45G-induced tumor cell senescence in vitro (A-B) Tet-on-GADD45G-Sk-Hep1 A. and Tet-on-GADD45G-SMMC-7721 cells (B) were transfected with siRNA targeting SIP1 (siSIP1) or with control siRNA ( siCon), then cultured for 3 days with or without GADD45G induction. Representative images of <t>SA-β-gal</t> staining (left panel) and the percentages of positive cells (right panel) are shown. Data shown are mean ± SD from three independent experiments (*** P
    Sa β Gal Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc senescence associated β galactosidase β gal activity
    FOXM1/Rb-dependent expression of <t>β-galactosidase</t> in myeloma cells. a Co-immunoprecipitation (Co-IP) result indicating physical interaction of Rb and FOXM1 in CAG and XG1 myeloma cells. Immunoblots using a specific IgG antibody (Ab) to FOXM1 (following IP with Ab to Rb) or Rb (following IP with Ab to FOXM1) are shown on top of each other. The IgG isotype control is labeled “IgG.” Whole cell lysates not subjected to Co-IP (“Input”) were included as an additional control. b Shown on top is a Western blot of total Rb and its activated, phosphorylated form (pRb) in FOXM1 Hi and FOXM1 N CAG and XG1 myeloma cells. A similar blot containing samples of FOXM1 KD and FOXM1 N H929 and ARP1 myeloma cells is presented at bottom. The abundance of Rb and pRb, relative to β-actin, was determined using densitometry. The ratios are indicated below the blots. c The upper panel illustrates the elevation of β-galactosidase (β-gal) activity, a classic phenotype of cellular senescence, in FOXM1 KD H929 cells (bottom) relative to FOXM1 N controls (top). Cells were not treated with drug. This result was confirmed using paired FOXM1 KD / FOXM1 N ARP1 samples (not shown). Depicted in the lower panel is the increased proportion of β-gal + XG1 cells following treatment with Dox. FOXM1 Hi cells exhibited a lesser increase than FOXM1 N cells (not shown). Cells were evaluated using an Olympus BX-51 Light Microscope equipped with an UPLSAPO objective (Olympus) of 40x magnification and 0.95 numerical aperture. The imaging medium was air. The light temperature of the microscope bulb varied between 3000 and 3400 K. Images were acquired with the help of a DP2 digital camera (Olympus) and DP2-BSW imaging software (Olympus), saved as TIF data files, and enhanced—with respect to brightness, contrast, and color balance—using the Adobe Photoshop CS2 Version 9.0.2 software (Adobe Systems, Inc)
    Senescence Associated β Galactosidase β Gal Activity, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson luminescent β gal detection assay
    <t>β-Gal</t> expression in the presence or absence of various concentrations of MT-affecting agents. Cells were inoculated with LacZ virus at an MOI of 350, incubated for 10 h before cells were harvested, and assayed for β-Gal activity. The mean
    Luminescent β Gal Detection Assay, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche β gal elisa kit
    <t>β-Gal</t> expression in the presence or absence of various concentrations of MT-affecting agents. Cells were inoculated with LacZ virus at an MOI of 350, incubated for 10 h before cells were harvested, and assayed for β-Gal activity. The mean
    β Gal Elisa Kit, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β gal activity
    Activity progress curves for <t>β-gal</t> immobilized individually on an SMA nanofibrous mat, and in combination with α-amylase, as well as a combination of α-amylase and protease. Activity was spectrophotometrically determined as a function of time using ONPG as substrate. All results are shown as the mean ± SD of triplicate experiments (n = 3).
    β Gal Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega luminescent β gal detection kit
    Activity progress curves for <t>β-gal</t> immobilized individually on an SMA nanofibrous mat, and in combination with α-amylase, as well as a combination of α-amylase and protease. Activity was spectrophotometrically determined as a function of time using ONPG as substrate. All results are shown as the mean ± SD of triplicate experiments (n = 3).
    Luminescent β Gal Detection Kit, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β gal staining
    Balding DPCs are more senescent than sex-, age-, and site-matched non-balding DPCs. (A) Non-balding DPCs isolated from the frontal scalp of normal individuals exhibited a relatively normal appearance at passage 2 compared with balding DPCs isolated from the frontal scalp of AGA patients of the same passage, which exhibited an enlarged, irregular, and flattened morphology. (B) <t>SA-β-Gal</t> activity was increased in balding DPCs. Scale bar = 100 µm. (C) Quantification of SA-β-Gal activity showed that the percentage of SA-β-Gal expression was increased in balding DPCs in all matched-pairs. (D) Balding DPCs exhibited an increase in cell size. (E) Prolongation of the cell doubling time were observed in balding DPCs. The pair 1–4 of the x axis in Figure 1C,D,E means the each age (20, 24, 27 and 40 years), sex (male), site (frontal) matched pairs of normal control (non-AGA males) and AGA patient. Values are means ± SDs from three determinations per experiment from three independent experiments using second-passage DPCs (* P
    β Gal Staining, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime sa β gal staining kit
    Mitochondria dysfunction precedes lysosomes dysfunction during SIPS development. NIH3T3 cells were treated with or without H 2 O 2 . (A) Quantification of intracellular ROS and lysosomal CTSB activity within 24 h after H 2 O 2 treatment. Intracellular ROS was measured by flow cytometry using the DCFH-DA probe. Lysosomal CTSB activity was measured using the fluorescence intensity of the Magic Red Cathepsin B probe. 0 h indicates the end time point of H 2 O 2 treatment. The data of untreated cells (time point −1 h) was set to 1 and other values were normalized. (B) Quantification of intracellular ROS and lysosomal CTSB activity at 0, 1, 3, 5 d after H 2 O 2 treatment. (C) Images of intracellular ROS probe of control or H 2 O 2 -treated cells at d 3. DMSO or 10 μM CCCP was added to the culture medium after H 2 O 2 treatment. Scale bars: 50 μm. (D) CTSB activity in control or H 2 O 2 -treated cells day 3 was visualized with the Magic Red Cathepsin B probe. DMSO or 50 μM ambroxol was added to the culture medium after H 2 O 2 treatment. Scale bars: 50 μm. (E) to (G) H 2 O 2 -treated cells were incubated with NAC (2 μM), CCCP (10 μM) or ambroxol (50 μM) at different periods of time. Cells were stained with the <t>SA-GLB1/SA-β-gal</t> assay kit at d 5. Scale bars: 100 μm. The percentages of SA-GLB1-positive cells in each group are quantified. The data are presented as means ± SD from 3 independent experiments. *p
    Sa β Gal Staining Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genlantis β gal kit
    a) Chemical structures of G0-AuNP , G1-AuNP , and G2-AuNP . b) Schematic illustration of AuNP/ <t>β-gal-siRNA</t> complexation and transfection into cells.
    β Gal Kit, supplied by Genlantis, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime sa β galactosidase
    MLN4924 induced apoptosis or senescence in a cell line-dependent manner (A and B) MLN4924 induced apoptosis in QBC939 cells and expression of pro-apoptotic proteins in MLN4924-treated QBC939 cells. Cells were treated with MLN4924 for 72 hours and then subjected to morphological observation, or immunoblotting analysis for proteins involved in apoptotic induction, using GAPDH as a loading control. (C) MLN4924 induced senescence in RBE cells. After 72-hour MLN4924 treatment, cells were subjected to morphological observation and staining of senescence-associated <t>β–galactosidase.</t> Scale bar, 100μm. (D) MLN4924 induced the accumulation of CRL substrates. Cells were treated with MLN4924 for 72 hours using indicated concentrations and subjected to immunoblotting analysis with GADPH as a loading control.
    Sa β Galactosidase, supplied by Beyotime, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Biolabs Inc fluorometric cellular senescence associated β galactosidase β gal activity assay
    MLN4924 induced apoptosis or senescence in a cell line-dependent manner (A and B) MLN4924 induced apoptosis in QBC939 cells and expression of pro-apoptotic proteins in MLN4924-treated QBC939 cells. Cells were treated with MLN4924 for 72 hours and then subjected to morphological observation, or immunoblotting analysis for proteins involved in apoptotic induction, using GAPDH as a loading control. (C) MLN4924 induced senescence in RBE cells. After 72-hour MLN4924 treatment, cells were subjected to morphological observation and staining of senescence-associated <t>β–galactosidase.</t> Scale bar, 100μm. (D) MLN4924 induced the accumulation of CRL substrates. Cells were treated with MLN4924 for 72 hours using indicated concentrations and subjected to immunoblotting analysis with GADPH as a loading control.
    Fluorometric Cellular Senescence Associated β Galactosidase β Gal Activity Assay, supplied by Cell Biolabs Inc, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genmed sa β gal staining kit
    SIRT1 regulated autophagy through physically interacted with and deacetylated BECN1 and LC3B in PD-NSCs. a – c Cell senescence of PD-NSCs treated with or without resveratrol or chloroquine was measured, including <t>SA-β-gal</t> staining ( a , b ) and expression of aging-related genes ( c ), analyzed by western blotting and densitometry. Cells were exposed to 10 Gy X-ray in the presence of 3 μM resveratrol or 3 μM chloroquine, and then incubated for 48 h. d , e Images of cellular senescence in PD-NSCs after knockdown of BECN1 were visualized using SA-β-gal assay. Cells were transfected with siBECN1, and then exposed to 10 Gy X-ray in the presence of 3 μM resveratrol, incubated for 48 h. f The interaction between SIRT1 and autophagy-related genes, LC3B and BECN1 , in PD-NSCs. Cell lysates were immunoprecipitated with anti-SIRT1 or control IgG antibody; and then, reciprocally probed with anti-BECN1 and anti-LC3B. Cell lysates were immunoprecipitated with anti-BECN1, anti-LC3B, or control IgG antibody; and then, reciprocally probed with anti-SIRT1. g Western blot and densitometric analysis of acetylation changes of BECN1 and LC3B after IR treatment in PD-NSCs. BECN1 and LC3B were immunoprecipitated from the total cell lysate of NSCs by BECN1 and LC3B antibody; and then, western blots were probed with anti-acetylated lysine antibody. h Western blot and densitometric analysis of acetylation changes of BECN1 and LC3B after PD-NSCs were treated with SIRT1 activator resveratrol (3 μM) and SIRT1 inhibitor nicotinamide (5 mM). BECN1 and LC3B were immunoprecipitated from the total cell lysate of NSCs by BECN1 and LC3B antibody; and then, western blots were probed with anti-acetylated lysine antibody. Data of the graph were expressed as the mean ± SD. * P
    Sa β Gal Staining Kit, supplied by Genmed, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega β gal enzyme assay kit
    T 3 signaling as determined by <t>β-gal</t> staining in areas of the P90 WT/FINDT3 and D3KO/FINDT3 cortex. Staining protocol 2 was used in A–F, and staining protocol 1 was used in G–L. Areas depicted are the motor cortex (A and B and G
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    R4-uAb-mediated proteolysis of β-gal in mammalian cells. a , immunoblots of soluble ( top panels ) or insoluble ( bottom panel ) fractions prepared from HEK293T cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing *

