microglial sa β gal activity  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Millipore microglial sa β gal activity
    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase <t>(SA-β-gal)</t> activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p
    Microglial Sa β Gal Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microglial sa β gal activity/product/Millipore
    Average 86 stars, based on 1828 article reviews
    Price from $9.99 to $1999.99
    microglial sa β gal activity - by Bioz Stars, 2020-08
    86/100 stars

    Images

    1) Product Images from "Microglia change from a reactive to an age-like phenotype with the time in culture"

    Article Title: Microglia change from a reactive to an age-like phenotype with the time in culture

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2014.00152

    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p
    Figure Legend Snippet: Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

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

    2) Product Images from "Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition"

    Article Title: Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.10

    CK2 inhibitor affects p53 target genes associated with cell cycle regulation and apoptosis. ( a ) CK2-I increases p21 expression in a p53-dependent manner. Representative blot is shown from three independent experiments with identical results. Blots were re-probed with c23 to establish equivalent loading. ( b ) CK2-I increases mRNA levels of pro-apoptotic molecules Noxa and GADD45 β in a p53-dependent manner. Total RNA was isolated from cells treated with different combinations of TNF α and CK2-I, and the mRNA levels for Noxa, GADD45 β and constitutive enzyme GAPDH were determined by RT-PCR. ( c ) Pifithrin- α reverses CK2-I-mediated G2/M phase arrest in A172 cells. FACS analysis was performed on A172 cells treated with different combinations of TNF α , CK2-Is and Pifithrin- α . Inset indicates percentage of cells in G1, S and G2/M phase of the cell cycle. C, T, Pf and A denote control, TNF α , Pifithrin- α and Apigenin, respectively
    Figure Legend Snippet: CK2 inhibitor affects p53 target genes associated with cell cycle regulation and apoptosis. ( a ) CK2-I increases p21 expression in a p53-dependent manner. Representative blot is shown from three independent experiments with identical results. Blots were re-probed with c23 to establish equivalent loading. ( b ) CK2-I increases mRNA levels of pro-apoptotic molecules Noxa and GADD45 β in a p53-dependent manner. Total RNA was isolated from cells treated with different combinations of TNF α and CK2-I, and the mRNA levels for Noxa, GADD45 β and constitutive enzyme GAPDH were determined by RT-PCR. ( c ) Pifithrin- α reverses CK2-I-mediated G2/M phase arrest in A172 cells. FACS analysis was performed on A172 cells treated with different combinations of TNF α , CK2-Is and Pifithrin- α . Inset indicates percentage of cells in G1, S and G2/M phase of the cell cycle. C, T, Pf and A denote control, TNF α , Pifithrin- α and Apigenin, respectively

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, FACS

    CK2 inhibition-induced p53 activation decreases telomerase activity and induces senescence but has no effect on NF- κ B activity. ( a ) CK2-Is decrease telomerase activity in glioma cells in a p53-dependent manner. Glioma cells were treated with TNF α in the presence or absence of CK2-Is and/or Pifithrin- α ; and T elo TAGGG Telomerase PCR ELISA was performed. Values represent the means±S.E.M. from three independent experiments. ( b ) CK2-I-induced senescence in glioma cells is p53-dependent as evident from senescence-specific β -gal staining. Images were taken at 20 × magnification. The graph represents fold increase in β -gal-positive cells upon treatment with different combinations of Apigenin, TNF α and Pifithrin- α . ( c ) Pifithrin- α does not affect the ability of DRB to abrogate TNF α -induced NF- κ B activation. The graph represents fold change in NF- κ B luciferase activity over control, in cells treated with different combinations of TNF α , DRB and Pifithrin- α . Values in ( a – c ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from control ( P
    Figure Legend Snippet: CK2 inhibition-induced p53 activation decreases telomerase activity and induces senescence but has no effect on NF- κ B activity. ( a ) CK2-Is decrease telomerase activity in glioma cells in a p53-dependent manner. Glioma cells were treated with TNF α in the presence or absence of CK2-Is and/or Pifithrin- α ; and T elo TAGGG Telomerase PCR ELISA was performed. Values represent the means±S.E.M. from three independent experiments. ( b ) CK2-I-induced senescence in glioma cells is p53-dependent as evident from senescence-specific β -gal staining. Images were taken at 20 × magnification. The graph represents fold increase in β -gal-positive cells upon treatment with different combinations of Apigenin, TNF α and Pifithrin- α . ( c ) Pifithrin- α does not affect the ability of DRB to abrogate TNF α -induced NF- κ B activation. The graph represents fold change in NF- κ B luciferase activity over control, in cells treated with different combinations of TNF α , DRB and Pifithrin- α . Values in ( a – c ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from control ( P

    Techniques Used: Inhibition, Activation Assay, Activity Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Staining, Luciferase

    CK2 inhibitors abrogate TNF α -induced NF- κ B activation. ( a ) Fas and FADD expression in glioma cells treated with either TNF α or CK2-I or both for 24 h as demonstrated by western blot. A representative blot is shown from three independent experiments with identical results. Blots were reprobed for β -actin to establish equivalent loading. ( b ) Caspase-8 activity in glioma cells treated with CK2-Is or TNF α or both for 24 h, as determined by the caspase-8 activity assay kit. ( c ) The graph represents the viable cells, percentage of control, observed when glioma cells were treated with different combinations of TNF α , CK2-Is and caspase-8 inhibitor, as determined by MTS assay. ( d ) The levels of NF- κ B, pI κ K α / β and I κ B α in glioma cells, treated with either TNF α or CK2-Is or both, as demonstrated by western blot. A representative blot is shown from three independent experiments with identical results. Blots were reprobed for β -actin and c23 to establish equivalent loading. ( e ) DRB and Apigenin decreases TNF α -mediated NF- κ B activation in glioma cells. The graph represents fold change in NF- κ B luciferase activity over control, in cells treated with TNF α in the presence and absence of CK2-Is. ( f ) siRNA-mediated knockdown of CK2 α / β decreases TNF α -induced NF- κ B activation in glioma cells. Values in ( b , c , e and f ) represent the mean ±S.E.M. from three independent experiments. * Denotes significant change from control ( P
    Figure Legend Snippet: CK2 inhibitors abrogate TNF α -induced NF- κ B activation. ( a ) Fas and FADD expression in glioma cells treated with either TNF α or CK2-I or both for 24 h as demonstrated by western blot. A representative blot is shown from three independent experiments with identical results. Blots were reprobed for β -actin to establish equivalent loading. ( b ) Caspase-8 activity in glioma cells treated with CK2-Is or TNF α or both for 24 h, as determined by the caspase-8 activity assay kit. ( c ) The graph represents the viable cells, percentage of control, observed when glioma cells were treated with different combinations of TNF α , CK2-Is and caspase-8 inhibitor, as determined by MTS assay. ( d ) The levels of NF- κ B, pI κ K α / β and I κ B α in glioma cells, treated with either TNF α or CK2-Is or both, as demonstrated by western blot. A representative blot is shown from three independent experiments with identical results. Blots were reprobed for β -actin and c23 to establish equivalent loading. ( e ) DRB and Apigenin decreases TNF α -mediated NF- κ B activation in glioma cells. The graph represents fold change in NF- κ B luciferase activity over control, in cells treated with TNF α in the presence and absence of CK2-Is. ( f ) siRNA-mediated knockdown of CK2 α / β decreases TNF α -induced NF- κ B activation in glioma cells. Values in ( b , c , e and f ) represent the mean ±S.E.M. from three independent experiments. * Denotes significant change from control ( P

    Techniques Used: Activation Assay, Expressing, Western Blot, Activity Assay, MTS Assay, Luciferase

    CK2 inhibitor-mediated downregulation of SIRT1 activity regulates p53. ( a ) CK2-Is decrease SIRT1 activity in glioma cells both in the presence and absence of TNF α . Glioma cells were treated with TNF α in the presence or absence of DRB/Api and subsequently SIRT1 activity was determined. Values represent the means±S.E.M. from three independent experiments. ( b ) SIRT1 immuno-localization in glioma tumor samples as revealed by IHC and western blot analysis. Cryosections of glioma and adjacent normal tissues were immunostained for SIRT1 as described in Materials and Methods. Images were taken at 40 × magnification. Western blot analysis also demonstrated elevated SIRT1 levels in GBM tumor as compared with surrounding non-neoplastic tissue. The figure shows blots from five independent tumor samples with identical results. ( c ) CK2-I-mediated decrease in SIRT1 activity regulates glioma cell viability. The viability of glioma cells transfected with SIRT1 over-expression vector and treated with Apigenin or TNF α or both for 24 h, was determined by MTS assay. The graph represents percentage viable cells of control. Inset shows the overexpressed SIRT1 levels as determined by immunoblotting. ( d ) CK2-I-mediated increase in p53 transcriptional activity is abrogated upon SIRT1 over-expression. Glioma cells co-transfected with SIRT1 over-expression vector and p53 luciferase reporter constructs were treated with Apigenin or TNF α or both and reporter assay was performed to determine p53 transcriptional activity. The graph represents fold change in p53 luciferase reporter activity over control. Values in ( a , c and d ) represent the means±S.E.M. from three independent experiments. * Denotes significant change from control ( P
    Figure Legend Snippet: CK2 inhibitor-mediated downregulation of SIRT1 activity regulates p53. ( a ) CK2-Is decrease SIRT1 activity in glioma cells both in the presence and absence of TNF α . Glioma cells were treated with TNF α in the presence or absence of DRB/Api and subsequently SIRT1 activity was determined. Values represent the means±S.E.M. from three independent experiments. ( b ) SIRT1 immuno-localization in glioma tumor samples as revealed by IHC and western blot analysis. Cryosections of glioma and adjacent normal tissues were immunostained for SIRT1 as described in Materials and Methods. Images were taken at 40 × magnification. Western blot analysis also demonstrated elevated SIRT1 levels in GBM tumor as compared with surrounding non-neoplastic tissue. The figure shows blots from five independent tumor samples with identical results. ( c ) CK2-I-mediated decrease in SIRT1 activity regulates glioma cell viability. The viability of glioma cells transfected with SIRT1 over-expression vector and treated with Apigenin or TNF α or both for 24 h, was determined by MTS assay. The graph represents percentage viable cells of control. Inset shows the overexpressed SIRT1 levels as determined by immunoblotting. ( d ) CK2-I-mediated increase in p53 transcriptional activity is abrogated upon SIRT1 over-expression. Glioma cells co-transfected with SIRT1 over-expression vector and p53 luciferase reporter constructs were treated with Apigenin or TNF α or both and reporter assay was performed to determine p53 transcriptional activity. The graph represents fold change in p53 luciferase reporter activity over control. Values in ( a , c and d ) represent the means±S.E.M. from three independent experiments. * Denotes significant change from control ( P

    Techniques Used: Activity Assay, Immunohistochemistry, Western Blot, Transfection, Over Expression, Plasmid Preparation, MTS Assay, Luciferase, Construct, Reporter Assay

    Proposed model demonstrating the role of CK2 in glioma cell apoptosis and resistance to therapy. CK2 inhibition induces glioma cell death via activation of p53 through SIRT1 inhibition and sensitizes glioma cell to TNF α via downregulation of TNF α -induced NF- κ B activity
    Figure Legend Snippet: Proposed model demonstrating the role of CK2 in glioma cell apoptosis and resistance to therapy. CK2 inhibition induces glioma cell death via activation of p53 through SIRT1 inhibition and sensitizes glioma cell to TNF α via downregulation of TNF α -induced NF- κ B activity

    Techniques Used: Inhibition, Activation Assay, Activity Assay

    CK2 inhibition triggers p53 expression and activation to induce glioma cell death. ( a ) Western blot indicated phosphorylated, acetylated and total p53 levels in cells treated with CK2-Is in the presence and absence of TNF α . A representative blot is shown from three independent experiments with identical results. c23 was used as loading control. ( b ) Increased binding of p53 to its DNA-binding sequence in cells treated with CK2-Is, either alone or in combination with TNF α , is abrogated by p53 inhibitor, Pifithrin- α , The graphs represent relative binding activity of p53 under different conditions, as compared with untreated controls. Values represent means ±S.E.M. from three individual experiments. ( c ) CK2-I-mediated increase in p53 transcriptional activity is abrogated upon p53 inhibition. The graphs represent fold change in p53 luciferase reporter activity over control in cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 12 h. ( d ) siRNA-mediated knockdown of CK2 α / β induces p53 transcriptional activity in p53 wild-type but not in mutant cell lines. The graph represents fold change in p53 luciferase reporter activity over NS-siRNA-transfected control. ( e ) CK2-I-mediated sensitization of glioma cells to TNF α -induced death is reversed by Pifithrin- α and ( f ) p53 siRNA. The graphs represent percentage viable glioma cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 24 h, as determined by MTS assay. (Inset) p53 siRNA abrogates CK2-I-induced p53 levels as determined by western blot analysis. ( g ) CK2-I reduced viability of p53 null cells lines only in presence of TNF α . The graph represents percentage viable SAOS2 and H1299 cells treated with different combinations of CK2-Is and TNF α . Values in ( b – g ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from untreated control, # denotes significant change from CK2-I-treated cells either alone or in presence of TNF α ( P
    Figure Legend Snippet: CK2 inhibition triggers p53 expression and activation to induce glioma cell death. ( a ) Western blot indicated phosphorylated, acetylated and total p53 levels in cells treated with CK2-Is in the presence and absence of TNF α . A representative blot is shown from three independent experiments with identical results. c23 was used as loading control. ( b ) Increased binding of p53 to its DNA-binding sequence in cells treated with CK2-Is, either alone or in combination with TNF α , is abrogated by p53 inhibitor, Pifithrin- α , The graphs represent relative binding activity of p53 under different conditions, as compared with untreated controls. Values represent means ±S.E.M. from three individual experiments. ( c ) CK2-I-mediated increase in p53 transcriptional activity is abrogated upon p53 inhibition. The graphs represent fold change in p53 luciferase reporter activity over control in cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 12 h. ( d ) siRNA-mediated knockdown of CK2 α / β induces p53 transcriptional activity in p53 wild-type but not in mutant cell lines. The graph represents fold change in p53 luciferase reporter activity over NS-siRNA-transfected control. ( e ) CK2-I-mediated sensitization of glioma cells to TNF α -induced death is reversed by Pifithrin- α and ( f ) p53 siRNA. The graphs represent percentage viable glioma cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 24 h, as determined by MTS assay. (Inset) p53 siRNA abrogates CK2-I-induced p53 levels as determined by western blot analysis. ( g ) CK2-I reduced viability of p53 null cells lines only in presence of TNF α . The graph represents percentage viable SAOS2 and H1299 cells treated with different combinations of CK2-Is and TNF α . Values in ( b – g ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from untreated control, # denotes significant change from CK2-I-treated cells either alone or in presence of TNF α ( P