    doi: 10.1074/jbc.M113.544825

    Figure Lengend Snippet: R4-uAb-mediated proteolysis of β-gal in mammalian cells. a , immunoblots of soluble ( top panels ) or insoluble ( bottom panel ) fractions prepared from HEK293T cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per

    Article Snippet: β-Gal activity was determined using a β-gal assay kit (Invitrogen) according to the manufacturer's instructions for the microtiter plate format.

    Techniques: Western Blot, Transfection, Plasmid Preparation

    R4-uAb-mediated proteolysis of β-gal in different cell lines. Immunoblots of extracts prepared from BHK21 ( left ) and COS-7 ( right ) cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per well (β-gal only)

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing *

    doi: 10.1074/jbc.M113.544825

    Figure Lengend Snippet: R4-uAb-mediated proteolysis of β-gal in different cell lines. Immunoblots of extracts prepared from BHK21 ( left ) and COS-7 ( right ) cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per well (β-gal only)

    Article Snippet: β-Gal activity was determined using a β-gal assay kit (Invitrogen) according to the manufacturer's instructions for the microtiter plate format.

    Techniques: Western Blot, Transfection, Plasmid Preparation

    In vitro ubiquitination of β-gal by engineered ubiquibodies. a , in vitro ubiquitination of β-gal in the presence of R4-uAb. At the indicated times, reactions were stopped by boiling and immunoblotted with anti-β-gal antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing *

    doi: 10.1074/jbc.M113.544825

    Figure Lengend Snippet: In vitro ubiquitination of β-gal by engineered ubiquibodies. a , in vitro ubiquitination of β-gal in the presence of R4-uAb. At the indicated times, reactions were stopped by boiling and immunoblotted with anti-β-gal antibodies.

    Article Snippet: β-Gal activity was determined using a β-gal assay kit (Invitrogen) according to the manufacturer's instructions for the microtiter plate format.

    Techniques: In Vitro

    Co-precipitation of ubiquitinated β-gal in HEK293T cells. Immunoblots of pulldown samples or extracts prepared from HEK293T cells transfected with pcDNA3-R4-uAb alone (R4-uAb only), pcDNA3-β-gal alone (β-gal only), or co-transfected

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing *

    doi: 10.1074/jbc.M113.544825

    Figure Lengend Snippet: Co-precipitation of ubiquitinated β-gal in HEK293T cells. Immunoblots of pulldown samples or extracts prepared from HEK293T cells transfected with pcDNA3-R4-uAb alone (R4-uAb only), pcDNA3-β-gal alone (β-gal only), or co-transfected

    Article Snippet: β-Gal activity was determined using a β-gal assay kit (Invitrogen) according to the manufacturer's instructions for the microtiter plate format.

    Techniques: Western Blot, Transfection

    Isolation of ubiquitinated β-gal. a , Coomassie-stained SDS-PAGE analysis of R4-uAb-mediated ubiquitination reactions in the presence and absence of E. coli β-gal at various times after initiation. Protein bands corresponding to unmodified

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing *

    doi: 10.1074/jbc.M113.544825

    Figure Lengend Snippet: Isolation of ubiquitinated β-gal. a , Coomassie-stained SDS-PAGE analysis of R4-uAb-mediated ubiquitination reactions in the presence and absence of E. coli β-gal at various times after initiation. Protein bands corresponding to unmodified

    Article Snippet: β-Gal activity was determined using a β-gal assay kit (Invitrogen) according to the manufacturer's instructions for the microtiter plate format.

    Techniques: Isolation, Staining, SDS Page

    TBI selectively increases p16 Ink4a expression and SA-β-gal activity in LKS + cells. (A) Quantification of p16 Ink4a mRNA expression in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice. Top panel: Amplification profiles of representative real-time RT-PCR assays. Bottom panel: A minimal estimate of the fold increase in p16 Ink4a mRNA expression is presented as the mean ± SE of 3 amplification assays. (B) Quantification of p16 Ink4a -expressing cells in LKS - and LKS + populations by flow cytometry. The expression of p16 Ink4a in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice was determined by flow cytometry as described in “Materials and methods.” Top panel: Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS - and LKS + cell gates are shown. The numbers marked in the plots are the percentage of p16 Ink4a -positive cells. Bottom panel: The percentage of p16 Ink4a-positive cells is presented as the mean ± SE (n = 3). (C) Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS + subpopulation from irradiated (day 14 after TBI) and control Ink4a/Arf KO or wild-type mice. (D) Representative immunofluorescent (IF) staining of p16 Ink4a in sorted LKS - and LKS + cells from irradiated and control mice. (E) Representative SA-β-gal staining in sorted LKS - and LKS + cells from irradiated and control mice. * P

    Journal: Blood

    Article Title: Total body irradiation selectively induces murine hematopoietic stem cell senescence

    doi: 10.1182/blood-2005-04-1418

    Figure Lengend Snippet: TBI selectively increases p16 Ink4a expression and SA-β-gal activity in LKS + cells. (A) Quantification of p16 Ink4a mRNA expression in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice. Top panel: Amplification profiles of representative real-time RT-PCR assays. Bottom panel: A minimal estimate of the fold increase in p16 Ink4a mRNA expression is presented as the mean ± SE of 3 amplification assays. (B) Quantification of p16 Ink4a -expressing cells in LKS - and LKS + populations by flow cytometry. The expression of p16 Ink4a in LKS - and LKS + cells from control (CTL) or irradiated (day 14 after TBI) mice was determined by flow cytometry as described in “Materials and methods.” Top panel: Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS - and LKS + cell gates are shown. The numbers marked in the plots are the percentage of p16 Ink4a -positive cells. Bottom panel: The percentage of p16 Ink4a-positive cells is presented as the mean ± SE (n = 3). (C) Representative flow cytometric analysis of the p16 Ink4a -expressing cells in LKS + subpopulation from irradiated (day 14 after TBI) and control Ink4a/Arf KO or wild-type mice. (D) Representative immunofluorescent (IF) staining of p16 Ink4a in sorted LKS - and LKS + cells from irradiated and control mice. (E) Representative SA-β-gal staining in sorted LKS - and LKS + cells from irradiated and control mice. * P

    Article Snippet: SA-β-gal activity in sorted LKS+ cells and LKS- cells was determined using a SA-β-gal staining kit from Cell Signaling Technology (Beverly, MA) according to the manufacturer's instructions and our previously reported procedures.

    Techniques: Expressing, Activity Assay, CTL Assay, Irradiation, Mouse Assay, Amplification, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining

    Interaction of nibrin subfragments with full-length Mre11 in the yeast two-hybrid system. Full-length nibrin and subfragments as indicated in the diagram were expressed as fusion proteins in pAS2-1 and individually tested for interaction with full-length Mre11 expressed in pACT2. Numbers flanking individual fragments indicate amino acid positions. β-Gal activity was measured by liquid assay as described in Materials and Methods. For each combination, three or more independent colonies were tested in duplicate for β-Gal production. The results are normalized to the β-Gal activity of the pAS2-1 vector alone. Growth was assessed by observing the viability of cotransformants on SD medium lacking uracil, lysine, tryptophan, leucine, and histidine and supplemented with 30 mM 3-amino-1,2,4-triazole.

    Journal: Molecular and Cellular Biology

    Article Title: Distinct Functional Domains of Nibrin Mediate Mre11 Binding, Focus Formation, and Nuclear Localization

    doi: 10.1128/MCB.21.6.2184-2191.2001

    Figure Lengend Snippet: Interaction of nibrin subfragments with full-length Mre11 in the yeast two-hybrid system. Full-length nibrin and subfragments as indicated in the diagram were expressed as fusion proteins in pAS2-1 and individually tested for interaction with full-length Mre11 expressed in pACT2. Numbers flanking individual fragments indicate amino acid positions. β-Gal activity was measured by liquid assay as described in Materials and Methods. For each combination, three or more independent colonies were tested in duplicate for β-Gal production. The results are normalized to the β-Gal activity of the pAS2-1 vector alone. Growth was assessed by observing the viability of cotransformants on SD medium lacking uracil, lysine, tryptophan, leucine, and histidine and supplemented with 30 mM 3-amino-1,2,4-triazole.

    Article Snippet: Activation of lacZ reporter gene expression was determined by a β-galactosidase (β-Gal) filter lift assay ( ) and quantitated by a liquid assay (Pierce Chemical Co., Rockford, Ill.).

    Techniques: Activity Assay, Plasmid Preparation

    Kallistatin ( KS ) deficiency in mouse lung endothelial cells increases senescence, oxidative stress and inflammation. Endothelial marker CD 31 expression in mouse endothelial cells determined by PCR (A) (n = 3/group). Representative images of immunostaining of CD 31 (green), with nuclei (Hoechst 33342, blue) and their merged images are shown (B) (n = 3/group). Identification of mouse kallistatin depletion (del) by target null gene DNA electrophoresis (C) (n = 3/group). Representative immunoblots of mouse KS protein (D) (n = 3/group), and mouse KS mRNA levels (E) (n = 6/group). Quantitative analysis of SA ‐β‐gal activity (F), p16 INK 4a and PAI ‐1 mRNA levels (G, H), superoxide formation (I), NADPH oxidase activity (J), VCAM ‐1, ICAM ‐1 and IL ‐6 mRNA levels (K‐M) in mouse endothelial cells with or without H 2 O 2 treatment (n = 6/group). Values are expressed as mean ± SEM. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐ SIRT1‐ eNOS pathway. Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

    doi: 10.1111/jcmm.13734

    Figure Lengend Snippet: Kallistatin ( KS ) deficiency in mouse lung endothelial cells increases senescence, oxidative stress and inflammation. Endothelial marker CD 31 expression in mouse endothelial cells determined by PCR (A) (n = 3/group). Representative images of immunostaining of CD 31 (green), with nuclei (Hoechst 33342, blue) and their merged images are shown (B) (n = 3/group). Identification of mouse kallistatin depletion (del) by target null gene DNA electrophoresis (C) (n = 3/group). Representative immunoblots of mouse KS protein (D) (n = 3/group), and mouse KS mRNA levels (E) (n = 6/group). Quantitative analysis of SA ‐β‐gal activity (F), p16 INK 4a and PAI ‐1 mRNA levels (G, H), superoxide formation (I), NADPH oxidase activity (J), VCAM ‐1, ICAM ‐1 and IL ‐6 mRNA levels (K‐M) in mouse endothelial cells with or without H 2 O 2 treatment (n = 6/group). Values are expressed as mean ± SEM. * P

    Article Snippet: 2.3 Senescence‐associated β‐galactosidase staining Cellular senescence of HUVECs or mouse lung endothelial cells in 12‐well plates was determined with senescence‐associated β‐galactosidase (SA‐β‐gal) staining kit (Cell Signaling, Danvers, MA, USA).

    Techniques: Marker, Expressing, Polymerase Chain Reaction, Immunostaining, Nucleic Acid Electrophoresis, Western Blot, Activity Assay

    Kallistatin ( KS ) inhibits H 2 O 2 ‐induced senescence and oxidative stress through SIRT 1‐ eNOS pathway in human endothelial cells. The effect of KS on SIRT 1, eNOS , catalase and SOD ‐2 mRNA levels analysed by qRT ‐ PCR (A‐D). The effects of SIRT 1 inhibitor NAM and NOS inhibitor L‐ NAME on KS ‐mediated eNOS and SIRT 1 expression (E, F). The effect of NAM on KS ‐regulated SA ‐β‐gal activity (G) and NADPH oxidase activity (H). Values are expressed as mean ± SEM. (n = 3 in each group). * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐ SIRT1‐ eNOS pathway. Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

    doi: 10.1111/jcmm.13734

    Figure Lengend Snippet: Kallistatin ( KS ) inhibits H 2 O 2 ‐induced senescence and oxidative stress through SIRT 1‐ eNOS pathway in human endothelial cells. The effect of KS on SIRT 1, eNOS , catalase and SOD ‐2 mRNA levels analysed by qRT ‐ PCR (A‐D). The effects of SIRT 1 inhibitor NAM and NOS inhibitor L‐ NAME on KS ‐mediated eNOS and SIRT 1 expression (E, F). The effect of NAM on KS ‐regulated SA ‐β‐gal activity (G) and NADPH oxidase activity (H). Values are expressed as mean ± SEM. (n = 3 in each group). * P

    Article Snippet: 2.3 Senescence‐associated β‐galactosidase staining Cellular senescence of HUVECs or mouse lung endothelial cells in 12‐well plates was determined with senescence‐associated β‐galactosidase (SA‐β‐gal) staining kit (Cell Signaling, Danvers, MA, USA).

    Techniques: Quantitative RT-PCR, Expressing, Activity Assay

    Kallistatin ( KS ) alleviates H 2 O 2 ‐induced endothelial senescence in human endothelial cells. Representative images of SA ‐β‐gal staining (A), and quantitative analysis of positive SA ‐β‐gal staining cells (B). p16 INK 4a and PAI ‐1 mRNA levels analysed by qRT ‐ PCR (C, D). Telomerase activity (E). Values are expressed as mean ± SEM. (n = 3 in each group). * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐ SIRT1‐ eNOS pathway. Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

    doi: 10.1111/jcmm.13734

    Figure Lengend Snippet: Kallistatin ( KS ) alleviates H 2 O 2 ‐induced endothelial senescence in human endothelial cells. Representative images of SA ‐β‐gal staining (A), and quantitative analysis of positive SA ‐β‐gal staining cells (B). p16 INK 4a and PAI ‐1 mRNA levels analysed by qRT ‐ PCR (C, D). Telomerase activity (E). Values are expressed as mean ± SEM. (n = 3 in each group). * P

    Article Snippet: 2.3 Senescence‐associated β‐galactosidase staining Cellular senescence of HUVECs or mouse lung endothelial cells in 12‐well plates was determined with senescence‐associated β‐galactosidase (SA‐β‐gal) staining kit (Cell Signaling, Danvers, MA, USA).

    Techniques: Staining, Quantitative RT-PCR, Activity Assay

    SIP1 inhibition attenuates GADD45G-induced tumor cell senescence in vitro (A-B) Tet-on-GADD45G-Sk-Hep1 A. and Tet-on-GADD45G-SMMC-7721 cells (B) were transfected with siRNA targeting SIP1 (siSIP1) or with control siRNA ( siCon), then cultured for 3 days with or without GADD45G induction. Representative images of SA-β-gal staining (left panel) and the percentages of positive cells (right panel) are shown. Data shown are mean ± SD from three independent experiments (*** P

    Journal: Oncotarget

    Article Title: SIP1 is a downstream effector of GADD45G in senescence induction and growth inhibition of liver tumor cells

    doi:

    Figure Lengend Snippet: SIP1 inhibition attenuates GADD45G-induced tumor cell senescence in vitro (A-B) Tet-on-GADD45G-Sk-Hep1 A. and Tet-on-GADD45G-SMMC-7721 cells (B) were transfected with siRNA targeting SIP1 (siSIP1) or with control siRNA ( siCon), then cultured for 3 days with or without GADD45G induction. Representative images of SA-β-gal staining (left panel) and the percentages of positive cells (right panel) are shown. Data shown are mean ± SD from three independent experiments (*** P

    Article Snippet: Senescence-associated β-galactosidase activity assay To detect the senescence ratio of cells after treatment, SA-β-gal activity was determined by using the SA-β-gal kit (Beyotime, Nantong, China) following the manufacturer's instructions.