    Techniques Used: Inhibition, Expressing, Activation Assay, Western Blot, Binding Assay, Sequencing, Activity Assay, Luciferase, Mutagenesis, Transfection, MTS Assay

    CK2 inhibition sensitizes glioma cells to TNF α -induced apoptosis. ( a ) CK2 immuno-localization in glioma tumor samples. Cryosections of glioma and adjacent normal tissues were immunostained for CK2 α as described in Materials and Methods. Images were taken at 40 × magnification. ( b ) Western blot analysis demonstrating elevated CK2 α expression in GBM tumor as compared with surrounding non-neoplastic tissue. The figure shows blots from five independent tumor samples with identical results. ( c ) Viability of glioma cells treated with CK2-Is in the presence and absence of TNF α was determined by MTS assay. The graphs represent the viable glioma cells, percentage of control, upon treatment with 50 ng/ml TNF α either alone or in combination with different concentrations of DRB or Apigenin for 24 h. ( d ) The graphs represent percentage of TUNEL-positive glioma cells upon treatment with either DRB/Api or TNF α or both, as counted from multiple fields. ( e ) siRNA-mediated knockdown of CK2 decreases glioma cell viability as determined by MTS assay. The graph represents the viable glioma cells, percentage of control, transfected with CK2 α / β siRNA when treated with TNF α for 24 h. Inset shows the knockdown efficiency of CK2 α siRNA. ( f ) Increase in caspase-3 activity in glioma cells treated with CK2-Is or TNF α or both, as determined by caspase-3 activity assay. Values ( c – f ) represent the means ±S.E.M. from three independent experiments. * Denotes significant change from control ( P
    Figure Legend Snippet: CK2 inhibition sensitizes glioma cells to TNF α -induced apoptosis. ( a ) CK2 immuno-localization in glioma tumor samples. Cryosections of glioma and adjacent normal tissues were immunostained for CK2 α as described in Materials and Methods. Images were taken at 40 × magnification. ( b ) Western blot analysis demonstrating elevated CK2 α expression in GBM tumor as compared with surrounding non-neoplastic tissue. The figure shows blots from five independent tumor samples with identical results. ( c ) Viability of glioma cells treated with CK2-Is in the presence and absence of TNF α was determined by MTS assay. The graphs represent the viable glioma cells, percentage of control, upon treatment with 50 ng/ml TNF α either alone or in combination with different concentrations of DRB or Apigenin for 24 h. ( d ) The graphs represent percentage of TUNEL-positive glioma cells upon treatment with either DRB/Api or TNF α or both, as counted from multiple fields. ( e ) siRNA-mediated knockdown of CK2 decreases glioma cell viability as determined by MTS assay. The graph represents the viable glioma cells, percentage of control, transfected with CK2 α / β siRNA when treated with TNF α for 24 h. Inset shows the knockdown efficiency of CK2 α siRNA. ( f ) Increase in caspase-3 activity in glioma cells treated with CK2-Is or TNF α or both, as determined by caspase-3 activity assay. Values ( c – f ) represent the means ±S.E.M. from three independent experiments. * Denotes significant change from control ( P

    Techniques Used: Inhibition, Western Blot, Expressing, MTS Assay, TUNEL Assay, Transfection, Activity Assay, Caspase-3 Activity Assay

    CK2 inhibition leads to p53 protein stabilization. ( a ) CK2-I prevents degradation of p53. Glioma cells, either untreated or treated with Apigenin for 12 h were further treated with CHX for the indicated times. Western blot analysis was performed to determine p53 levels in the lysates. Representative blot is shown from three independent experiments with identical results. Blots were reprobed for GAPDH to establish equivalent loading. Densitometric analysis shows p53 levels upon CHX treatment in the presence and absence of Apigenin. ( b ) CK2-I decreases MDM2–p53 interaction and p53 ubiquitination in glioma cells. Lysates from cells treated with Apigenin were immunoprecipitated with p53 and probed with antibodies against MDM2 and ubiquitin. IgG levels are shown to establish equivalent loading. Representative blot is shown from two independent experiments with identical results. * Denotes significant change from control ( P
    Figure Legend Snippet: CK2 inhibition leads to p53 protein stabilization. ( a ) CK2-I prevents degradation of p53. Glioma cells, either untreated or treated with Apigenin for 12 h were further treated with CHX for the indicated times. Western blot analysis was performed to determine p53 levels in the lysates. Representative blot is shown from three independent experiments with identical results. Blots were reprobed for GAPDH to establish equivalent loading. Densitometric analysis shows p53 levels upon CHX treatment in the presence and absence of Apigenin. ( b ) CK2-I decreases MDM2–p53 interaction and p53 ubiquitination in glioma cells. Lysates from cells treated with Apigenin were immunoprecipitated with p53 and probed with antibodies against MDM2 and ubiquitin. IgG levels are shown to establish equivalent loading. Representative blot is shown from two independent experiments with identical results. * Denotes significant change from control ( P

    Techniques Used: Inhibition, Western Blot, Immunoprecipitation

    3) Product Images from "Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?"

    Article Title: Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137188

    Senescence and inflammation induced by T-oligos in CD-1 pregnant mice. (A-D) Senescence associated β-galactosidase (SA-β-gal) staining of murine amniotic sac. Single blue stained cells indicate β-gal activity. A. saline, B. Cont-oligo, C. T-oligo and D. T-oligo+SB203580 (p38MAPK inhibitor) treated mice. SA-β-gal staining is pronounced in T-oligo treated mice. (E) Concentration of interleukin (IL)-8 protein in murine amniotic fluid. Higher levels of IL-8 were found in T-oligo treated animals compared to controls (saline and Cont-oligo). The production of IL-8 was inhibited by simultaneous treatment with SB203580. (*ANOVA, p
    Figure Legend Snippet: Senescence and inflammation induced by T-oligos in CD-1 pregnant mice. (A-D) Senescence associated β-galactosidase (SA-β-gal) staining of murine amniotic sac. Single blue stained cells indicate β-gal activity. A. saline, B. Cont-oligo, C. T-oligo and D. T-oligo+SB203580 (p38MAPK inhibitor) treated mice. SA-β-gal staining is pronounced in T-oligo treated mice. (E) Concentration of interleukin (IL)-8 protein in murine amniotic fluid. Higher levels of IL-8 were found in T-oligo treated animals compared to controls (saline and Cont-oligo). The production of IL-8 was inhibited by simultaneous treatment with SB203580. (*ANOVA, p

    Techniques Used: Mouse Assay, Staining, Activity Assay, Concentration Assay

    4) Product Images from "Tumor Necrosis Factor-alpha (TNF-α) Enhances miR-155-Mediated Endothelial Senescence by Targeting Sirtuin1 (SIRT1)"

    Article Title: Tumor Necrosis Factor-alpha (TNF-α) Enhances miR-155-Mediated Endothelial Senescence by Targeting Sirtuin1 (SIRT1)

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    doi: 10.12659/MSM.919721

    Effects of TNF-α on HUVECs senescence. ( A ) Impact of miR-155 on HUVECs senescence. Cells were measured by SA β-Gal activity and SMP-30. (n=3, * p
    Figure Legend Snippet: Effects of TNF-α on HUVECs senescence. ( A ) Impact of miR-155 on HUVECs senescence. Cells were measured by SA β-Gal activity and SMP-30. (n=3, * p

    Techniques Used: Activity Assay

    Valsartan and simvastatin ameliorated TNF-α-induced HUVECs senescence through inhibition of miR-155 expression. HUVECs were transfected using miR-155 inhibitor for 36 h followed by valsartan (40 umol/L) or simvastatin (1 μmol/L) stimulation for 3 h, and subsequently stimulated with TNF-α for 8 h, then changes in the expression of SIRT1 were assessed by WB ( A ). Cells senescence was measured by SA β-Gal activity and SMP-30 ( B ) and ( C ). (n=3, ** P
    Figure Legend Snippet: Valsartan and simvastatin ameliorated TNF-α-induced HUVECs senescence through inhibition of miR-155 expression. HUVECs were transfected using miR-155 inhibitor for 36 h followed by valsartan (40 umol/L) or simvastatin (1 μmol/L) stimulation for 3 h, and subsequently stimulated with TNF-α for 8 h, then changes in the expression of SIRT1 were assessed by WB ( A ). Cells senescence was measured by SA β-Gal activity and SMP-30 ( B ) and ( C ). (n=3, ** P

    Techniques Used: Inhibition, Expressing, Transfection, Western Blot, Activity Assay

    miR-155 regulated proliferation and senescence through the SIRT1/FoxO-1/P53/P21-dependent pathway. ( A ) The proliferation effects of miR-155 downregulation were mostly abolished after SIRT1 knockdown. ( B ) SA β-Gal activity and Western blot analysis of SMP-30 protein levels in HUVECs after miR-155 knockdown. (n=3, * p
    Figure Legend Snippet: miR-155 regulated proliferation and senescence through the SIRT1/FoxO-1/P53/P21-dependent pathway. ( A ) The proliferation effects of miR-155 downregulation were mostly abolished after SIRT1 knockdown. ( B ) SA β-Gal activity and Western blot analysis of SMP-30 protein levels in HUVECs after miR-155 knockdown. (n=3, * p

    Techniques Used: Activity Assay, Western Blot

    Effect of valsartan and simvastatin on senescence and miR-155 and SIRT1 expression in HUVECs treated with TNF-α. ( A–C ) HUVECs were pretreated with or without valsartan (40 umol/L) or simvastatin (1μmol/L) for 3 h and subsequently stimulated with TNF-α for 8 h, and then changes in the expression of miR-155 ( A ) and SIRT1 ( B ) were assessed by WB and qPCR. Cells senescence was measured by SA β-Gal activity and SMP-30 ( C ). (n=3, ** p
    Figure Legend Snippet: Effect of valsartan and simvastatin on senescence and miR-155 and SIRT1 expression in HUVECs treated with TNF-α. ( A–C ) HUVECs were pretreated with or without valsartan (40 umol/L) or simvastatin (1μmol/L) for 3 h and subsequently stimulated with TNF-α for 8 h, and then changes in the expression of miR-155 ( A ) and SIRT1 ( B ) were assessed by WB and qPCR. Cells senescence was measured by SA β-Gal activity and SMP-30 ( C ). (n=3, ** p

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

    5) Product Images from "Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation"

    Article Title: Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0030-4

    Senescence-associated β-galactosidase expression of human adipose tissue-derived stem cells. (A-B) Single cell senescence-associated β-galactosidase (SA-β-gal) staining showing the formation of a blue precipitate in senescent cells (scale bar 50 μm). Representative images of human adipose tissue-derived stem cells ( hASCs) cultivated in (C) allogeneic human serum (aHS) and (D) fetal bovine serum (FBS) at passage 10 showing earlier β-gal expression by hASCs cultivated in medium with FBS (scale bar 100 μm). (E) Percentage of β-gal-positive cells/total cells at each passage evaluated, expressed as the mean ± SEM. Analysis of variance with Bonferroni’s post-test was performed. * P
    Figure Legend Snippet: Senescence-associated β-galactosidase expression of human adipose tissue-derived stem cells. (A-B) Single cell senescence-associated β-galactosidase (SA-β-gal) staining showing the formation of a blue precipitate in senescent cells (scale bar 50 μm). Representative images of human adipose tissue-derived stem cells ( hASCs) cultivated in (C) allogeneic human serum (aHS) and (D) fetal bovine serum (FBS) at passage 10 showing earlier β-gal expression by hASCs cultivated in medium with FBS (scale bar 100 μm). (E) Percentage of β-gal-positive cells/total cells at each passage evaluated, expressed as the mean ± SEM. Analysis of variance with Bonferroni’s post-test was performed. * P

    Techniques Used: Expressing, Derivative Assay, Staining

    6) Product Images from "3?-Deoxy-3?-[18F]Fluorothymidine Positron Emission Tomography Is a Sensitive Method for Imaging the Response of BRAF-Dependent Tumors to MEK Inhibition"

    Article Title: 3?-Deoxy-3?-[18F]Fluorothymidine Positron Emission Tomography Is a Sensitive Method for Imaging the Response of BRAF-Dependent Tumors to MEK Inhibition

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-07-2976

    MEK inhibition causes a decrease in thymidine uptake in SKMEL-28 (V600E BRAF) cells. A, SKMEL-28 and BT-474 cells were treated with PD0325901 for 48 h and thymidine uptake was measured. Thymidine uptake was inhibited by > 90% in SKMEL-28 (V600E BRAF) cells but minimally affected by PD0325901 treatment in BT-474 cells. B, on washout of drug, thymidine uptake was restored to pretreatment levels in SKMEL-28 cells. C and D, treatment of SKMEL-28 cells with PD0325901 (25 nmol/L) resulted in an increase in the fraction of cells staining positive for SA-β-Gal. The photomicrograph in D shows representative fields from control and PD0325901-treated cells at the 48-h time point.
    Figure Legend Snippet: MEK inhibition causes a decrease in thymidine uptake in SKMEL-28 (V600E BRAF) cells. A, SKMEL-28 and BT-474 cells were treated with PD0325901 for 48 h and thymidine uptake was measured. Thymidine uptake was inhibited by > 90% in SKMEL-28 (V600E BRAF) cells but minimally affected by PD0325901 treatment in BT-474 cells. B, on washout of drug, thymidine uptake was restored to pretreatment levels in SKMEL-28 cells. C and D, treatment of SKMEL-28 cells with PD0325901 (25 nmol/L) resulted in an increase in the fraction of cells staining positive for SA-β-Gal. The photomicrograph in D shows representative fields from control and PD0325901-treated cells at the 48-h time point.