    Techniques: Inhibition, In Vitro, Transfection, Cell Culture, Staining

    GADD45G and SIP1 are required for the proteasome inhibitor MG132-induced tumor cell senescence (A-B) Sk-Hep1 (A) and SMMC-7721 (B) cells were transfected with siRNA targeting GADD45G or a control siRNA for 24 hours, then treated with or without 3 μM MG132 for 2 hours. Cells were harvested at 24 hours after MG132 treatment. Western blot analysis shows levels of SIP1 and GADD45G proteins. (C) Sk-Hep1 and SMMC-7721 cells were treated as indicated above. SA-β-gal staining analysis was performed for senescent cells. Data shown are mean ± SD from three independent experiments (*** P

    Journal: Oncotarget

    Article Title: SIP1 is a downstream effector of GADD45G in senescence induction and growth inhibition of liver tumor cells

    doi:

    Figure Lengend Snippet: GADD45G and SIP1 are required for the proteasome inhibitor MG132-induced tumor cell senescence (A-B) Sk-Hep1 (A) and SMMC-7721 (B) cells were transfected with siRNA targeting GADD45G or a control siRNA for 24 hours, then treated with or without 3 μM MG132 for 2 hours. Cells were harvested at 24 hours after MG132 treatment. Western blot analysis shows levels of SIP1 and GADD45G proteins. (C) Sk-Hep1 and SMMC-7721 cells were treated as indicated above. SA-β-gal staining analysis was performed for senescent cells. Data shown are mean ± SD from three independent experiments (*** P

    Article Snippet: Senescence-associated β-galactosidase activity assay To detect the senescence ratio of cells after treatment, SA-β-gal activity was determined by using the SA-β-gal kit (Beyotime, Nantong, China) following the manufacturer's instructions.

    Techniques: Transfection, Western Blot, Staining

    SIP1 activation in GADD45G-induced tumor cell senescence (A) Tet-on-GADD45G-Sk-Hep1 and Tet-on-GADD45G-SMMC-7721 cells were cultured for the indicated times in the presence of 2.5μg/mL DOX (Tet-on) for GADD45G induction. The representative images of SA-β-gal staining (left panel) and the percentage of SA-β-gal positive cells (right panel) are shown. Data shown are mean ± SD from three independent experiments (** P

    Journal: Oncotarget

    Article Title: SIP1 is a downstream effector of GADD45G in senescence induction and growth inhibition of liver tumor cells

    doi:

    Figure Lengend Snippet: SIP1 activation in GADD45G-induced tumor cell senescence (A) Tet-on-GADD45G-Sk-Hep1 and Tet-on-GADD45G-SMMC-7721 cells were cultured for the indicated times in the presence of 2.5μg/mL DOX (Tet-on) for GADD45G induction. The representative images of SA-β-gal staining (left panel) and the percentage of SA-β-gal positive cells (right panel) are shown. Data shown are mean ± SD from three independent experiments (** P

    Article Snippet: Senescence-associated β-galactosidase activity assay To detect the senescence ratio of cells after treatment, SA-β-gal activity was determined by using the SA-β-gal kit (Beyotime, Nantong, China) following the manufacturer's instructions.

    Techniques: Activation Assay, Cell Culture, Staining

    FOXM1/Rb-dependent expression of β-galactosidase in myeloma cells. a Co-immunoprecipitation (Co-IP) result indicating physical interaction of Rb and FOXM1 in CAG and XG1 myeloma cells. Immunoblots using a specific IgG antibody (Ab) to FOXM1 (following IP with Ab to Rb) or Rb (following IP with Ab to FOXM1) are shown on top of each other. The IgG isotype control is labeled “IgG.” Whole cell lysates not subjected to Co-IP (“Input”) were included as an additional control. b Shown on top is a Western blot of total Rb and its activated, phosphorylated form (pRb) in FOXM1 Hi and FOXM1 N CAG and XG1 myeloma cells. A similar blot containing samples of FOXM1 KD and FOXM1 N H929 and ARP1 myeloma cells is presented at bottom. The abundance of Rb and pRb, relative to β-actin, was determined using densitometry. The ratios are indicated below the blots. c The upper panel illustrates the elevation of β-galactosidase (β-gal) activity, a classic phenotype of cellular senescence, in FOXM1 KD H929 cells (bottom) relative to FOXM1 N controls (top). Cells were not treated with drug. This result was confirmed using paired FOXM1 KD / FOXM1 N ARP1 samples (not shown). Depicted in the lower panel is the increased proportion of β-gal + XG1 cells following treatment with Dox. FOXM1 Hi cells exhibited a lesser increase than FOXM1 N cells (not shown). Cells were evaluated using an Olympus BX-51 Light Microscope equipped with an UPLSAPO objective (Olympus) of 40x magnification and 0.95 numerical aperture. The imaging medium was air. The light temperature of the microscope bulb varied between 3000 and 3400 K. Images were acquired with the help of a DP2 digital camera (Olympus) and DP2-BSW imaging software (Olympus), saved as TIF data files, and enhanced—with respect to brightness, contrast, and color balance—using the Adobe Photoshop CS2 Version 9.0.2 software (Adobe Systems, Inc)

    Journal: BMC Cancer

    Article Title: Upregulation of FOXM1 leads to diminished drug sensitivity in myeloma

    doi: 10.1186/s12885-018-5015-0

    Figure Lengend Snippet: FOXM1/Rb-dependent expression of β-galactosidase in myeloma cells. a Co-immunoprecipitation (Co-IP) result indicating physical interaction of Rb and FOXM1 in CAG and XG1 myeloma cells. Immunoblots using a specific IgG antibody (Ab) to FOXM1 (following IP with Ab to Rb) or Rb (following IP with Ab to FOXM1) are shown on top of each other. The IgG isotype control is labeled “IgG.” Whole cell lysates not subjected to Co-IP (“Input”) were included as an additional control. b Shown on top is a Western blot of total Rb and its activated, phosphorylated form (pRb) in FOXM1 Hi and FOXM1 N CAG and XG1 myeloma cells. A similar blot containing samples of FOXM1 KD and FOXM1 N H929 and ARP1 myeloma cells is presented at bottom. The abundance of Rb and pRb, relative to β-actin, was determined using densitometry. The ratios are indicated below the blots. c The upper panel illustrates the elevation of β-galactosidase (β-gal) activity, a classic phenotype of cellular senescence, in FOXM1 KD H929 cells (bottom) relative to FOXM1 N controls (top). Cells were not treated with drug. This result was confirmed using paired FOXM1 KD / FOXM1 N ARP1 samples (not shown). Depicted in the lower panel is the increased proportion of β-gal + XG1 cells following treatment with Dox. FOXM1 Hi cells exhibited a lesser increase than FOXM1 N cells (not shown). Cells were evaluated using an Olympus BX-51 Light Microscope equipped with an UPLSAPO objective (Olympus) of 40x magnification and 0.95 numerical aperture. The imaging medium was air. The light temperature of the microscope bulb varied between 3000 and 3400 K. Images were acquired with the help of a DP2 digital camera (Olympus) and DP2-BSW imaging software (Olympus), saved as TIF data files, and enhanced—with respect to brightness, contrast, and color balance—using the Adobe Photoshop CS2 Version 9.0.2 software (Adobe Systems, Inc)

    Article Snippet: For determination of senescence-associated β-galactosidase (β-gal) activity, a kit from Cell Signaling Technology (Cat# 9860) was used.

    Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Labeling, Activity Assay, Light Microscopy, Imaging, Microscopy, Software

    β-Gal expression in the presence or absence of various concentrations of MT-affecting agents. Cells were inoculated with LacZ virus at an MOI of 350, incubated for 10 h before cells were harvested, and assayed for β-Gal activity. The mean

    Journal:

    Article Title: Improvement in Nuclear Entry and Transgene Expression of Baculoviruses by Disintegration of Microtubules in Human Hepatocytes

    doi: 10.1128/JVI.79.5.2720-2728.2005

    Figure Lengend Snippet: β-Gal expression in the presence or absence of various concentrations of MT-affecting agents. Cells were inoculated with LacZ virus at an MOI of 350, incubated for 10 h before cells were harvested, and assayed for β-Gal activity. The mean

    Article Snippet: The β-Gal activity of cells inoculated with LacZ virus was determined by using the luminescent β-Gal detection assay (BD Biosciences Clontech, Palo Alto, Calif.).