    Techniques Used: Inhibition, Staining

    7) Product Images from "Inhibition of chronic prostate inflammation by hyaluronic acid through an immortalized human prostate stromal cell line model"

    Article Title: Inhibition of chronic prostate inflammation by hyaluronic acid through an immortalized human prostate stromal cell line model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0178152

    Cell growth and senescence of ihPSC. (A) A representative growth curve, (B) population doubling time (hrs), and (C) Senescence associated β-galactosidase (SA-β-gal) activity in ihPSC were compared to hPSC. Results are shown as the mean ±SD for three independent experimental cultures. * indicates a significant difference with P
    Figure Legend Snippet: Cell growth and senescence of ihPSC. (A) A representative growth curve, (B) population doubling time (hrs), and (C) Senescence associated β-galactosidase (SA-β-gal) activity in ihPSC were compared to hPSC. Results are shown as the mean ±SD for three independent experimental cultures. * indicates a significant difference with P

    Techniques Used: Activity Assay

    8) Product Images from "Identification of Salvia haenkei as gerosuppressant agent by using an integrated senescence-screening assay"

    Article Title: Identification of Salvia haenkei as gerosuppressant agent by using an integrated senescence-screening assay

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101076

    Effect of S. haenkei treatment on growth arrest and senescence in Pten−/− MEFs ( a ) Proliferation of Pten−/− MEFs in culture after 5 days of treatment with S. haenkei extract. Pten−/− MEFs were plated in concentration of 2×10 4 cells/ml and treated for 5 days with 10μM MDM2i (Nutlin-3) or 10μg/ml SH extract. After this period, the proliferation was determined using Crystal violet staining. ( c ) Results are expressed as mean values (+SEM) of absorbance at 590nm for duplicates treated with SH and triplicate for control and Nutlin-3 treated groups, from one representative experiment out of 3 independent experiments. ( b-d ) Senescence of Pten−/− MEFs in culture after 5 days of treatment with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture upon 5 day treatment with 10μM MDM2i (Nutlin-3) or 10μg/ml S. haenkei extract. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in three independent experiments.
    Figure Legend Snippet: Effect of S. haenkei treatment on growth arrest and senescence in Pten−/− MEFs ( a ) Proliferation of Pten−/− MEFs in culture after 5 days of treatment with S. haenkei extract. Pten−/− MEFs were plated in concentration of 2×10 4 cells/ml and treated for 5 days with 10μM MDM2i (Nutlin-3) or 10μg/ml SH extract. After this period, the proliferation was determined using Crystal violet staining. ( c ) Results are expressed as mean values (+SEM) of absorbance at 590nm for duplicates treated with SH and triplicate for control and Nutlin-3 treated groups, from one representative experiment out of 3 independent experiments. ( b-d ) Senescence of Pten−/− MEFs in culture after 5 days of treatment with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture upon 5 day treatment with 10μM MDM2i (Nutlin-3) or 10μg/ml S. haenkei extract. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in three independent experiments.

    Techniques Used: Concentration Assay, Staining, Cell Counting

    Effect of S. haenkei treatment on replicative senescence in human fibroblasts ( a ) Growth curve of human WI38 fibroblasts treated with S . haenkei extract. WI-CCL75 human fibroblasts were plated 300.000 cells per 10cm dish, and subsequently passed and replated in the same number every 3 days for total of 24 passages up to the point when treatment with S. haenkei was initiated. At passage 25, cells were plated at the same number 300.000 cells per plate, and treated with 10μg/ml SH extract. Every 3 days cell number was determined by Trypan blue staining and cells replated 300.000 per plate and re-treated. Results are expressed as fold change in cell number from one representative experiment out of 4 independent experiments. ( b ) Senescence of human WI38 fibroblasts treated with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture at each passage. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in four independent experiments. ( c ) Cell death in culture of human WI38 fibroblasts upon treatment with S. haenkei extract. The graph represents percentage of Trypan blue positive (dead) cells revealed in culture at each passage. Quantifications were done on one experimental image (roughly 100 cells) in one representative experiment.
    Figure Legend Snippet: Effect of S. haenkei treatment on replicative senescence in human fibroblasts ( a ) Growth curve of human WI38 fibroblasts treated with S . haenkei extract. WI-CCL75 human fibroblasts were plated 300.000 cells per 10cm dish, and subsequently passed and replated in the same number every 3 days for total of 24 passages up to the point when treatment with S. haenkei was initiated. At passage 25, cells were plated at the same number 300.000 cells per plate, and treated with 10μg/ml SH extract. Every 3 days cell number was determined by Trypan blue staining and cells replated 300.000 per plate and re-treated. Results are expressed as fold change in cell number from one representative experiment out of 4 independent experiments. ( b ) Senescence of human WI38 fibroblasts treated with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture at each passage. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in four independent experiments. ( c ) Cell death in culture of human WI38 fibroblasts upon treatment with S. haenkei extract. The graph represents percentage of Trypan blue positive (dead) cells revealed in culture at each passage. Quantifications were done on one experimental image (roughly 100 cells) in one representative experiment.

    Techniques Used: Staining, Cell Counting

    Effect of S. haenkei treatment on photo ageing of human fibroblasts WICCL75 human fibroblasts were irradiated with 30J/m 2 UVB and 3h later treated with 10μg/ml S. haenkei extract. ( a ) Proliferation of irradiated human WI38 fibroblasts treated with S. haenkei extract. Cell proliferation was measured by Crystal violet staining at time points treatment (10μg/ml) 24h and 48h and represented as fold change in growth (compared to untreated control). Results are expressed as mean values (+SEM) for duplicate in each group in one representative experiment out of three independent experiments. ( b ) Senescence of irradiated human WI38 fibroblasts treated with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture at time points 24h and 48h. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in three independent experiments.
    Figure Legend Snippet: Effect of S. haenkei treatment on photo ageing of human fibroblasts WICCL75 human fibroblasts were irradiated with 30J/m 2 UVB and 3h later treated with 10μg/ml S. haenkei extract. ( a ) Proliferation of irradiated human WI38 fibroblasts treated with S. haenkei extract. Cell proliferation was measured by Crystal violet staining at time points treatment (10μg/ml) 24h and 48h and represented as fold change in growth (compared to untreated control). Results are expressed as mean values (+SEM) for duplicate in each group in one representative experiment out of three independent experiments. ( b ) Senescence of irradiated human WI38 fibroblasts treated with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture at time points 24h and 48h. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in three independent experiments.

    Techniques Used: Irradiation, Staining, Cell Counting

    Toxicity and irritability evaluation of S. haenkei extract in reconstituted human epidermis Skin issues were cultured in 12 well plates containing 37°C pre-warmed maintenance media (2 ml/well) and incubated overnight at 37°C, 5% CO 2 and 95% humidity, prior to the experiment. EpiSkin tissues were irradiated with UVB (30J/m 2 ) and 3 hours later treated by topical application with 10μg/ml Salvia haenkei extract and 5% SDS for positive control. 4h later, the epidermis was washed with PBS and left for incubation at 37°C, 5% CO 2 . ( a ) 22h after topical application of 10μg/ml of SH on EpiSkin tissues, senescence (bars) was calculated as a percentage of the control for β-galactosidase positive cells. Here, the treatment with UV was used as positive control. Results are expressed as the mean (+SEM) of triplicates in one representative experiment. ( b ) IL1ɑ production by EpiSkin tissue in response to S. haenkei treatment in the presence or absence of UV irradiation. Treatment with SDS was used as positive control. 22h after topical application of 10μg/ml of S . haenkei extract on EpiSkin tissues, supernatants were collected and samples stored at −80°C. The levels of IL1α were tested by ELISA. Results are represented in logarithmic values (pg/ml) and expressed as mean value±SEM, from triplicates in one experiment.
    Figure Legend Snippet: Toxicity and irritability evaluation of S. haenkei extract in reconstituted human epidermis Skin issues were cultured in 12 well plates containing 37°C pre-warmed maintenance media (2 ml/well) and incubated overnight at 37°C, 5% CO 2 and 95% humidity, prior to the experiment. EpiSkin tissues were irradiated with UVB (30J/m 2 ) and 3 hours later treated by topical application with 10μg/ml Salvia haenkei extract and 5% SDS for positive control. 4h later, the epidermis was washed with PBS and left for incubation at 37°C, 5% CO 2 . ( a ) 22h after topical application of 10μg/ml of SH on EpiSkin tissues, senescence (bars) was calculated as a percentage of the control for β-galactosidase positive cells. Here, the treatment with UV was used as positive control. Results are expressed as the mean (+SEM) of triplicates in one representative experiment. ( b ) IL1ɑ production by EpiSkin tissue in response to S. haenkei treatment in the presence or absence of UV irradiation. Treatment with SDS was used as positive control. 22h after topical application of 10μg/ml of S . haenkei extract on EpiSkin tissues, supernatants were collected and samples stored at −80°C. The levels of IL1α were tested by ELISA. Results are represented in logarithmic values (pg/ml) and expressed as mean value±SEM, from triplicates in one experiment.

    Techniques Used: Cell Culture, Incubation, Irradiation, Positive Control, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen"

    Article Title: Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen

    Journal: Journal of Assisted Reproduction and Genetics

    doi: 10.1007/s10815-014-0299-8

    Percentage of blastocysts positive for senescence-associated (SA-) β-galactosidase activity, stratified by oxygen concentration and protein supplementation. Data shown are mean ± standard error. All culture conditions were
    Figure Legend Snippet: Percentage of blastocysts positive for senescence-associated (SA-) β-galactosidase activity, stratified by oxygen concentration and protein supplementation. Data shown are mean ± standard error. All culture conditions were

    Techniques Used: Activity Assay, Concentration Assay

    A, Representative images of blastocysts (left: in vivo; right: in vitro) after staining for senescence-associated (SA-) β-galactosidase (blue color; original magnification, ×400). B, Percentage of blastocysts positive for SA-β-galactosidase
    Figure Legend Snippet: A, Representative images of blastocysts (left: in vivo; right: in vitro) after staining for senescence-associated (SA-) β-galactosidase (blue color; original magnification, ×400). B, Percentage of blastocysts positive for SA-β-galactosidase

    Techniques Used: In Vivo, In Vitro, Staining

    10) Product Images from "Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis"

    Article Title: Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2016.03.004

    Depletion of HAS2 expression induces fibroblast senescence. Fibroblasts transfected with HAS2 siRNA and control siRNA. (A). Cell numbers were counted at indicated time after transfection. Data shown is the mean of triplicates ± s. d. of one representative
    Figure Legend Snippet: Depletion of HAS2 expression induces fibroblast senescence. Fibroblasts transfected with HAS2 siRNA and control siRNA. (A). Cell numbers were counted at indicated time after transfection. Data shown is the mean of triplicates ± s. d. of one representative

    Techniques Used: Expressing, Transfection

    HAS2 depletion induced fibroblast senescence is associated with increased p27-CDK2-SKP2 expression. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western
    Figure Legend Snippet: HAS2 depletion induced fibroblast senescence is associated with increased p27-CDK2-SKP2 expression. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western

    Techniques Used: Expressing, Transfection, Western Blot

    Stress responses mediate HAS2 deficiency-induced senescence. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western blots. The expression of proteins
    Figure Legend Snippet: Stress responses mediate HAS2 deficiency-induced senescence. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western blots. The expression of proteins

    Techniques Used: Transfection, Western Blot, Expressing

    11) Product Images from "Inflammation-induced Gro1 triggers senescence in neuronal progenitors: effects of estradiol"

    Article Title: Inflammation-induced Gro1 triggers senescence in neuronal progenitors: effects of estradiol

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1298-y

    Gro1 induces senescence and arrests proliferation of murine hippocampal neurons. a Western blot analysis of markers of neurogenesis, senescence, apoptosis, and proliferation in HT-22 cells treated with mGro1. Three independent experiments were performed, and representative blots are shown. b Intensities of protein bands were quantified from three individual experiments, normalized to GAPDH, and presented as percent of control (untreated cells). c SA-β-gal enzymatic activity (blue) in HT-22 cells treated with 80 ng/mL Gro1. d Percent SA-β-gal positivity in HT-22 cells treated with Gro1 assessed in 6-well plates in triplicate, with 1000 cells/field counted in three fields/well. Control cells were untreated. * p
    Figure Legend Snippet: Gro1 induces senescence and arrests proliferation of murine hippocampal neurons. a Western blot analysis of markers of neurogenesis, senescence, apoptosis, and proliferation in HT-22 cells treated with mGro1. Three independent experiments were performed, and representative blots are shown. b Intensities of protein bands were quantified from three individual experiments, normalized to GAPDH, and presented as percent of control (untreated cells). c SA-β-gal enzymatic activity (blue) in HT-22 cells treated with 80 ng/mL Gro1. d Percent SA-β-gal positivity in HT-22 cells treated with Gro1 assessed in 6-well plates in triplicate, with 1000 cells/field counted in three fields/well. Control cells were untreated. * p

    Techniques Used: Western Blot, Activity Assay

    12) Product Images from "Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence"

    Article Title: Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-65732-4

    Continuous exposure to 1.7 GHz LTE RF-EMF induced cell cycle delay at G1 phase in ASCs and Huh7 cells. ( A–B ) ASC and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The DNA content of the 1.7 GHz RF-EMF-exposed and the unexposed sham control cells was analyzed by flow cytometry (BD Bioscience) after PI staining. 100,000 cells were counted for each experiment. The FACS results were analyzed using Flowing software 2 and the distribution of cells in each stage of the cell cycle was calculated from the FACS results shown in the left panel using Flowing software 2. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF induced cell cycle delay at G1 phase in ASCs and Huh7 cells. ( A–B ) ASC and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The DNA content of the 1.7 GHz RF-EMF-exposed and the unexposed sham control cells was analyzed by flow cytometry (BD Bioscience) after PI staining. 100,000 cells were counted for each experiment. The FACS results were analyzed using Flowing software 2 and the distribution of cells in each stage of the cell cycle was calculated from the FACS results shown in the left panel using Flowing software 2. P

    Techniques Used: Flow Cytometry, Staining, FACS, Software

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.