    Techniques: Expressing, Incubation, Activity Assay

    Production of the β-galactosidase in hepatic cells inoculated with LacZ virus. (A) Accumulation of β-Gal in the nucleus of cells (arrows). Inoculated cells were incubated for 12 h prior to fixation and staining for β-Gal with a

    Journal:

    Article Title: Improvement in Nuclear Entry and Transgene Expression of Baculoviruses by Disintegration of Microtubules in Human Hepatocytes

    doi: 10.1128/JVI.79.5.2720-2728.2005

    Figure Lengend Snippet: Production of the β-galactosidase in hepatic cells inoculated with LacZ virus. (A) Accumulation of β-Gal in the nucleus of cells (arrows). Inoculated cells were incubated for 12 h prior to fixation and staining for β-Gal with a

    Article Snippet: The β-Gal activity of cells inoculated with LacZ virus was determined by using the luminescent β-Gal detection assay (BD Biosciences Clontech, Palo Alto, Calif.).

    Techniques: Incubation, Staining

    Baculovirus-mediated expression of β-Gal at various times and in the presence or absence of MT-affecting drugs. Cells were inoculated with LacZ virus prior incubation and assaying for β-Gal activity. (A) Kinetics of β-Gal expression

    Journal:

    Article Title: Improvement in Nuclear Entry and Transgene Expression of Baculoviruses by Disintegration of Microtubules in Human Hepatocytes

    doi: 10.1128/JVI.79.5.2720-2728.2005

    Figure Lengend Snippet: Baculovirus-mediated expression of β-Gal at various times and in the presence or absence of MT-affecting drugs. Cells were inoculated with LacZ virus prior incubation and assaying for β-Gal activity. (A) Kinetics of β-Gal expression

    Article Snippet: The β-Gal activity of cells inoculated with LacZ virus was determined by using the luminescent β-Gal detection assay (BD Biosciences Clontech, Palo Alto, Calif.).

    Techniques: Expressing, Incubation, Activity Assay

    Activity progress curves for β-gal immobilized individually on an SMA nanofibrous mat, and in combination with α-amylase, as well as a combination of α-amylase and protease. Activity was spectrophotometrically determined as a function of time using ONPG as substrate. All results are shown as the mean ± SD of triplicate experiments (n = 3).

    Journal: Molecules

    Article Title: Degradation of Proteins and Starch by Combined Immobilization of Protease, α-Amylase and β-Galactosidase on a Single Electrospun Nanofibrous Membrane

    doi: 10.3390/molecules24030508

    Figure Lengend Snippet: Activity progress curves for β-gal immobilized individually on an SMA nanofibrous mat, and in combination with α-amylase, as well as a combination of α-amylase and protease. Activity was spectrophotometrically determined as a function of time using ONPG as substrate. All results are shown as the mean ± SD of triplicate experiments (n = 3).

    Article Snippet: The o-nitrophenol-β-d -galoctoside (ONPG) used for determination of β-gal activity was 99.0% pure and was obtained from Sigma-Aldrich, Schnelldorf, Germany.

    Techniques: Activity Assay

    Balding DPCs are more senescent than sex-, age-, and site-matched non-balding DPCs. (A) Non-balding DPCs isolated from the frontal scalp of normal individuals exhibited a relatively normal appearance at passage 2 compared with balding DPCs isolated from the frontal scalp of AGA patients of the same passage, which exhibited an enlarged, irregular, and flattened morphology. (B) SA-β-Gal activity was increased in balding DPCs. Scale bar = 100 µm. (C) Quantification of SA-β-Gal activity showed that the percentage of SA-β-Gal expression was increased in balding DPCs in all matched-pairs. (D) Balding DPCs exhibited an increase in cell size. (E) Prolongation of the cell doubling time were observed in balding DPCs. The pair 1–4 of the x axis in Figure 1C,D,E means the each age (20, 24, 27 and 40 years), sex (male), site (frontal) matched pairs of normal control (non-AGA males) and AGA patient. Values are means ± SDs from three determinations per experiment from three independent experiments using second-passage DPCs (* P

    Journal: PLoS ONE

    Article Title: Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

    doi: 10.1371/journal.pone.0079434

    Figure Lengend Snippet: Balding DPCs are more senescent than sex-, age-, and site-matched non-balding DPCs. (A) Non-balding DPCs isolated from the frontal scalp of normal individuals exhibited a relatively normal appearance at passage 2 compared with balding DPCs isolated from the frontal scalp of AGA patients of the same passage, which exhibited an enlarged, irregular, and flattened morphology. (B) SA-β-Gal activity was increased in balding DPCs. Scale bar = 100 µm. (C) Quantification of SA-β-Gal activity showed that the percentage of SA-β-Gal expression was increased in balding DPCs in all matched-pairs. (D) Balding DPCs exhibited an increase in cell size. (E) Prolongation of the cell doubling time were observed in balding DPCs. The pair 1–4 of the x axis in Figure 1C,D,E means the each age (20, 24, 27 and 40 years), sex (male), site (frontal) matched pairs of normal control (non-AGA males) and AGA patient. Values are means ± SDs from three determinations per experiment from three independent experiments using second-passage DPCs (* P

    Article Snippet: SA β-gal Staining The SA-β-Gal activity was determined by using a SA-β-Gal staining kit (Sigma).

    Techniques: Isolation, Activity Assay, Expressing

    Overexpression of the AR promotes androgen-accelerated premature senescence in DPCs. (A) Non-balding DPCs of frontal scalp were transfected with pcDNA3-hAR, or pcDNA3 empty vector and cultured in the presence of DHT or ethanol (vehicle control) for 3 days. Premature senescence of DPCs was evaluated on day 5. Scale bar = 100 µm. DHT increased SA-β-Gal activity (B), cell size (C), and the number of SAHF-containing DPCs. (D) Overexpression of AR enhanced the statistical significance of DHT effects. Values are means ± SDs from three independent experiments (* P

    Journal: PLoS ONE

    Article Title: Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

    doi: 10.1371/journal.pone.0079434

    Figure Lengend Snippet: Overexpression of the AR promotes androgen-accelerated premature senescence in DPCs. (A) Non-balding DPCs of frontal scalp were transfected with pcDNA3-hAR, or pcDNA3 empty vector and cultured in the presence of DHT or ethanol (vehicle control) for 3 days. Premature senescence of DPCs was evaluated on day 5. Scale bar = 100 µm. DHT increased SA-β-Gal activity (B), cell size (C), and the number of SAHF-containing DPCs. (D) Overexpression of AR enhanced the statistical significance of DHT effects. Values are means ± SDs from three independent experiments (* P

    Article Snippet: SA β-gal Staining The SA-β-Gal activity was determined by using a SA-β-Gal staining kit (Sigma).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Activity Assay

    Androgen promotes senescence in earlier-passage DPCs of frontal scalp. DPCs of various origin and DU145 cells were treated with 0.1 µM of DHT for 3 days and then stained for SA-β-Gal 5 days after DHT stimulation. (A–C) Early and late passages of non-balding DPCs are shown for comparison. SA-β-Gal activity increased after DHT stimulation. A quantitative analysis revealed a significant increase in the percentage of senescent DPCs in DHT-treated, earlier-passage groups. DHT-induced premature senescence was also evaluated by measuring cell size. The bar graph shows quantification results. (D–F) DHT accelerated premature senescence in non-balding and transitional zone of balding DPCs only. Scale bar = 200 µm. Values are means ± SDs from three independent experiments (* P

    Journal: PLoS ONE

    Article Title: Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

    doi: 10.1371/journal.pone.0079434

    Figure Lengend Snippet: Androgen promotes senescence in earlier-passage DPCs of frontal scalp. DPCs of various origin and DU145 cells were treated with 0.1 µM of DHT for 3 days and then stained for SA-β-Gal 5 days after DHT stimulation. (A–C) Early and late passages of non-balding DPCs are shown for comparison. SA-β-Gal activity increased after DHT stimulation. A quantitative analysis revealed a significant increase in the percentage of senescent DPCs in DHT-treated, earlier-passage groups. DHT-induced premature senescence was also evaluated by measuring cell size. The bar graph shows quantification results. (D–F) DHT accelerated premature senescence in non-balding and transitional zone of balding DPCs only. Scale bar = 200 µm. Values are means ± SDs from three independent experiments (* P

    Article Snippet: SA β-gal Staining The SA-β-Gal activity was determined by using a SA-β-Gal staining kit (Sigma).