    Techniques Used: Incubation, Staining, Positive Control, Fluorescence, Microscopy

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased ASC and Huh7 cell proliferation. ( A,B ) ASCs and Huh7 cells prepared as described in Materials and Method were exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, cells were collected, and counted with a cell counter (Nexcelom Bioscience). Three independent experiments were performed and the cell number was plotted as mean ± S.D. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased ASC and Huh7 cell proliferation. ( A,B ) ASCs and Huh7 cells prepared as described in Materials and Method were exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, cells were collected, and counted with a cell counter (Nexcelom Bioscience). Three independent experiments were performed and the cell number was plotted as mean ± S.D. P

    Techniques Used: Incubation

    Continuous 1.7 GHz LTE RF-EMF exposure did not induce DNA damage and cellular apoptosis in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells were continuously exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A,B ) or 2 SAR ( C,D ) and collected. Cells were lysed with lysis buffer and histones were extracted, which were separated by 8–15% SDS-polyacrylamide gel electrophoresis (PAGE) for western analyses. Western blots were performed with ( A,C ) anti γ-H2AX and anti-histone H3 antibodies, ( B,D ) anti-PARP and actin antibodies. Cells exposed to UV or treated with 3 μM doxorubicin (DOX) were used as positive controls for DNA damage and apoptosis. ( A,C ) Histone H3 and ( B,D ) β-actin from the same blot were used as a loading control. More than three independent replicates of all experiments were performed. The original full-length blots/gels were presented in the Supplementary Information.
    Figure Legend Snippet: Continuous 1.7 GHz LTE RF-EMF exposure did not induce DNA damage and cellular apoptosis in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells were continuously exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A,B ) or 2 SAR ( C,D ) and collected. Cells were lysed with lysis buffer and histones were extracted, which were separated by 8–15% SDS-polyacrylamide gel electrophoresis (PAGE) for western analyses. Western blots were performed with ( A,C ) anti γ-H2AX and anti-histone H3 antibodies, ( B,D ) anti-PARP and actin antibodies. Cells exposed to UV or treated with 3 μM doxorubicin (DOX) were used as positive controls for DNA damage and apoptosis. ( A,C ) Histone H3 and ( B,D ) β-actin from the same blot were used as a loading control. More than three independent replicates of all experiments were performed. The original full-length blots/gels were presented in the Supplementary Information.

    Techniques Used: Lysis, Polyacrylamide Gel Electrophoresis, Western Blot

    Continuous exposure to 1.7 GHz LTE RF-EMF induced cellular senescence in ASCs and Huh7 cells. ( A,B ) ASCs and Huh7 cells exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR (A) or 2 SAR ( B ). Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde, and incubated with 0.1% X-gal for 30 h. ASCs treated with 200 μM H 2 O 2 for 1 h and Huh7 cells treated with 300 μM H 2 O 2 for 2 h were respectively used as a positive control. Images were taken with a Nikon microscope (ECLIPSE Ts2) under a 10× objective. Scale bar, 50 μm. A total 200 cells were counted for each experiment. The percentages are SA-β-gal positive cells over total cells counted. The experiment was performed in triplicate and the cell percentage was plotted as mean ± S.D. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF induced cellular senescence in ASCs and Huh7 cells. ( A,B ) ASCs and Huh7 cells exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR (A) or 2 SAR ( B ). Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde, and incubated with 0.1% X-gal for 30 h. ASCs treated with 200 μM H 2 O 2 for 1 h and Huh7 cells treated with 300 μM H 2 O 2 for 2 h were respectively used as a positive control. Images were taken with a Nikon microscope (ECLIPSE Ts2) under a 10× objective. Scale bar, 50 μm. A total 200 cells were counted for each experiment. The percentages are SA-β-gal positive cells over total cells counted. The experiment was performed in triplicate and the cell percentage was plotted as mean ± S.D. P

    Techniques Used: Incubation, Positive Control, Microscopy

    13) Product Images from "Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence"

    Article Title: Continuous Exposure to 1.7 GHz LTE Electromagnetic Fields Increases Intracellular Reactive Oxygen Species to Decrease Human Cell Proliferation and Induce Senescence

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-65732-4

    1.7 GHz LTE RF-EMF cell exposure device and its water cooling system. ( A ) The 1.7 GHz LTE RF-EMF cell exposure device used. ( B ) A water cooling system for the incubator to forcibly lower the heated water temperature by 1.7 GHz RF-EMF. ( C ) The chamber of the incubator with a 1.7 GHz RF-EMF LTE antenna. ( D ) A plate for cell culture dishes in ( C ) are located 13.6 cm from the conical antenna in the center of the exposure chamber. ( E ) A diagram of ( D ) designating the position of the cell dishes for accurate SAR exposure. ( F ) The SAR conversion table for this RF-EMF exposure device. SAR values for precise exposure conditions were obtained through engineering calculations. ( G ) The X-axis in the upper and lower graphs represents the real-time at which the RF-EMF is being exposed to cells. The Y-axis in the upper graph represents the SAR value (Watt) of RF-EMF during the exposure. The Y-axis in the bottom graph shows the temperature of the incubator (yellow line) and the temperature of the refrigerated water-cooling system (red line) of the RF-EMF exposure device during experiment.
    Figure Legend Snippet: 1.7 GHz LTE RF-EMF cell exposure device and its water cooling system. ( A ) The 1.7 GHz LTE RF-EMF cell exposure device used. ( B ) A water cooling system for the incubator to forcibly lower the heated water temperature by 1.7 GHz RF-EMF. ( C ) The chamber of the incubator with a 1.7 GHz RF-EMF LTE antenna. ( D ) A plate for cell culture dishes in ( C ) are located 13.6 cm from the conical antenna in the center of the exposure chamber. ( E ) A diagram of ( D ) designating the position of the cell dishes for accurate SAR exposure. ( F ) The SAR conversion table for this RF-EMF exposure device. SAR values for precise exposure conditions were obtained through engineering calculations. ( G ) The X-axis in the upper and lower graphs represents the real-time at which the RF-EMF is being exposed to cells. The Y-axis in the upper graph represents the SAR value (Watt) of RF-EMF during the exposure. The Y-axis in the bottom graph shows the temperature of the incubator (yellow line) and the temperature of the refrigerated water-cooling system (red line) of the RF-EMF exposure device during experiment.

    Techniques Used: Cell Culture

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased the proliferation of various human cells. ( A,B ) Hep3B, HeLa, SH-SY5Y, and IMR 90 cells were exposed to LTE 1.7 GHz RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, cells were collected and counted with a cell counter (Nexcelom Bioscience). At least three independent experiments were performed and the cell numbers were plotted as mean ± S.D. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased the proliferation of various human cells. ( A,B ) Hep3B, HeLa, SH-SY5Y, and IMR 90 cells were exposed to LTE 1.7 GHz RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, cells were collected and counted with a cell counter (Nexcelom Bioscience). At least three independent experiments were performed and the cell numbers were plotted as mean ± S.D. P

    Techniques Used: Incubation

    1.7 GHz LTE RF-EMF cell exposure device and its water cooling system. ( A ) The 1.7 GHz LTE RF-EMF cell exposure device used. ( B ) A water cooling system for the incubator to forcibly lower the heated water temperature by 1.7 GHz RF-EMF. ( C ) The chamber of the incubator with a 1.7 GHz RF-EMF LTE antenna. ( D ) A plate for cell culture dishes in ( C ) are located 13.6 cm from the conical antenna in the center of the exposure chamber. ( E ) A diagram of ( D ) designating the position of the cell dishes for accurate SAR exposure. ( F ) The SAR conversion table for this RF-EMF exposure device. SAR values for precise exposure conditions were obtained through engineering calculations. ( G ) The X-axis in the upper and lower graphs represents the real-time at which the RF-EMF is being exposed to cells. The Y-axis in the upper graph represents the SAR value (Watt) of RF-EMF during the exposure. The Y-axis in the bottom graph shows the temperature of the incubator (yellow line) and the temperature of the refrigerated water-cooling system (red line) of the RF-EMF exposure device during experiment.
    Figure Legend Snippet: 1.7 GHz LTE RF-EMF cell exposure device and its water cooling system. ( A ) The 1.7 GHz LTE RF-EMF cell exposure device used. ( B ) A water cooling system for the incubator to forcibly lower the heated water temperature by 1.7 GHz RF-EMF. ( C ) The chamber of the incubator with a 1.7 GHz RF-EMF LTE antenna. ( D ) A plate for cell culture dishes in ( C ) are located 13.6 cm from the conical antenna in the center of the exposure chamber. ( E ) A diagram of ( D ) designating the position of the cell dishes for accurate SAR exposure. ( F ) The SAR conversion table for this RF-EMF exposure device. SAR values for precise exposure conditions were obtained through engineering calculations. ( G ) The X-axis in the upper and lower graphs represents the real-time at which the RF-EMF is being exposed to cells. The Y-axis in the upper graph represents the SAR value (Watt) of RF-EMF during the exposure. The Y-axis in the bottom graph shows the temperature of the incubator (yellow line) and the temperature of the refrigerated water-cooling system (red line) of the RF-EMF exposure device during experiment.

    Techniques Used: Cell Culture

    Continuous exposure to 1.7 GHz LTE RF-EMF induced cell cycle delay at G1 phase in ASCs and Huh7 cells. ( A–B ) ASC and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The DNA content of the 1.7 GHz RF-EMF-exposed and the unexposed sham control cells was analyzed by flow cytometry (BD Bioscience) after PI staining. 100,000 cells were counted for each experiment. The FACS results were analyzed using Flowing software 2 and the distribution of cells in each stage of the cell cycle was calculated from the FACS results shown in the left panel using Flowing software 2. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF induced cell cycle delay at G1 phase in ASCs and Huh7 cells. ( A–B ) ASC and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The DNA content of the 1.7 GHz RF-EMF-exposed and the unexposed sham control cells was analyzed by flow cytometry (BD Bioscience) after PI staining. 100,000 cells were counted for each experiment. The FACS results were analyzed using Flowing software 2 and the distribution of cells in each stage of the cell cycle was calculated from the FACS results shown in the left panel using Flowing software 2. P

    Techniques Used: Flow Cytometry, Staining, FACS, Software

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased cell proliferation by inducing intracellular ROS in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells pre-treated or not with 100 μM NAC were exposed to 1.7 GHz RF-EMF for 72 h at 2 SAR, while the sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, ( A,C ) the cells were collected and counted with a cell counter (Nexcelom Bioscience). Huh7 cells ( B ) and ASCs ( D ) were stained with carboxy-H 2 DCFDA. Cells treat with TBHP were used as a positive control for intracellular ROS generation. ( E,F ) ASCs and Huh7 cells were exposed to 1.7 GHz RF-EMF for 72 h at 1 SAR or 2 SAR, and were stained with MitoSOX. ( B,D–F ) Nuclei were stained with Hoechst 33342. Images were taken with an Axioplan2 fluorescence microscope (Zeiss) under a 200× objective. Scale bar, 25 μm. All experiments consisted of three independent replicates.

    Techniques Used: Incubation, Staining, Positive Control, Fluorescence, Microscopy

    Continuous exposure to 1.7 GHz LTE RF-EMF decreased ASC and Huh7 cell proliferation. ( A,B ) ASCs and Huh7 cells prepared as described in Materials and Method were exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, cells were collected, and counted with a cell counter (Nexcelom Bioscience). Three independent experiments were performed and the cell number was plotted as mean ± S.D. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF decreased ASC and Huh7 cell proliferation. ( A,B ) ASCs and Huh7 cells prepared as described in Materials and Method were exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A ) or 2 SAR ( B ). The sham control cells were incubated for 72 h without RF-EMF exposure. After the exposure, cells were collected, and counted with a cell counter (Nexcelom Bioscience). Three independent experiments were performed and the cell number was plotted as mean ± S.D. P

    Techniques Used: Incubation

    Continuous 1.7 GHz LTE RF-EMF exposure did not induce DNA damage and cellular apoptosis in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells were continuously exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A,B ) or 2 SAR ( C,D ) and collected. Cells were lysed with lysis buffer and histones were extracted, which were separated by 8–15% SDS-polyacrylamide gel electrophoresis (PAGE) for western analyses. Western blots were performed with ( A,C ) anti γ-H2AX and anti-histone H3 antibodies, ( B,D ) anti-PARP and actin antibodies. Cells exposed to UV or treated with 3 μM doxorubicin (DOX) were used as positive controls for DNA damage and apoptosis. ( A,C ) Histone H3 and ( B,D ) β-actin from the same blot were used as a loading control. More than three independent replicates of all experiments were performed. The original full-length blots/gels were presented in the Supplementary Information.
    Figure Legend Snippet: Continuous 1.7 GHz LTE RF-EMF exposure did not induce DNA damage and cellular apoptosis in ASCs and Huh7 cells. ( A–D ) ASCs and Huh7 cells were continuously exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR ( A,B ) or 2 SAR ( C,D ) and collected. Cells were lysed with lysis buffer and histones were extracted, which were separated by 8–15% SDS-polyacrylamide gel electrophoresis (PAGE) for western analyses. Western blots were performed with ( A,C ) anti γ-H2AX and anti-histone H3 antibodies, ( B,D ) anti-PARP and actin antibodies. Cells exposed to UV or treated with 3 μM doxorubicin (DOX) were used as positive controls for DNA damage and apoptosis. ( A,C ) Histone H3 and ( B,D ) β-actin from the same blot were used as a loading control. More than three independent replicates of all experiments were performed. The original full-length blots/gels were presented in the Supplementary Information.

    Techniques Used: Lysis, Polyacrylamide Gel Electrophoresis, Western Blot

    Design of the 1.7 GHz LTE RF-EMF cell exposure system. ( A ) A schematic diagram of the radial transmission line (RTL) exposure system. ( B ) Cross-sectional view of the RTL exposure chamber. ( C ) Return loss characteristics of the RTL exposure chamber. ( D ) Antenna and the measurement points in each culture plate. ( E ) Temperature and linear fitting for the center point at the LTE 1.7 GHz frequency. Temperature was measured without circulating water during RF exposure.
    Figure Legend Snippet: Design of the 1.7 GHz LTE RF-EMF cell exposure system. ( A ) A schematic diagram of the radial transmission line (RTL) exposure system. ( B ) Cross-sectional view of the RTL exposure chamber. ( C ) Return loss characteristics of the RTL exposure chamber. ( D ) Antenna and the measurement points in each culture plate. ( E ) Temperature and linear fitting for the center point at the LTE 1.7 GHz frequency. Temperature was measured without circulating water during RF exposure.