    Techniques: Staining, Activity Assay

    AR knockdown suppresses DHT-mediated senescence in DPCs. (A) DPCs infected with AR-shRNA or scrambled shRNA were cultured in the presence of 0.1 µM of DHT or ethanol for 3 days. Premature senescence of DPCs was evaluated on day 5. The senescence-promoting effect of DHT was inhibited by AR knockdown. Scale bar = 100 µm. (B–D) The percentage of SA-β-Gal activity and Cell size was statistically unchanged in AR-knockdown DPCs after exposure to DHT. DHT induced SAHF formation was suppressed by AR downregulation. Values are means ± SDs from three independent experiments (* P

    Journal: PLoS ONE

    Article Title: Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

    doi: 10.1371/journal.pone.0079434

    Figure Lengend Snippet: AR knockdown suppresses DHT-mediated senescence in DPCs. (A) DPCs infected with AR-shRNA or scrambled shRNA were cultured in the presence of 0.1 µM of DHT or ethanol for 3 days. Premature senescence of DPCs was evaluated on day 5. The senescence-promoting effect of DHT was inhibited by AR knockdown. Scale bar = 100 µm. (B–D) The percentage of SA-β-Gal activity and Cell size was statistically unchanged in AR-knockdown DPCs after exposure to DHT. DHT induced SAHF formation was suppressed by AR downregulation. Values are means ± SDs from three independent experiments (* P

    Article Snippet: SA β-gal Staining The SA-β-Gal activity was determined by using a SA-β-Gal staining kit (Sigma).

    Techniques: Infection, shRNA, Cell Culture, Activity Assay

    Mitochondria dysfunction precedes lysosomes dysfunction during SIPS development. NIH3T3 cells were treated with or without H 2 O 2 . (A) Quantification of intracellular ROS and lysosomal CTSB activity within 24 h after H 2 O 2 treatment. Intracellular ROS was measured by flow cytometry using the DCFH-DA probe. Lysosomal CTSB activity was measured using the fluorescence intensity of the Magic Red Cathepsin B probe. 0 h indicates the end time point of H 2 O 2 treatment. The data of untreated cells (time point −1 h) was set to 1 and other values were normalized. (B) Quantification of intracellular ROS and lysosomal CTSB activity at 0, 1, 3, 5 d after H 2 O 2 treatment. (C) Images of intracellular ROS probe of control or H 2 O 2 -treated cells at d 3. DMSO or 10 μM CCCP was added to the culture medium after H 2 O 2 treatment. Scale bars: 50 μm. (D) CTSB activity in control or H 2 O 2 -treated cells day 3 was visualized with the Magic Red Cathepsin B probe. DMSO or 50 μM ambroxol was added to the culture medium after H 2 O 2 treatment. Scale bars: 50 μm. (E) to (G) H 2 O 2 -treated cells were incubated with NAC (2 μM), CCCP (10 μM) or ambroxol (50 μM) at different periods of time. Cells were stained with the SA-GLB1/SA-β-gal assay kit at d 5. Scale bars: 100 μm. The percentages of SA-GLB1-positive cells in each group are quantified. The data are presented as means ± SD from 3 independent experiments. *p

    Journal: Autophagy

    Article Title: Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

    doi: 10.1080/15548627.2016.1247143

    Figure Lengend Snippet: Mitochondria dysfunction precedes lysosomes dysfunction during SIPS development. NIH3T3 cells were treated with or without H 2 O 2 . (A) Quantification of intracellular ROS and lysosomal CTSB activity within 24 h after H 2 O 2 treatment. Intracellular ROS was measured by flow cytometry using the DCFH-DA probe. Lysosomal CTSB activity was measured using the fluorescence intensity of the Magic Red Cathepsin B probe. 0 h indicates the end time point of H 2 O 2 treatment. The data of untreated cells (time point −1 h) was set to 1 and other values were normalized. (B) Quantification of intracellular ROS and lysosomal CTSB activity at 0, 1, 3, 5 d after H 2 O 2 treatment. (C) Images of intracellular ROS probe of control or H 2 O 2 -treated cells at d 3. DMSO or 10 μM CCCP was added to the culture medium after H 2 O 2 treatment. Scale bars: 50 μm. (D) CTSB activity in control or H 2 O 2 -treated cells day 3 was visualized with the Magic Red Cathepsin B probe. DMSO or 50 μM ambroxol was added to the culture medium after H 2 O 2 treatment. Scale bars: 50 μm. (E) to (G) H 2 O 2 -treated cells were incubated with NAC (2 μM), CCCP (10 μM) or ambroxol (50 μM) at different periods of time. Cells were stained with the SA-GLB1/SA-β-gal assay kit at d 5. Scale bars: 100 μm. The percentages of SA-GLB1-positive cells in each group are quantified. The data are presented as means ± SD from 3 independent experiments. *p

    Article Snippet: SA-GLB1 activity was determined using the SA-β-gal staining kit (Beyotime, C0602) according to a standard protocol.

    Techniques: Activity Assay, Flow Cytometry, Cytometry, Fluorescence, Incubation, Staining, β-Gal Assay

    a) Chemical structures of G0-AuNP , G1-AuNP , and G2-AuNP . b) Schematic illustration of AuNP/ β-gal-siRNA complexation and transfection into cells.

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: Dendronized gold nanoparticles for siRNA delivery

    doi: 10.1002/smll.201201141

    Figure Lengend Snippet: a) Chemical structures of G0-AuNP , G1-AuNP , and G2-AuNP . b) Schematic illustration of AuNP/ β-gal-siRNA complexation and transfection into cells.

    Article Snippet: After centrifugation of lysates, β-gal levels were determined by a β-gal kit (Genlantis, CA).

    Techniques: Transfection

    Cell viability determined by Alamar blue® assay. Cell viability was determined after the treatment of a) G2-AuNP and b) G2-AuNP /β-gal-siRNA complex at various molar ratios (β-gal siRNA=75 pmol).

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: Dendronized gold nanoparticles for siRNA delivery

    doi: 10.1002/smll.201201141

    Figure Lengend Snippet: Cell viability determined by Alamar blue® assay. Cell viability was determined after the treatment of a) G2-AuNP and b) G2-AuNP /β-gal-siRNA complex at various molar ratios (β-gal siRNA=75 pmol).

    Article Snippet: After centrifugation of lysates, β-gal levels were determined by a β-gal kit (Genlantis, CA).

    Techniques: Alamar Blue Assay

    β-gal gene silencing in SVR-bag4 cell. a) Gene silencing effect of G2-AuNP/β-gal-siRNA with different concentration from 0.021 to 0.084 μM (NP/siRNA=2). b) Gene silencing effect of naked β-gal siRNA, naked nonsense siRNA,

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: Dendronized gold nanoparticles for siRNA delivery

    doi: 10.1002/smll.201201141

    Figure Lengend Snippet: β-gal gene silencing in SVR-bag4 cell. a) Gene silencing effect of G2-AuNP/β-gal-siRNA with different concentration from 0.021 to 0.084 μM (NP/siRNA=2). b) Gene silencing effect of naked β-gal siRNA, naked nonsense siRNA,

    Article Snippet: After centrifugation of lysates, β-gal levels were determined by a β-gal kit (Genlantis, CA).

    Techniques: Concentration Assay

    Hydrodynamic diameter and zeta potential of G0-AuNP , G1-AuNP , and G2-AuNP . Gel retardation assay of AuNP/β-gal-siRNA complexation at different molar ratios showed a decrease in band intensity due to the fluorescence quenching by complexation with

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: Dendronized gold nanoparticles for siRNA delivery

    doi: 10.1002/smll.201201141

    Figure Lengend Snippet: Hydrodynamic diameter and zeta potential of G0-AuNP , G1-AuNP , and G2-AuNP . Gel retardation assay of AuNP/β-gal-siRNA complexation at different molar ratios showed a decrease in band intensity due to the fluorescence quenching by complexation with

    Article Snippet: After centrifugation of lysates, β-gal levels were determined by a β-gal kit (Genlantis, CA).

    Techniques: Electrophoretic Mobility Shift Assay, Fluorescence

    MLN4924 induced apoptosis or senescence in a cell line-dependent manner (A and B) MLN4924 induced apoptosis in QBC939 cells and expression of pro-apoptotic proteins in MLN4924-treated QBC939 cells. Cells were treated with MLN4924 for 72 hours and then subjected to morphological observation, or immunoblotting analysis for proteins involved in apoptotic induction, using GAPDH as a loading control. (C) MLN4924 induced senescence in RBE cells. After 72-hour MLN4924 treatment, cells were subjected to morphological observation and staining of senescence-associated β–galactosidase. Scale bar, 100μm. (D) MLN4924 induced the accumulation of CRL substrates. Cells were treated with MLN4924 for 72 hours using indicated concentrations and subjected to immunoblotting analysis with GADPH as a loading control.