    Techniques Used: Transmission Assay

    Continuous exposure to 1.7 GHz LTE RF-EMF induced cellular senescence in ASCs and Huh7 cells. ( A,B ) ASCs and Huh7 cells exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR (A) or 2 SAR ( B ). Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde, and incubated with 0.1% X-gal for 30 h. ASCs treated with 200 μM H 2 O 2 for 1 h and Huh7 cells treated with 300 μM H 2 O 2 for 2 h were respectively used as a positive control. Images were taken with a Nikon microscope (ECLIPSE Ts2) under a 10× objective. Scale bar, 50 μm. A total 200 cells were counted for each experiment. The percentages are SA-β-gal positive cells over total cells counted. The experiment was performed in triplicate and the cell percentage was plotted as mean ± S.D. P
    Figure Legend Snippet: Continuous exposure to 1.7 GHz LTE RF-EMF induced cellular senescence in ASCs and Huh7 cells. ( A,B ) ASCs and Huh7 cells exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 SAR (A) or 2 SAR ( B ). Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde, and incubated with 0.1% X-gal for 30 h. ASCs treated with 200 μM H 2 O 2 for 1 h and Huh7 cells treated with 300 μM H 2 O 2 for 2 h were respectively used as a positive control. Images were taken with a Nikon microscope (ECLIPSE Ts2) under a 10× objective. Scale bar, 50 μm. A total 200 cells were counted for each experiment. The percentages are SA-β-gal positive cells over total cells counted. The experiment was performed in triplicate and the cell percentage was plotted as mean ± S.D. P

    Techniques Used: Incubation, Positive Control, Microscopy

    14) Product Images from "FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence"

    Article Title: FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05258-6

    FoxM1 governs aneuploidization-driven cellular senescence in elderly cells. a – d FACS sorting of senescent cells from neonatal a , elderly b , FoxM1 siRNA-depleted neonatal c , and 84 y/87 y with FoxM1-dNdK d cell populations with high β-galactosidase activity. The gates were defined accordingly to the respective auto-fluorescent control. e Relative intensity levels of the fluorogenic substrate DDAOG in the sorted cell populations. f Aneuploidy index in FACS-sorted β-gal-positive fibroblast subpopulations (β-gal +) vs. unsorted populations (∅) as determined by FISH analysis for three chromosome pairs. g Experimental layout of live-cell/fixed-cell correlative microscopy analysis shown in h , i . Mitotic elderly fibroblasts expressing H2B–GFP and respective daughter cells were imaged for 72 h. SA-β-Gal assay and immunostaining for 53BP1/p21 were performed at the end of imaging. h Movie frames of representative phenotypes observed for elderly cells expressing H2B–GFP. Top panel, correct chromosome segregation, with cycling daughter cells (Supplementary Movie 9 ). Middle panel, correct chromosome segregation with non-cycling daughter cells staining negative for SA-β-Gal and 53BP1/p21 (Supplementary Movie 10 ). Bottom panel, chromosome mis-segregation leading to micronuclei formation (arrowheads), with non-cycling daughter cells staining positive for senescence markers (SA-β-Gal and 53BP1/p21) (Supplementary Movie 11 ). Dashed line indicates the tracked daughter cell. Time, hour:minute. Scale bar, 30 µm (movie frames) and 15 µm (immunostaining). i Single-cell analysis of daughter cell fate (cell death, cell cycle arrest, and cell senescence) from mitoses with apparent correct chromosome segregation or with mis-segregation (leading to micronuclei formation). Non-cycling daughter cells were stained for senescence markers (β-gal and 53BP1/p21). Values are mean ± SD of two independent experiments. Sample size ( n ) is indicated for f and i . NS, p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 by two-tailed χ 2 statistical tests
    Figure Legend Snippet: FoxM1 governs aneuploidization-driven cellular senescence in elderly cells. a – d FACS sorting of senescent cells from neonatal a , elderly b , FoxM1 siRNA-depleted neonatal c , and 84 y/87 y with FoxM1-dNdK d cell populations with high β-galactosidase activity. The gates were defined accordingly to the respective auto-fluorescent control. e Relative intensity levels of the fluorogenic substrate DDAOG in the sorted cell populations. f Aneuploidy index in FACS-sorted β-gal-positive fibroblast subpopulations (β-gal +) vs. unsorted populations (∅) as determined by FISH analysis for three chromosome pairs. g Experimental layout of live-cell/fixed-cell correlative microscopy analysis shown in h , i . Mitotic elderly fibroblasts expressing H2B–GFP and respective daughter cells were imaged for 72 h. SA-β-Gal assay and immunostaining for 53BP1/p21 were performed at the end of imaging. h Movie frames of representative phenotypes observed for elderly cells expressing H2B–GFP. Top panel, correct chromosome segregation, with cycling daughter cells (Supplementary Movie 9 ). Middle panel, correct chromosome segregation with non-cycling daughter cells staining negative for SA-β-Gal and 53BP1/p21 (Supplementary Movie 10 ). Bottom panel, chromosome mis-segregation leading to micronuclei formation (arrowheads), with non-cycling daughter cells staining positive for senescence markers (SA-β-Gal and 53BP1/p21) (Supplementary Movie 11 ). Dashed line indicates the tracked daughter cell. Time, hour:minute. Scale bar, 30 µm (movie frames) and 15 µm (immunostaining). i Single-cell analysis of daughter cell fate (cell death, cell cycle arrest, and cell senescence) from mitoses with apparent correct chromosome segregation or with mis-segregation (leading to micronuclei formation). Non-cycling daughter cells were stained for senescence markers (β-gal and 53BP1/p21). Values are mean ± SD of two independent experiments. Sample size ( n ) is indicated for f and i . NS, p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 by two-tailed χ 2 statistical tests

    Techniques Used: FACS, Activity Assay, Fluorescence In Situ Hybridization, Microscopy, Expressing, β-Gal Assay, Immunostaining, Imaging, Staining, Single-cell Analysis, Two Tailed Test

    15) Product Images from "Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast"

    Article Title: Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast

    Journal: Korean Journal for Food Science of Animal Resources

    doi: 10.5851/kosfa.2016.36.2.237

    The effect of the Hanwoo bone ( leg bone, foot and tail ) infusions on the senescence of skin fibroblast by senescent cells histochemical staining kit. (A) The senescent skin fibroblast weas observed under a fluorescent microscope after cells were stained with senescent cells histochemical staining kit (×200). (B) Aged cells were numbered.
    Figure Legend Snippet: The effect of the Hanwoo bone ( leg bone, foot and tail ) infusions on the senescence of skin fibroblast by senescent cells histochemical staining kit. (A) The senescent skin fibroblast weas observed under a fluorescent microscope after cells were stained with senescent cells histochemical staining kit (×200). (B) Aged cells were numbered.

    Techniques Used: Staining, Microscopy

    The effect of the bovine collagen on skin fibroblast metabolism. (A) Collagen synthesis. (B) The senescent skin fibroblast were observed under a fluorescent microscope after cells were stained with senescent cells histochemical staining kit (×200). (C) Aged cells were numbered.
    Figure Legend Snippet: The effect of the bovine collagen on skin fibroblast metabolism. (A) Collagen synthesis. (B) The senescent skin fibroblast were observed under a fluorescent microscope after cells were stained with senescent cells histochemical staining kit (×200). (C) Aged cells were numbered.

    Techniques Used: Microscopy, Staining

    16) Product Images from "A role for mitochondrial oxidants in stress-induced premature senescence of human vascular smooth muscle cells"

    Article Title: A role for mitochondrial oxidants in stress-induced premature senescence of human vascular smooth muscle cells

    Journal: Redox Biology

    doi: 10.1016/j.redox.2013.08.004

    Ang II induction of premature senescence in hVSMC is dependent upon cellular oxidants, mtETC activity and mitochondrial superoxide. Quiescent cells were pre-incubated with catalase (A), NAC (B), SOD (C) or inhibitors of the mtETC, rotenone (D) or TTFA (E) or the mitochondrial O 2 − scavenger mitoTEMPO (F) for 4 h prior to induction of senescence with 1×10 −8 mol/L Ang II. Following treatment, senescence was assessed by staining for SA-β-gal activity. Bars represent mean+SD; n =3–5. ⁎⁎ p
    Figure Legend Snippet: Ang II induction of premature senescence in hVSMC is dependent upon cellular oxidants, mtETC activity and mitochondrial superoxide. Quiescent cells were pre-incubated with catalase (A), NAC (B), SOD (C) or inhibitors of the mtETC, rotenone (D) or TTFA (E) or the mitochondrial O 2 − scavenger mitoTEMPO (F) for 4 h prior to induction of senescence with 1×10 −8 mol/L Ang II. Following treatment, senescence was assessed by staining for SA-β-gal activity. Bars represent mean+SD; n =3–5. ⁎⁎ p

    Techniques Used: Activity Assay, Incubation, Staining

    Tert-BHP and Ang II induce SIPS in hVSMC. A, Successive tert-BHP exposures induce SIPS in hVSMC. Sub-confluent cells were submitted to 3 stresses of 4×10 −5 mol/L tert-BHP for 2 h, over 3 days. Senescence was determined by SA-β-gal activity on the third day after the final recovery period. Bars represent mean+SD; n =6 ( *** p
    Figure Legend Snippet: Tert-BHP and Ang II induce SIPS in hVSMC. A, Successive tert-BHP exposures induce SIPS in hVSMC. Sub-confluent cells were submitted to 3 stresses of 4×10 −5 mol/L tert-BHP for 2 h, over 3 days. Senescence was determined by SA-β-gal activity on the third day after the final recovery period. Bars represent mean+SD; n =6 ( *** p

    Techniques Used: Activity Assay

    17) Product Images from "FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice"

    Article Title: FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice

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

    doi: 10.1073/pnas.1607079113

    Aging-associated features in donor fibroblasts. ( A ) Representative images indicating age-dependent decrease of EdU incorporation (EdU staining in red, Top ) and increase in senescence associated β-Gal activity (black staining, Bottom ) in fibroblasts from different age groups (MEFs, 4D, 3M, and 25M, Left to Right ) counterstained with DAPI (blue). ( B ) Average bar graphs indicate means ± SEM of percentages of EdU + ( i ), SA-β-Gal + ( ii ) fibroblasts and average relative mRNA levels for senescence markers p16 ( iii ) and p19 ( iv ) measured by qRT-PCR. Asterisks indicate significant difference ( n = 3 independent batches; * P
    Figure Legend Snippet: Aging-associated features in donor fibroblasts. ( A ) Representative images indicating age-dependent decrease of EdU incorporation (EdU staining in red, Top ) and increase in senescence associated β-Gal activity (black staining, Bottom ) in fibroblasts from different age groups (MEFs, 4D, 3M, and 25M, Left to Right ) counterstained with DAPI (blue). ( B ) Average bar graphs indicate means ± SEM of percentages of EdU + ( i ), SA-β-Gal + ( ii ) fibroblasts and average relative mRNA levels for senescence markers p16 ( iii ) and p19 ( iv ) measured by qRT-PCR. Asterisks indicate significant difference ( n = 3 independent batches; * P

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

    18) Product Images from "Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation"

    Article Title: Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0030-4

    Senescence-associated β-galactosidase expression of human adipose tissue-derived stem cells. (A-B) Single cell senescence-associated β-galactosidase (SA-β-gal) staining showing the formation of a blue precipitate in senescent cells (scale bar 50 μm). Representative images of human adipose tissue-derived stem cells ( hASCs) cultivated in (C) allogeneic human serum (aHS) and (D) fetal bovine serum (FBS) at passage 10 showing earlier β-gal expression by hASCs cultivated in medium with FBS (scale bar 100 μm). (E) Percentage of β-gal-positive cells/total cells at each passage evaluated, expressed as the mean ± SEM. Analysis of variance with Bonferroni’s post-test was performed. * P
    Figure Legend Snippet: Senescence-associated β-galactosidase expression of human adipose tissue-derived stem cells. (A-B) Single cell senescence-associated β-galactosidase (SA-β-gal) staining showing the formation of a blue precipitate in senescent cells (scale bar 50 μm). Representative images of human adipose tissue-derived stem cells ( hASCs) cultivated in (C) allogeneic human serum (aHS) and (D) fetal bovine serum (FBS) at passage 10 showing earlier β-gal expression by hASCs cultivated in medium with FBS (scale bar 100 μm). (E) Percentage of β-gal-positive cells/total cells at each passage evaluated, expressed as the mean ± SEM. Analysis of variance with Bonferroni’s post-test was performed. * P

    Techniques Used: Expressing, Derivative Assay, Staining

    19) Product Images from "The Natural Polyphenol Epigallocatechin Gallate Protects Intervertebral Disc Cells from Oxidative Stress"

    Article Title: The Natural Polyphenol Epigallocatechin Gallate Protects Intervertebral Disc Cells from Oxidative Stress

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2016/7031397

    As an antioxidant, EGCG inhibited senescence-associated β -galactosidase accumulation. (a) EGCG exhibited increasing 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity between 10 and 100 μ M, which confirmed its antioxidant properties. Ascorbic acid in the same concentration was used as positive control ( n = 3). ((b)–(d)) Oxidative stress was induced with 50 μ M H 2 O 2 for 2 hours and cellular senescence was measured during the following 10 days. 10 μ M EGCG inhibited SA β -gal accumulation when added to the oxidative stress phase, when its antioxidant activity was confirmed ( n = 5). (c) 10 μ M EGCG added to the recovery phase did not influence SA β -gal accumulation compared to the H 2 O 2 -only group ( n = 5). (d) EGCG combined in both phases (5 + 5 μ M) did not significantly inhibit SA β -gal accumulation, although a trend is visible ( n = 5). Asterisks indicate statistical significance at p
    Figure Legend Snippet: As an antioxidant, EGCG inhibited senescence-associated β -galactosidase accumulation. (a) EGCG exhibited increasing 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity between 10 and 100 μ M, which confirmed its antioxidant properties. Ascorbic acid in the same concentration was used as positive control ( n = 3). ((b)–(d)) Oxidative stress was induced with 50 μ M H 2 O 2 for 2 hours and cellular senescence was measured during the following 10 days. 10 μ M EGCG inhibited SA β -gal accumulation when added to the oxidative stress phase, when its antioxidant activity was confirmed ( n = 5). (c) 10 μ M EGCG added to the recovery phase did not influence SA β -gal accumulation compared to the H 2 O 2 -only group ( n = 5). (d) EGCG combined in both phases (5 + 5 μ M) did not significantly inhibit SA β -gal accumulation, although a trend is visible ( n = 5). Asterisks indicate statistical significance at p