    Journal: Oncotarget

    Article Title: Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target

    doi:

    Figure Lengend Snippet: MLN4924 induced apoptosis or senescence in a cell line-dependent manner (A and B) MLN4924 induced apoptosis in QBC939 cells and expression of pro-apoptotic proteins in MLN4924-treated QBC939 cells. Cells were treated with MLN4924 for 72 hours and then subjected to morphological observation, or immunoblotting analysis for proteins involved in apoptotic induction, using GAPDH as a loading control. (C) MLN4924 induced senescence in RBE cells. After 72-hour MLN4924 treatment, cells were subjected to morphological observation and staining of senescence-associated β–galactosidase. Scale bar, 100μm. (D) MLN4924 induced the accumulation of CRL substrates. Cells were treated with MLN4924 for 72 hours using indicated concentrations and subjected to immunoblotting analysis with GADPH as a loading control.

    Article Snippet: SA-β-Galactosidase Staining The expression of senescence-associated β-galactosidase was determined by SA-β-Galactosidase (SA-β-Gal) staining (Beyotime) according to the manufacturer's instructions and as previously described [ , ].

    Techniques: Expressing, Staining

    SIRT1 regulated autophagy through physically interacted with and deacetylated BECN1 and LC3B in PD-NSCs. a – c Cell senescence of PD-NSCs treated with or without resveratrol or chloroquine was measured, including SA-β-gal staining ( a , b ) and expression of aging-related genes ( c ), analyzed by western blotting and densitometry. Cells were exposed to 10 Gy X-ray in the presence of 3 μM resveratrol or 3 μM chloroquine, and then incubated for 48 h. d , e Images of cellular senescence in PD-NSCs after knockdown of BECN1 were visualized using SA-β-gal assay. Cells were transfected with siBECN1, and then exposed to 10 Gy X-ray in the presence of 3 μM resveratrol, incubated for 48 h. f The interaction between SIRT1 and autophagy-related genes, LC3B and BECN1 , in PD-NSCs. Cell lysates were immunoprecipitated with anti-SIRT1 or control IgG antibody; and then, reciprocally probed with anti-BECN1 and anti-LC3B. Cell lysates were immunoprecipitated with anti-BECN1, anti-LC3B, or control IgG antibody; and then, reciprocally probed with anti-SIRT1. g Western blot and densitometric analysis of acetylation changes of BECN1 and LC3B after IR treatment in PD-NSCs. BECN1 and LC3B were immunoprecipitated from the total cell lysate of NSCs by BECN1 and LC3B antibody; and then, western blots were probed with anti-acetylated lysine antibody. h Western blot and densitometric analysis of acetylation changes of BECN1 and LC3B after PD-NSCs were treated with SIRT1 activator resveratrol (3 μM) and SIRT1 inhibitor nicotinamide (5 mM). BECN1 and LC3B were immunoprecipitated from the total cell lysate of NSCs by BECN1 and LC3B antibody; and then, western blots were probed with anti-acetylated lysine antibody. Data of the graph were expressed as the mean ± SD. * P

    Journal: Cell Death & Disease

    Article Title: Stress-induced precocious aging in PD-patient iPSC-derived NSCs may underlie the pathophysiology of Parkinson’s disease

    doi: 10.1038/s41419-019-1313-y

    Figure Lengend Snippet: SIRT1 regulated autophagy through physically interacted with and deacetylated BECN1 and LC3B in PD-NSCs. a – c Cell senescence of PD-NSCs treated with or without resveratrol or chloroquine was measured, including SA-β-gal staining ( a , b ) and expression of aging-related genes ( c ), analyzed by western blotting and densitometry. Cells were exposed to 10 Gy X-ray in the presence of 3 μM resveratrol or 3 μM chloroquine, and then incubated for 48 h. d , e Images of cellular senescence in PD-NSCs after knockdown of BECN1 were visualized using SA-β-gal assay. Cells were transfected with siBECN1, and then exposed to 10 Gy X-ray in the presence of 3 μM resveratrol, incubated for 48 h. f The interaction between SIRT1 and autophagy-related genes, LC3B and BECN1 , in PD-NSCs. Cell lysates were immunoprecipitated with anti-SIRT1 or control IgG antibody; and then, reciprocally probed with anti-BECN1 and anti-LC3B. Cell lysates were immunoprecipitated with anti-BECN1, anti-LC3B, or control IgG antibody; and then, reciprocally probed with anti-SIRT1. g Western blot and densitometric analysis of acetylation changes of BECN1 and LC3B after IR treatment in PD-NSCs. BECN1 and LC3B were immunoprecipitated from the total cell lysate of NSCs by BECN1 and LC3B antibody; and then, western blots were probed with anti-acetylated lysine antibody. h Western blot and densitometric analysis of acetylation changes of BECN1 and LC3B after PD-NSCs were treated with SIRT1 activator resveratrol (3 μM) and SIRT1 inhibitor nicotinamide (5 mM). BECN1 and LC3B were immunoprecipitated from the total cell lysate of NSCs by BECN1 and LC3B antibody; and then, western blots were probed with anti-acetylated lysine antibody. Data of the graph were expressed as the mean ± SD. * P

    Article Snippet: SA-β-gal assay Cellular senescence was determined using the SA-β-gal staining Kit (Genmed Scientifics).

    Techniques: Staining, Expressing, Western Blot, Incubation, β-Gal Assay, Transfection, Immunoprecipitation

    Autophagy was implicated in the IR-induced premature aging of PD-NSCs. a , b Cell senescence induced by irradiation was detected by SA-β-gal staining in the presence of rapamycin or chloroquine. Cells were exposed to 10 Gy X-ray in the presence of 0.04 μM rapamycin or 3 μM chloroquine, incubated for 48 h, and then stained with SA-β-gal. c p16, p21, and p53 expression from rapamycin- or chloroquine-treated NSCs were detected by western blotting and densitometry. d , e The effect of rapamycin or chloroquine on Ki67 expression was visualized by immunostaining. Data represent as the mean ± SD of three independent experiments. Statistical analysis was performed by Student’s t -test, * P

    Journal: Cell Death & Disease

    Article Title: Stress-induced precocious aging in PD-patient iPSC-derived NSCs may underlie the pathophysiology of Parkinson’s disease

    doi: 10.1038/s41419-019-1313-y

    Figure Lengend Snippet: Autophagy was implicated in the IR-induced premature aging of PD-NSCs. a , b Cell senescence induced by irradiation was detected by SA-β-gal staining in the presence of rapamycin or chloroquine. Cells were exposed to 10 Gy X-ray in the presence of 0.04 μM rapamycin or 3 μM chloroquine, incubated for 48 h, and then stained with SA-β-gal. c p16, p21, and p53 expression from rapamycin- or chloroquine-treated NSCs were detected by western blotting and densitometry. d , e The effect of rapamycin or chloroquine on Ki67 expression was visualized by immunostaining. Data represent as the mean ± SD of three independent experiments. Statistical analysis was performed by Student’s t -test, * P

    Article Snippet: SA-β-gal assay Cellular senescence was determined using the SA-β-gal staining Kit (Genmed Scientifics).

    Techniques: Irradiation, Staining, Incubation, Expressing, Western Blot, Immunostaining

    NSCs derived from patient-specific PD iPSCs manifested premature aging phenotypes and were sensitive to irradiation. a iPSC-derived neural stem cells were stained positively for NSC-specific markers Nestin and SOX2. Scale bar: 100 μm. b , c Neural differentiation capacity was severely impaired in PD iPSCs. b The representative images for spontaneous differentiation of PD- and WT-NSCs toward neurons and astrocytes as determined by the neuronal marker (Tuj1) and the astrocyte marker (GFAP). c Statistic results showed the percentage of Tuj1 and GFAP cells out of all cells derived from WT- and PD iPSCs. Scale bar: 100 μm. d – f The proliferative capacity of PD-NSCs was compromised as revealed by CCK8 assays and Ki67 staining. d Cell growth curve was detected by CCK8 assays at 24, 48, 72, 96, and 120 h. e Proliferation capacity was determined by Ki67 staining. f Graph showed the percentage of Ki67 cells. Scale bar: 100 μm. g , h Cellular senescence was significantly aggravated by IR treatment as evaluated with the SA-β-gal assay. g Representative images from at least three independent experiments were presented. h The statistical results showed the percent of SA-β-gal-positive cells (%). Scale bar: 100 μm. i , j ROS level increased significantly in PD-NSC with IR treatment. i Cellular ROS was stained with DCF-DA and observed under a fluorescence microscope. j ROS level was detected by fluorescence microplate reader. Scale bar: 100 μm. k , l Proliferative capacity was further decreased in PD-NSCs after IR treatment as revealed by Ki67 staining. k Expression of Ki67 in cells with or without IR treatment. l Graph showed the ratio of Ki67-positive cells. Scale bar: 100 μm. m , n DNA damage accumulation increased dramatically in PD-NSCs by IR as revealed by γH2AX staining (red) at 48 h post IR treatment. m Formation of DNA double-strand breaks in NSCs post irradiation. n Graphical depiction indicated the number of γH 2 AX foci in NSCs. Scale bar: 10 μm. o Cell proliferation curve of WT-NSCs and PD-NSCs with or without IR. Cell numbers were analyzed at 24, 48, 72, 96, and 120 h. p The expression level of cell senescence markers p53, p21, and p16 was augmented in PD-NSCs with IR treatment, analyzed by western blotting and densitometry. NSCs were treated with 10 Gy of IR and incubated for the indicated periods. β-actin was used as the loading control. All the data were expressed as mean ± SD. * P