    Techniques Used: Activity Assay, Concentration Assay, Positive Control, Antioxidant Activity Assay

    In vitro model system of stress-induced premature senescence. Sublethal oxidative stress (50 μ M H 2 O 2 ) with subsequent recovery period activated premature senescence of IVD cells in vitro . (a) Percentage of SA β -gal-positive cells in the H 2 O 2 treatment group gradually increased during 15 days after stress ( n = 5). (b) Upper part: representative images of SA β -gal staining of the untreated (ctrl) and the H 2 O 2 -treated cells on day 8 after stress, showing senescent (blue) cells. (b) Lower part: representative images of reseeded cells on day 9, confirming general cellular fitness. (c) Phosphorylation of p53 (Ser15) and expression of p21 in the H 2 O 2 treatment group on day 15 after stress indicated cellular senescence. ((d), (e)) Proliferative capacity, displayed as number of cells on days 8 and 15 after stress, was reduced in the H 2 O 2 groups. On day 15, the number of cells in the H 2 O 2 treatment group decreased below the seeding number (1 × 10 5 cells per well, depicted as red line), suggesting ongoing cell death. Asterisks indicate statistical significance at p
    Figure Legend Snippet: In vitro model system of stress-induced premature senescence. Sublethal oxidative stress (50 μ M H 2 O 2 ) with subsequent recovery period activated premature senescence of IVD cells in vitro . (a) Percentage of SA β -gal-positive cells in the H 2 O 2 treatment group gradually increased during 15 days after stress ( n = 5). (b) Upper part: representative images of SA β -gal staining of the untreated (ctrl) and the H 2 O 2 -treated cells on day 8 after stress, showing senescent (blue) cells. (b) Lower part: representative images of reseeded cells on day 9, confirming general cellular fitness. (c) Phosphorylation of p53 (Ser15) and expression of p21 in the H 2 O 2 treatment group on day 15 after stress indicated cellular senescence. ((d), (e)) Proliferative capacity, displayed as number of cells on days 8 and 15 after stress, was reduced in the H 2 O 2 groups. On day 15, the number of cells in the H 2 O 2 treatment group decreased below the seeding number (1 × 10 5 cells per well, depicted as red line), suggesting ongoing cell death. Asterisks indicate statistical significance at p

    Techniques Used: In Vitro, Staining, Expressing

    20) Product Images from "TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer’s disease mouse models"

    Article Title: TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer’s disease mouse models

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20142322

    TREM2 is expressed in plaque-associated myeloid cells. (a) In situ hybridization with TREM2 probes colocalized with Iba1 ( n = 2). (b) X-gal staining of brain tissue from 4-mo-old APPPS1; Trem2 LacZ/+ mice colocalized with fluorescent IHC for Iba1 and 6E10 ( n = 3). (c–f) Confocal microscopy was used to assess TREM2 colocalization with 6E10 + plaque-associated myeloid cells (c; Iba1), astrocytes (d; GFAP), neurons (e; MAP2), or oligodendrocytes (f; MBP; n = 8). At least two independent experiments were performed for all analyses. Bars: (a) 20 µm; (b–f) 50 µm.
    Figure Legend Snippet: TREM2 is expressed in plaque-associated myeloid cells. (a) In situ hybridization with TREM2 probes colocalized with Iba1 ( n = 2). (b) X-gal staining of brain tissue from 4-mo-old APPPS1; Trem2 LacZ/+ mice colocalized with fluorescent IHC for Iba1 and 6E10 ( n = 3). (c–f) Confocal microscopy was used to assess TREM2 colocalization with 6E10 + plaque-associated myeloid cells (c; Iba1), astrocytes (d; GFAP), neurons (e; MAP2), or oligodendrocytes (f; MBP; n = 8). At least two independent experiments were performed for all analyses. Bars: (a) 20 µm; (b–f) 50 µm.

    Techniques Used: In Situ Hybridization, Staining, Mouse Assay, Immunohistochemistry, Confocal Microscopy

    21) Product Images from "Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells"

    Article Title: Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells

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

    doi: 10.1073/pnas.0911446107

    A high concentration of FCS induces mOPC senescence. ( A ) Morphology of mOPCs cultured in low FCS, high FCS, and reversion medium. (Scale bar, 10 μm.) ( B – D ) Data from cells cultured as in A . ( B ) SA-β-gal staining of mOPCs. (Scale
    Figure Legend Snippet: A high concentration of FCS induces mOPC senescence. ( A ) Morphology of mOPCs cultured in low FCS, high FCS, and reversion medium. (Scale bar, 10 μm.) ( B – D ) Data from cells cultured as in A . ( B ) SA-β-gal staining of mOPCs. (Scale

    Techniques Used: Concentration Assay, Cell Culture, Staining

    22) Product Images from "Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity"

    Article Title: Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity

    Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

    doi: 10.1093/gerona/glt057

    ( A – E ) Representative micrographs showing that γ-irradiation, in a dose-dependent manner (0–8 Gy), increases the number of senescent cerebromicrovascular endothelial cells with positive senescent-associated β-galactosidase
    Figure Legend Snippet: ( A – E ) Representative micrographs showing that γ-irradiation, in a dose-dependent manner (0–8 Gy), increases the number of senescent cerebromicrovascular endothelial cells with positive senescent-associated β-galactosidase

    Techniques Used: Irradiation

    23) Product Images from "Lack of Fetuin-A (?2-HS-Glycoprotein) Reduces Mammary Tumor Incidence and Prolongs Tumor Latency via the Transforming Growth Factor-? Signaling Pathway in a Mouse Model of Breast Cancer"

    Article Title: Lack of Fetuin-A (?2-HS-Glycoprotein) Reduces Mammary Tumor Incidence and Prolongs Tumor Latency via the Transforming Growth Factor-? Signaling Pathway in a Mouse Model of Breast Cancer

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.100177

    Proliferation, apoptotic, and senescence markers in mammary tumors from PyMT/Fet −/− and PyMT/Fet +/+ animals. Mammary tumors (120 days old) from fetuin-A null and qjwild-type PyMT animals were sectioned and processed for immunohistochemical analysis as described in Materials and Methods . The slides were stained for PCNA (proliferation) ( A ) and cleaved caspase-3 (cas-3, apoptosis) ( B ). C: The slides were also stained for β-galactosidase activity that was quantified as number of cells positive for β-galactosidase (β-gal) activity per field (* P
    Figure Legend Snippet: Proliferation, apoptotic, and senescence markers in mammary tumors from PyMT/Fet −/− and PyMT/Fet +/+ animals. Mammary tumors (120 days old) from fetuin-A null and qjwild-type PyMT animals were sectioned and processed for immunohistochemical analysis as described in Materials and Methods . The slides were stained for PCNA (proliferation) ( A ) and cleaved caspase-3 (cas-3, apoptosis) ( B ). C: The slides were also stained for β-galactosidase activity that was quantified as number of cells positive for β-galactosidase (β-gal) activity per field (* P

    Techniques Used: Immunohistochemistry, Staining, Activity Assay

    24) Product Images from "FL118 Induces p53-Dependent Senescence in Colorectal Cancer Cells by Promoting Degradation of MdmX"

    Article Title: FL118 Induces p53-Dependent Senescence in Colorectal Cancer Cells by Promoting Degradation of MdmX

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-14-0683

    FL118 induces p53/p21-dependent senescence and p53/p21-independent apoptosis. A, FL118 effects on SA-β-gal positivity. HCT8 cells without treatment or treated with 10 nmol/L FL118 for 3 days followed by another 10-day culture in drug-free medium
    Figure Legend Snippet: FL118 induces p53/p21-dependent senescence and p53/p21-independent apoptosis. A, FL118 effects on SA-β-gal positivity. HCT8 cells without treatment or treated with 10 nmol/L FL118 for 3 days followed by another 10-day culture in drug-free medium

    Techniques Used:

    25) Product Images from "Roles of Progesterone Receptor Membrane Component 1 in Oxidative Stress–Induced Aging in Chorion Cells"

    Article Title: Roles of Progesterone Receptor Membrane Component 1 in Oxidative Stress–Induced Aging in Chorion Cells

    Journal: Reproductive Sciences

    doi: 10.1177/1933719118776790

    The increased cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and downregulated sirtuin 3 (SIRT3) expression induced by hydrogen peroxide (H 2 O2) were enhanced in PGRMC1 knockdown chorion cells. A, Senescence cell histochemical staining of chorion cells treated with 0 and 300 μM H 2 O2 for 24 hours in scrambled small-interfering RNA (siRNA)-transfected chorion cells (CsiRNA) and in PGRMC1 knockdown cells (PsiRNA). SA-β-Gal-positive chorion cells are blue in representative images. B, The proportion of SA-β-Gal-positive chorion cells is summarized in a bar graph. C, Representative Western blot image to demonstrate the p38 MAPK phosphorylation under 300 μM H 2 O 2 treatment at 30 minutes in CsiRNA and PsiRNA groups. Densitometry analysis was summarized in a bar graph (N = 8). D, Representative Western blot image to demonstrate SIRT3 protein expression in CsiRNA and PsiRNA groups and densitometry analysis was summarized in a bar graph (N = 10). MAPK indicates mitogen-activated protein kinases; H 2 O2, hydrogen peroxide; PGRMC1, progesterone receptor membrane component 1; siRNA, small-interfering RNA; SIRT3, sirtuin 3.
    Figure Legend Snippet: The increased cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and downregulated sirtuin 3 (SIRT3) expression induced by hydrogen peroxide (H 2 O2) were enhanced in PGRMC1 knockdown chorion cells. A, Senescence cell histochemical staining of chorion cells treated with 0 and 300 μM H 2 O2 for 24 hours in scrambled small-interfering RNA (siRNA)-transfected chorion cells (CsiRNA) and in PGRMC1 knockdown cells (PsiRNA). SA-β-Gal-positive chorion cells are blue in representative images. B, The proportion of SA-β-Gal-positive chorion cells is summarized in a bar graph. C, Representative Western blot image to demonstrate the p38 MAPK phosphorylation under 300 μM H 2 O 2 treatment at 30 minutes in CsiRNA and PsiRNA groups. Densitometry analysis was summarized in a bar graph (N = 8). D, Representative Western blot image to demonstrate SIRT3 protein expression in CsiRNA and PsiRNA groups and densitometry analysis was summarized in a bar graph (N = 10). MAPK indicates mitogen-activated protein kinases; H 2 O2, hydrogen peroxide; PGRMC1, progesterone receptor membrane component 1; siRNA, small-interfering RNA; SIRT3, sirtuin 3.

    Techniques Used: Expressing, Staining, Small Interfering RNA, Transfection, Western Blot

    Hydrogen peroxide (H 2 O2)-induced cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and downregulated sirtuin 3 (SIRT3) expression in chorion cells. A, Senescence cell histochemical staining of chorion cells treated with 0, 100, 300, and 500 μM H 2 O2 for 24 hours. SA-β-Gal-16 positive chorion cells are blue in representative images. B, The proportion of SA-β-Gal-positive chorion cells were summarized in a bar graph. C, Representative Western blot image to demonstrate the p38 MAPK phosphorylation under 300 μM H 2 O 2 treatment at 15, 30, and 60 minutes. Densitometry analysis was summarized in a bar graph (N = 8). D, SIRT3 mRNA expression level in fold change calculated using the ΔΔCt method after normalization (24-hour treatments, N = 9). E, Representative Western blot image to demonstrate SIRT3 protein expression and densitometry analysis was summarized in a bar graph (24-hour treatments, N = 9).
    Figure Legend Snippet: Hydrogen peroxide (H 2 O2)-induced cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and downregulated sirtuin 3 (SIRT3) expression in chorion cells. A, Senescence cell histochemical staining of chorion cells treated with 0, 100, 300, and 500 μM H 2 O2 for 24 hours. SA-β-Gal-16 positive chorion cells are blue in representative images. B, The proportion of SA-β-Gal-positive chorion cells were summarized in a bar graph. C, Representative Western blot image to demonstrate the p38 MAPK phosphorylation under 300 μM H 2 O 2 treatment at 15, 30, and 60 minutes. Densitometry analysis was summarized in a bar graph (N = 8). D, SIRT3 mRNA expression level in fold change calculated using the ΔΔCt method after normalization (24-hour treatments, N = 9). E, Representative Western blot image to demonstrate SIRT3 protein expression and densitometry analysis was summarized in a bar graph (24-hour treatments, N = 9).

    Techniques Used: Expressing, Staining, Western Blot

    Hydrogen peroxide (H 2 O2)-induced cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and downregulated sirtuin 3 (SIRT3) expression in full-thickness fetal membrane explants. A, Senescence cell histochemical staining of full-thickness fetal membrane explants treated with 0, 100, 300, and 500 μM H 2 O2 for 24 hours. SA-β-Gal-positive cells are blue in representative images. B, The proportion of SA-β-Gal-positive cells was summarized in a bar graph. C, Representative Western blot image to demonstrate the p38 MAPK phosphorylation under 500 μM H 2 O 2 treatment at 15, 30, and 60 minutes. Densitometry analysis was summarized in a bar graph (N = 6). D, SIRT3 mRNA expression level in fold change calculated using the ΔΔCt method after normalization (24-hour treatments, N = 6). E, Representative Western blot image to demonstrate SIRT3 protein expression and densitometry analysis was summarized in a bar graph (24-hour treatments, N = 6).
    Figure Legend Snippet: Hydrogen peroxide (H 2 O2)-induced cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and downregulated sirtuin 3 (SIRT3) expression in full-thickness fetal membrane explants. A, Senescence cell histochemical staining of full-thickness fetal membrane explants treated with 0, 100, 300, and 500 μM H 2 O2 for 24 hours. SA-β-Gal-positive cells are blue in representative images. B, The proportion of SA-β-Gal-positive cells was summarized in a bar graph. C, Representative Western blot image to demonstrate the p38 MAPK phosphorylation under 500 μM H 2 O 2 treatment at 15, 30, and 60 minutes. Densitometry analysis was summarized in a bar graph (N = 6). D, SIRT3 mRNA expression level in fold change calculated using the ΔΔCt method after normalization (24-hour treatments, N = 6). E, Representative Western blot image to demonstrate SIRT3 protein expression and densitometry analysis was summarized in a bar graph (24-hour treatments, N = 6).

    Techniques Used: Expressing, Staining, Western Blot

    26) Product Images from "Inducing and exploiting vulnerabilities for the treatment of liver cancer"

    Article Title: Inducing and exploiting vulnerabilities for the treatment of liver cancer

    Journal: Nature

    doi: 10.1038/s41586-019-1607-3

    Pharmacological or genetic inhibition of CDC7 induces a senescent phenotype in TP53 mutant liver cancer cells. a, b, TP53 mutant liver cancer cell lines (Hep3B, Huh7, SNU398, MHCC97H and HCCLM3; blue) and TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6; red) were seeded at low confluence and grown in the absence or presence of the CDC7 inhibitors LY3177833 or TAK-931 at the indicated concentrations, in long-term colony formation assays. Cells were fixed, stained, and photographed after 10-14 days of culture. c, d, Growth curves measured by Incucyte live cell analyses of TP53 mutant liver cancer cell lines (Hep3B and Huh7; blue) and TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6; red) exposed to LY3177833 or TAK-931 (Graphs represent mean ± s.d. from four technical replicates). e, f, Liver cancer cells were cultured in the presence of the CDC7 inhibitors LY3177833 or TAK-931 at the indicated concentration for 4 days. SA-β-Gal staining revealed that CDC7 inhibitors (LY3177833 or TAK-931) selectively induced senescence in TP53 mutant liver cancer cells (blue) and not in TP53 wild-type liver cancer cells (red). g, TP53 mutant liver cancer cell lines (Hep3B and Huh7) and TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6) were stably transduced with control pLKO vector or with two independent shRNAs targeting CDC7 (shCDC7#1, shCDC7#2) and the efficiency of CDC7 knockdown in liver cancer cell lines was evaluated by western blot. h, Colony formation assays of TP53 mutant (blue) and TP53 wild-type liver cancer cell lines (red) with and without CDC7 knockdown were performed. Cells were fixed, stained, and photographed after 10 days of culture. i, CDC7 knockdown induced senescence in TP53 mutant Hep3B and Huh7 cells, but not in TP53 . Data in a-i are representative of three independent biological experiments.
    Figure Legend Snippet: Pharmacological or genetic inhibition of CDC7 induces a senescent phenotype in TP53 mutant liver cancer cells. a, b, TP53 mutant liver cancer cell lines (Hep3B, Huh7, SNU398, MHCC97H and HCCLM3; blue) and TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6; red) were seeded at low confluence and grown in the absence or presence of the CDC7 inhibitors LY3177833 or TAK-931 at the indicated concentrations, in long-term colony formation assays. Cells were fixed, stained, and photographed after 10-14 days of culture. c, d, Growth curves measured by Incucyte live cell analyses of TP53 mutant liver cancer cell lines (Hep3B and Huh7; blue) and TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6; red) exposed to LY3177833 or TAK-931 (Graphs represent mean ± s.d. from four technical replicates). e, f, Liver cancer cells were cultured in the presence of the CDC7 inhibitors LY3177833 or TAK-931 at the indicated concentration for 4 days. SA-β-Gal staining revealed that CDC7 inhibitors (LY3177833 or TAK-931) selectively induced senescence in TP53 mutant liver cancer cells (blue) and not in TP53 wild-type liver cancer cells (red). g, TP53 mutant liver cancer cell lines (Hep3B and Huh7) and TP53 wild-type liver cancer cell lines (SK-Hep1 and Huh6) were stably transduced with control pLKO vector or with two independent shRNAs targeting CDC7 (shCDC7#1, shCDC7#2) and the efficiency of CDC7 knockdown in liver cancer cell lines was evaluated by western blot. h, Colony formation assays of TP53 mutant (blue) and TP53 wild-type liver cancer cell lines (red) with and without CDC7 knockdown were performed. Cells were fixed, stained, and photographed after 10 days of culture. i, CDC7 knockdown induced senescence in TP53 mutant Hep3B and Huh7 cells, but not in TP53 . Data in a-i are representative of three independent biological experiments.

    Techniques Used: Inhibition, Mutagenesis, Staining, Cell Culture, Concentration Assay, Stable Transfection, Transduction, Plasmid Preparation, Western Blot

    CDC7 inhibition induces senescence selectively in TP53 mutant liver cancer cells. a, TP53 mutant liver cancer cell lines were cultured in the presence of 10 μM XL413 for 4 days, which induces senescence as detected by SA-β-gal staining. b, Growth curves measured by Incucyte live cell analyses of TP53 mutant liver cancer cell lines either untreated, continuously treated with XL413, or treated with 10 μM of XL413 for 5 or 6 days prior to treatment withdrawal (Graphs represent mean ± s.d. from five technical replicates). c, Representative images of H3K9Me3 staining in TP53 mutant liver cancer cell lines exposed to 10 μM XL413 for 7 days. d, XL413 treatment induces a senescence-associated secretory phenotype (SASP) in Hep3B and Huh7 cells treated with 10 μM XL413 for 7 days. mRNA expression of SASP genes was determined by qRT-PCR analysis (Graphs represent mean ± s.d. from four technical replicates). e, Liver cancer cells were cultured in the presence of 10 μM XL413 for 4 days and apoptotic cells were visualized by caspase-3/7 apoptosis assay. Data in a are representative of three independent biological experiments. Data in b-e are representative of two independent biological experiments.
    Figure Legend Snippet: CDC7 inhibition induces senescence selectively in TP53 mutant liver cancer cells. a, TP53 mutant liver cancer cell lines were cultured in the presence of 10 μM XL413 for 4 days, which induces senescence as detected by SA-β-gal staining. b, Growth curves measured by Incucyte live cell analyses of TP53 mutant liver cancer cell lines either untreated, continuously treated with XL413, or treated with 10 μM of XL413 for 5 or 6 days prior to treatment withdrawal (Graphs represent mean ± s.d. from five technical replicates). c, Representative images of H3K9Me3 staining in TP53 mutant liver cancer cell lines exposed to 10 μM XL413 for 7 days. d, XL413 treatment induces a senescence-associated secretory phenotype (SASP) in Hep3B and Huh7 cells treated with 10 μM XL413 for 7 days. mRNA expression of SASP genes was determined by qRT-PCR analysis (Graphs represent mean ± s.d. from four technical replicates). e, Liver cancer cells were cultured in the presence of 10 μM XL413 for 4 days and apoptotic cells were visualized by caspase-3/7 apoptosis assay. Data in a are representative of three independent biological experiments. Data in b-e are representative of two independent biological experiments.

    Techniques Used: Inhibition, Mutagenesis, Cell Culture, Staining, Expressing, Quantitative RT-PCR, Apoptosis Assay

    Pro-senescence treatment combined with mTOR inhibitor suppresses tumour growth in liver cancer xenografts. a, Representative images of γH2AX and SA-β-Gal staining performed on formalin-fixed, paraffin-embedded or frozen sections from subcutaneous Huh7 xenografts treated with vehicle, XL413, AZD8055 or combination for 12 days. b, Representative images of SA-β-Gal staining performed on frozen sections from subcutaneous SK-Hep1 xenografts treated either with vehicle or XL413 for 21 days. c, Tumour volume measurements in vehicle, XL413, AZD8055 or combination-treated mice bearing Huh7 and MHCC97H xenografts at endpoint (12 days and 22 days, respectively, for sample size see ). One mouse in the vehicle group and one mouse in the XL413 group were excluded in the analysis, since the maximum permitted tumour volumes (2,000 mm 3 ) were reached prior to trial endpoint. Graph shows mean ± s.e.m., analysed with two-sided unpaired Student t -test. d, e, Longitudinal tumour volume progression in Huh7 and MHCC97H tumour-bearing mice treated with vehicle or sorafenib for 16 or 22 days, revealing that sorafenib therapy displays limited efficacy in these two different xenograft models. Graph shows mean ± s.e.m. f, g, Representative images of H E, PCNA, cleaved caspase-3 and p-4EBP1 staining performed on formalin-fixed, paraffin-embedded Huh7 and MHCC97H xenografts from mice sacrificed after the last dose of vehicle, XL413, AZD8055 or combination treatment. Data in a, b, f, g are representative of three independent biological experiments. Figure 4a
    Figure Legend Snippet: Pro-senescence treatment combined with mTOR inhibitor suppresses tumour growth in liver cancer xenografts. a, Representative images of γH2AX and SA-β-Gal staining performed on formalin-fixed, paraffin-embedded or frozen sections from subcutaneous Huh7 xenografts treated with vehicle, XL413, AZD8055 or combination for 12 days. b, Representative images of SA-β-Gal staining performed on frozen sections from subcutaneous SK-Hep1 xenografts treated either with vehicle or XL413 for 21 days. c, Tumour volume measurements in vehicle, XL413, AZD8055 or combination-treated mice bearing Huh7 and MHCC97H xenografts at endpoint (12 days and 22 days, respectively, for sample size see ). One mouse in the vehicle group and one mouse in the XL413 group were excluded in the analysis, since the maximum permitted tumour volumes (2,000 mm 3 ) were reached prior to trial endpoint. Graph shows mean ± s.e.m., analysed with two-sided unpaired Student t -test. d, e, Longitudinal tumour volume progression in Huh7 and MHCC97H tumour-bearing mice treated with vehicle or sorafenib for 16 or 22 days, revealing that sorafenib therapy displays limited efficacy in these two different xenograft models. Graph shows mean ± s.e.m. f, g, Representative images of H E, PCNA, cleaved caspase-3 and p-4EBP1 staining performed on formalin-fixed, paraffin-embedded Huh7 and MHCC97H xenografts from mice sacrificed after the last dose of vehicle, XL413, AZD8055 or combination treatment. Data in a, b, f, g are representative of three independent biological experiments. Figure 4a

    Techniques Used: Staining, Formalin-fixed Paraffin-Embedded, Mouse Assay

    CDC7 inhibition induces senescence selectively in TP53 mutant cancer cells. a, TP53 mutant lung cancer cell lines (blue) and TP53 wild-type lung cancer cell lines (red) were seeded at low confluence and grown in the absence or presence of XL413 at the indicated concentration for 10-14 days in colony formation assays. b, Lung cancer cells were exposed to 10 μM XL413 for 4 days, which induces senescence selectively in TP53 mutant cells as detected by SA-β-gal staining. c , p53 expression was assessed in isogenic TP53 -/- and TP53 +/+ HCT116 colon cancer cell lines by western blot. d, HCT116 TP53 +/+ and HCT116 TP53 -/- cells were seeded at low confluence and grown in the absence or presence of XL413 at the indicated concentration for 7 days in a colony formation assay to assess their proliferation capacity. e, HCT116 TP53 +/+ and HCT116 TP53 -/- cells were cultured in the presence of 10 μM XL413 for 4 days, and senescence was selectively induced in TP53 -/- . Data in a-e are representative of two independent biological experiments.
    Figure Legend Snippet: CDC7 inhibition induces senescence selectively in TP53 mutant cancer cells. a, TP53 mutant lung cancer cell lines (blue) and TP53 wild-type lung cancer cell lines (red) were seeded at low confluence and grown in the absence or presence of XL413 at the indicated concentration for 10-14 days in colony formation assays. b, Lung cancer cells were exposed to 10 μM XL413 for 4 days, which induces senescence selectively in TP53 mutant cells as detected by SA-β-gal staining. c , p53 expression was assessed in isogenic TP53 -/- and TP53 +/+ HCT116 colon cancer cell lines by western blot. d, HCT116 TP53 +/+ and HCT116 TP53 -/- cells were seeded at low confluence and grown in the absence or presence of XL413 at the indicated concentration for 7 days in a colony formation assay to assess their proliferation capacity. e, HCT116 TP53 +/+ and HCT116 TP53 -/- cells were cultured in the presence of 10 μM XL413 for 4 days, and senescence was selectively induced in TP53 -/- . Data in a-e are representative of two independent biological experiments.

    Techniques Used: Inhibition, Mutagenesis, Concentration Assay, Staining, Expressing, Western Blot, Colony Assay, Cell Culture

    CDC7 inhibition selectively induces senescence in TP53 mutant liver cancer cells. a, Long-term colony formation assay was performed over 10-14 days on TP53 mutant liver cancer cell lines (blue), TP53 wild-type liver cancer cell lines (red), and non-transformed cell lines (purple) cultured with the indicated concentrations of XL413. b, Liver cancer cells and non-transformed cell lines were cultured in the presence of 10 μM XL413 for 4 days and induction of senescence was assessed by staining for SA-β-Gal activity. c, for more detailed information). d, Proteins were extracted from TP53 mutant and wild-type liver cancer cell lines treated with XL413 (10 μM) for 7 days and analysed by western blotting. e, Neutral comet assays were performed on Hep3B, Huh7, SK-Hep1 and Huh6 cells cultured with 10 μM XL413 for 7 days. The value of tail moments in each treatment group were normalized based on the mean value of the control cells. (n=50 per cell line/condition, data in graphs are mean ± s.d. and analysed by unpaired two-sided t -test). f, GSEA in TP53 mutant (Hep3B and Huh7) and TP53 . Data in a, b are representative of three independent biological experiments. Data in d, e are representative of two independent biological experiments.
    Figure Legend Snippet: CDC7 inhibition selectively induces senescence in TP53 mutant liver cancer cells. a, Long-term colony formation assay was performed over 10-14 days on TP53 mutant liver cancer cell lines (blue), TP53 wild-type liver cancer cell lines (red), and non-transformed cell lines (purple) cultured with the indicated concentrations of XL413. b, Liver cancer cells and non-transformed cell lines were cultured in the presence of 10 μM XL413 for 4 days and induction of senescence was assessed by staining for SA-β-Gal activity. c, for more detailed information). d, Proteins were extracted from TP53 mutant and wild-type liver cancer cell lines treated with XL413 (10 μM) for 7 days and analysed by western blotting. e, Neutral comet assays were performed on Hep3B, Huh7, SK-Hep1 and Huh6 cells cultured with 10 μM XL413 for 7 days. The value of tail moments in each treatment group were normalized based on the mean value of the control cells. (n=50 per cell line/condition, data in graphs are mean ± s.d. and analysed by unpaired two-sided t -test). f, GSEA in TP53 mutant (Hep3B and Huh7) and TP53 . Data in a, b are representative of three independent biological experiments. Data in d, e are representative of two independent biological experiments.