    Journal: Cell Death & Disease

    Article Title: Stress-induced precocious aging in PD-patient iPSC-derived NSCs may underlie the pathophysiology of Parkinson’s disease

    doi: 10.1038/s41419-019-1313-y

    Figure Lengend Snippet: NSCs derived from patient-specific PD iPSCs manifested premature aging phenotypes and were sensitive to irradiation. a iPSC-derived neural stem cells were stained positively for NSC-specific markers Nestin and SOX2. Scale bar: 100 μm. b , c Neural differentiation capacity was severely impaired in PD iPSCs. b The representative images for spontaneous differentiation of PD- and WT-NSCs toward neurons and astrocytes as determined by the neuronal marker (Tuj1) and the astrocyte marker (GFAP). c Statistic results showed the percentage of Tuj1 and GFAP cells out of all cells derived from WT- and PD iPSCs. Scale bar: 100 μm. d – f The proliferative capacity of PD-NSCs was compromised as revealed by CCK8 assays and Ki67 staining. d Cell growth curve was detected by CCK8 assays at 24, 48, 72, 96, and 120 h. e Proliferation capacity was determined by Ki67 staining. f Graph showed the percentage of Ki67 cells. Scale bar: 100 μm. g , h Cellular senescence was significantly aggravated by IR treatment as evaluated with the SA-β-gal assay. g Representative images from at least three independent experiments were presented. h The statistical results showed the percent of SA-β-gal-positive cells (%). Scale bar: 100 μm. i , j ROS level increased significantly in PD-NSC with IR treatment. i Cellular ROS was stained with DCF-DA and observed under a fluorescence microscope. j ROS level was detected by fluorescence microplate reader. Scale bar: 100 μm. k , l Proliferative capacity was further decreased in PD-NSCs after IR treatment as revealed by Ki67 staining. k Expression of Ki67 in cells with or without IR treatment. l Graph showed the ratio of Ki67-positive cells. Scale bar: 100 μm. m , n DNA damage accumulation increased dramatically in PD-NSCs by IR as revealed by γH2AX staining (red) at 48 h post IR treatment. m Formation of DNA double-strand breaks in NSCs post irradiation. n Graphical depiction indicated the number of γH 2 AX foci in NSCs. Scale bar: 10 μm. o Cell proliferation curve of WT-NSCs and PD-NSCs with or without IR. Cell numbers were analyzed at 24, 48, 72, 96, and 120 h. p The expression level of cell senescence markers p53, p21, and p16 was augmented in PD-NSCs with IR treatment, analyzed by western blotting and densitometry. NSCs were treated with 10 Gy of IR and incubated for the indicated periods. β-actin was used as the loading control. All the data were expressed as mean ± SD. * P

    Article Snippet: SA-β-gal assay Cellular senescence was determined using the SA-β-gal staining Kit (Genmed Scientifics).

    Techniques: Derivative Assay, Irradiation, Staining, Marker, β-Gal Assay, Fluorescence, Microscopy, Expressing, Western Blot, Incubation

    T 3 signaling as determined by β-gal staining in areas of the P90 WT/FINDT3 and D3KO/FINDT3 cortex. Staining protocol 2 was used in A–F, and staining protocol 1 was used in G–L. Areas depicted are the motor cortex (A and B and G

    Journal: Endocrinology

    Article Title: Type 3 Deiodinase Deficiency Causes Spatial and Temporal Alterations in Brain T3 Signaling that Are Dissociated from Serum Thyroid Hormone Levels

    doi: 10.1210/en.2010-0450

    Figure Lengend Snippet: T 3 signaling as determined by β-gal staining in areas of the P90 WT/FINDT3 and D3KO/FINDT3 cortex. Staining protocol 2 was used in A–F, and staining protocol 1 was used in G–L. Areas depicted are the motor cortex (A and B and G

    Article Snippet: β-Gal activities were determined in tissue homogenates using the β-gal enzyme assay kit from Promega (Madison, WI), according to the manufacturer’s instructions.

    Techniques: Staining

    Abnormal patterns of β-gal staining in certain regions of the P60 D3KO brain. A, and B, Prefrontal cortex. C and D, Septal diagonal band. E and F, Motor cortex. Arrows point to differences in staining patterns (A–D), and white lines of

    Journal: Endocrinology

    Article Title: Type 3 Deiodinase Deficiency Causes Spatial and Temporal Alterations in Brain T3 Signaling that Are Dissociated from Serum Thyroid Hormone Levels

    doi: 10.1210/en.2010-0450

    Figure Lengend Snippet: Abnormal patterns of β-gal staining in certain regions of the P60 D3KO brain. A, and B, Prefrontal cortex. C and D, Septal diagonal band. E and F, Motor cortex. Arrows point to differences in staining patterns (A–D), and white lines of

    Article Snippet: β-Gal activities were determined in tissue homogenates using the β-gal enzyme assay kit from Promega (Madison, WI), according to the manufacturer’s instructions.

    Techniques: Staining

    Effect of T 3 treatment on brain T 3 signaling and serum T 3 . A–D, T 3 signaling as determined by β-gal staining in the P60 insular cortex in WT/FINDT3 and D3KO/FINDT3 animals treated (B and D) or not (A and C) for 7 d with 0.25 μg/ml

    Journal: Endocrinology

    Article Title: Type 3 Deiodinase Deficiency Causes Spatial and Temporal Alterations in Brain T3 Signaling that Are Dissociated from Serum Thyroid Hormone Levels

    doi: 10.1210/en.2010-0450

    Figure Lengend Snippet: Effect of T 3 treatment on brain T 3 signaling and serum T 3 . A–D, T 3 signaling as determined by β-gal staining in the P60 insular cortex in WT/FINDT3 and D3KO/FINDT3 animals treated (B and D) or not (A and C) for 7 d with 0.25 μg/ml

    Article Snippet: β-Gal activities were determined in tissue homogenates using the β-gal enzyme assay kit from Promega (Madison, WI), according to the manufacturer’s instructions.

    Techniques: Staining

    T 3 signaling as determined by β-gal staining in septal and cortical areas of P5 WT/FINDT3 (A and D), D3KO/FINDT3 (B and E), and WT (C and F) neonates. Protocol 2 was used to detect β-gal expression (see Materials and Methods ). No staining

    Journal: Endocrinology

    Article Title: Type 3 Deiodinase Deficiency Causes Spatial and Temporal Alterations in Brain T3 Signaling that Are Dissociated from Serum Thyroid Hormone Levels

    doi: 10.1210/en.2010-0450

    Figure Lengend Snippet: T 3 signaling as determined by β-gal staining in septal and cortical areas of P5 WT/FINDT3 (A and D), D3KO/FINDT3 (B and E), and WT (C and F) neonates. Protocol 2 was used to detect β-gal expression (see Materials and Methods ). No staining

    Article Snippet: β-Gal activities were determined in tissue homogenates using the β-gal enzyme assay kit from Promega (Madison, WI), according to the manufacturer’s instructions.

    Techniques: Staining, Expressing

    T 3 availability as determined by β-gal staining in areas of the P15 WT/FINDT3 and D3KO/FINDT3 brains. A and B, Orbital (OCx) and prefrontal cortices. C and D, Septum (S). E and F, Pyramidal cell layer of hippocampus (Py). G and H, nuclei in anterior

    Journal: Endocrinology

    Article Title: Type 3 Deiodinase Deficiency Causes Spatial and Temporal Alterations in Brain T3 Signaling that Are Dissociated from Serum Thyroid Hormone Levels

    doi: 10.1210/en.2010-0450

    Figure Lengend Snippet: T 3 availability as determined by β-gal staining in areas of the P15 WT/FINDT3 and D3KO/FINDT3 brains. A and B, Orbital (OCx) and prefrontal cortices. C and D, Septum (S). E and F, Pyramidal cell layer of hippocampus (Py). G and H, nuclei in anterior

    Article Snippet: β-Gal activities were determined in tissue homogenates using the β-gal enzyme assay kit from Promega (Madison, WI), according to the manufacturer’s instructions.

    Techniques: Staining