    Techniques Used: Inhibition, Mutagenesis, Colony Assay, Transformation Assay, Cell Culture, Staining, Activity Assay, Western Blot

    AZD8055 does not induce apoptosis in Cisplatin or Alisertib-induced senescent cells a, BJ/ET/Ras V12 cells were treated with 100 nm 4-OHT for 21 days to induce senescence, as detected by SA-β-gal staining. b, Control or senescent BJ/ET/Ras V12 cells were treated with either vehicle or 400 nM AZD8055 for 96 hours and apoptotic cells were visualized by caspase-3/7 apoptosis assay. c, Hep3B cells were cultured in the presence of Cisplatin or Alisertib (Aurora-A kinase inhibitor) for 4 days at the indicated concentrations and senescence induction was detected by SA-β-gal staining. d, Hep3B cells were treated with Cisplatin (1 μg/ml) or Alisertib (250 nM) for 4 days and subsequently exposed to vehicle or 400 nM AZD8055 for 96 hours. Apoptotic cells were visualized by caspase-3/7 apoptosis assay. e, . Data in a-e are representative of two independent biological experiments.
    Figure Legend Snippet: AZD8055 does not induce apoptosis in Cisplatin or Alisertib-induced senescent cells a, BJ/ET/Ras V12 cells were treated with 100 nm 4-OHT for 21 days to induce senescence, as detected by SA-β-gal staining. b, Control or senescent BJ/ET/Ras V12 cells were treated with either vehicle or 400 nM AZD8055 for 96 hours and apoptotic cells were visualized by caspase-3/7 apoptosis assay. c, Hep3B cells were cultured in the presence of Cisplatin or Alisertib (Aurora-A kinase inhibitor) for 4 days at the indicated concentrations and senescence induction was detected by SA-β-gal staining. d, Hep3B cells were treated with Cisplatin (1 μg/ml) or Alisertib (250 nM) for 4 days and subsequently exposed to vehicle or 400 nM AZD8055 for 96 hours. Apoptotic cells were visualized by caspase-3/7 apoptosis assay. e, . Data in a-e are representative of two independent biological experiments.

    Techniques Used: Staining, Apoptosis Assay, Cell Culture

    CDC7 inhibition induces senescence in vivo and suppresses tumour growth when combined with mTOR inhibition in multiple liver cancer models. a, Huh7 and MHCC97H cells were grown as tumour xenografts in BALB/c nude mice. Longitudinal tumour volume progression in Huh7 and MHCC97H tumour-bearing mice treated with vehicle, XL413, AZD8055 or combined therapies for 12 or 22 days, respectively. Graph shows mean ± s.e.m. b-g, Analyses of combination therapy response in the Myc OE ; Trp53 KO HCC somatic murine model. b, Representative MRI images out of 9 independent experimental cohorts, of day 0 and day 14 of mice enrolled in vehicle, XL413, AZD8055 or combination treatment. The yellow line indicates the visible tumour area used to calculate the tumour volume. c, Tumour volumes were calculated based on MRI images from HCC-bearing mice with matched initial tumour volume. Graph shows mean ± s.e.m. from mice treated with vehicle (n=5), XL413 (n=8), AZD8055 (n=11) or combination (n=8) at the intermediate time point post-treatment initiation (day 14-16 in matched treatment groups, unpaired two-sided t -test). d, Survival curve generated from Myc OE ; Trp53 KO -tumour bearing mice treated with vehicle (n=11; median survival 17 days), XL413 (n=10; median survival 22.5 days), AZD8055 (n=11; median survival 20 days) or combination (n=11; median survival 33 days). e, Survival curve generated from independent cohorts of Myc OE ; Trp53 KO -HCC bearing mice treated with sorafenib (n=4; median survival 19.5 days) or combination of XL413 and AZD8055 (n=8; median survival 41.5 days). Vehicle group from Fig. 4d is used as a reference. d, e, Statistical significance was calculated using a two-sided Log-rank test. f, g, Graphs show mean ± s.e.m. of number of SA-β-Gal + ( f ) or p16 + ( g ) cells per tumour nodule/mm 2 (unpaired two-sided t ).
    Figure Legend Snippet: CDC7 inhibition induces senescence in vivo and suppresses tumour growth when combined with mTOR inhibition in multiple liver cancer models. a, Huh7 and MHCC97H cells were grown as tumour xenografts in BALB/c nude mice. Longitudinal tumour volume progression in Huh7 and MHCC97H tumour-bearing mice treated with vehicle, XL413, AZD8055 or combined therapies for 12 or 22 days, respectively. Graph shows mean ± s.e.m. b-g, Analyses of combination therapy response in the Myc OE ; Trp53 KO HCC somatic murine model. b, Representative MRI images out of 9 independent experimental cohorts, of day 0 and day 14 of mice enrolled in vehicle, XL413, AZD8055 or combination treatment. The yellow line indicates the visible tumour area used to calculate the tumour volume. c, Tumour volumes were calculated based on MRI images from HCC-bearing mice with matched initial tumour volume. Graph shows mean ± s.e.m. from mice treated with vehicle (n=5), XL413 (n=8), AZD8055 (n=11) or combination (n=8) at the intermediate time point post-treatment initiation (day 14-16 in matched treatment groups, unpaired two-sided t -test). d, Survival curve generated from Myc OE ; Trp53 KO -tumour bearing mice treated with vehicle (n=11; median survival 17 days), XL413 (n=10; median survival 22.5 days), AZD8055 (n=11; median survival 20 days) or combination (n=11; median survival 33 days). e, Survival curve generated from independent cohorts of Myc OE ; Trp53 KO -HCC bearing mice treated with sorafenib (n=4; median survival 19.5 days) or combination of XL413 and AZD8055 (n=8; median survival 41.5 days). Vehicle group from Fig. 4d is used as a reference. d, e, Statistical significance was calculated using a two-sided Log-rank test. f, g, Graphs show mean ± s.e.m. of number of SA-β-Gal + ( f ) or p16 + ( g ) cells per tumour nodule/mm 2 (unpaired two-sided t ).

    Techniques Used: Inhibition, In Vivo, Mouse Assay, Magnetic Resonance Imaging, Generated

    Pro-senescence treatment combined with mTOR inhibitor suppresses tumour growth in p53-deficient, immunocompetent somatic murine models of HCC a, Schematic representation of hydrodynamic tail vein gene delivery of the c-Myc proto-oncogene transposon system and a CRISPR-Cas9 vector targeting either Trp53 or Pten tumour suppressor, used to induce HCC 2-3 weeks post-HDTV injection (HDTVi). b, Quantification of SA-β-Gal staining performed on frozen sections from Myc OE ; Pten KO or Myc OE ; Trp53 KO HCC 14 days post treatment with vehicle or XL413 monotherapy ( Myc OE ; Trp53 KO also showed in ). For Myc OE ; Pten KO analyses: vehicle, n=9 biologically independent nodules out of 3 mice; XL413, n=16 biologically independent nodules out of 3 mice. For Myc OE ; Trp53 KO , vehicle, n=41 biologically independent nodules out of 7 mice; XL413, n=81 biologically independent nodules out of 11 mice. Graph shows the mean ± s.e.m. of the number of SA-β-Gal positive cells per tumour nodule/mm 2 . Statistics were calculated by two-sided unpaired Student t -test. c, Trial design to evaluate the efficacy of the pro-senescence treatment combined with mTOR inhibitor in Myc OE ; Trp53 KO HCC-bearing mice: Animals were monitored by weekly MRI post-HDTVi and enrolled into vehicle, XL413 (100mg/kg, daily gavage), AZD8055 (20mg/kg, daily gavage) or XL413+AZD8055 combination treatment groups at first signs of tumour development by MRI. Drugs were administered 6 days / week, and mice were sacrificed when symptomatic. Immunohistochemical (IHC) analyses confirmed Myc expression and p53 knockout in endpoint Myc OE ; Trp53 KO HCC. d, Longitudinal individual body weight curves from Myc OE ; Trp53 KO tumour-bearing mice treated with the combination of XL413+AZD8055 therapies. e, Individual tumour growth curves from mice treated with vehicle, XL413, AZD8055 or combination treatment were calculated based on MRI images from Myc OE ; Trp53 KO tumour-bearing mice. f, Myc OE ; Trp53 KO ), Sorafenib (n=4) or XL413 + AZD8055 (n=6) at day 0 and day 14. Graphs show mean ± s.e.m. analysed with two-sided unpaired Student t -test. g, h, Representative images of SA-β-Gal ( g ) and p16 ( h ) staining performed on frozen and paraffin-embedded sections, respectively, from Myc OE ; Trp53 KO ). Scale bar, 50 µm. i, Myc OE ; Trp53 KO tumour-bearing mice treated with vehicle, XL413, AZD8055 or combination were sacrificed at the indicated time point post-treatment. Tumours were dissociated as single cell suspension and flow cytometry analyses were performed to determine the content of tumour-associated macrophages (CD45 + CD11b + Ly6C - Ly6G - ), CD8 T cells (CD45 + CD3 + CD19 - NK1.1 - CD8 + ) and CD4 T cells (CD45 + CD3 + CD19 - NK1.1 - CD4 + ) relative to total CD45 + leucocytes and cell proliferation (Ki67 + ) was determined within CD8 T cells and CD4 T cell populations. Graphs show mean ± s.e.m., analysed with two-sided unpaired Student t ). j, Myc OE ; Trp53 KO HCC-bearing mice were treated with XL413 (n=20) or XL413+AZD8055 combination (n=8) for 14 days. Among the XL413-treated mice, a subset (n=10) was sacrificed directly 14 days post treatment, concomitantly to the combination-treated group. The rest of the XL413-treated mice (n=10) underwent XL413-drug withdrawal for 4 days. The absolute number of senescent cells per tumour nodule were visualized by SA-β-Gal staining performed on frozen sections and quantified for each treatment group (XL413 n=60 biologically independent nodules out of 10 mice; XL413 withdrawn n=63 biologically independent nodules out of 10 mice; XL413+AZD8055 n=57 biologically independent nodules out of 7 mice). Graphs show mean ± s.e.m. analysed with two-sided unpaired Student t -test. Data in c are representative of three independent biological experiments. Fig. 4f
    Figure Legend Snippet: Pro-senescence treatment combined with mTOR inhibitor suppresses tumour growth in p53-deficient, immunocompetent somatic murine models of HCC a, Schematic representation of hydrodynamic tail vein gene delivery of the c-Myc proto-oncogene transposon system and a CRISPR-Cas9 vector targeting either Trp53 or Pten tumour suppressor, used to induce HCC 2-3 weeks post-HDTV injection (HDTVi). b, Quantification of SA-β-Gal staining performed on frozen sections from Myc OE ; Pten KO or Myc OE ; Trp53 KO HCC 14 days post treatment with vehicle or XL413 monotherapy ( Myc OE ; Trp53 KO also showed in ). For Myc OE ; Pten KO analyses: vehicle, n=9 biologically independent nodules out of 3 mice; XL413, n=16 biologically independent nodules out of 3 mice. For Myc OE ; Trp53 KO , vehicle, n=41 biologically independent nodules out of 7 mice; XL413, n=81 biologically independent nodules out of 11 mice. Graph shows the mean ± s.e.m. of the number of SA-β-Gal positive cells per tumour nodule/mm 2 . Statistics were calculated by two-sided unpaired Student t -test. c, Trial design to evaluate the efficacy of the pro-senescence treatment combined with mTOR inhibitor in Myc OE ; Trp53 KO HCC-bearing mice: Animals were monitored by weekly MRI post-HDTVi and enrolled into vehicle, XL413 (100mg/kg, daily gavage), AZD8055 (20mg/kg, daily gavage) or XL413+AZD8055 combination treatment groups at first signs of tumour development by MRI. Drugs were administered 6 days / week, and mice were sacrificed when symptomatic. Immunohistochemical (IHC) analyses confirmed Myc expression and p53 knockout in endpoint Myc OE ; Trp53 KO HCC. d, Longitudinal individual body weight curves from Myc OE ; Trp53 KO tumour-bearing mice treated with the combination of XL413+AZD8055 therapies. e, Individual tumour growth curves from mice treated with vehicle, XL413, AZD8055 or combination treatment were calculated based on MRI images from Myc OE ; Trp53 KO tumour-bearing mice. f, Myc OE ; Trp53 KO ), Sorafenib (n=4) or XL413 + AZD8055 (n=6) at day 0 and day 14. Graphs show mean ± s.e.m. analysed with two-sided unpaired Student t -test. g, h, Representative images of SA-β-Gal ( g ) and p16 ( h ) staining performed on frozen and paraffin-embedded sections, respectively, from Myc OE ; Trp53 KO ). Scale bar, 50 µm. i, Myc OE ; Trp53 KO tumour-bearing mice treated with vehicle, XL413, AZD8055 or combination were sacrificed at the indicated time point post-treatment. Tumours were dissociated as single cell suspension and flow cytometry analyses were performed to determine the content of tumour-associated macrophages (CD45 + CD11b + Ly6C - Ly6G - ), CD8 T cells (CD45 + CD3 + CD19 - NK1.1 - CD8 + ) and CD4 T cells (CD45 + CD3 + CD19 - NK1.1 - CD4 + ) relative to total CD45 + leucocytes and cell proliferation (Ki67 + ) was determined within CD8 T cells and CD4 T cell populations. Graphs show mean ± s.e.m., analysed with two-sided unpaired Student t ). j, Myc OE ; Trp53 KO HCC-bearing mice were treated with XL413 (n=20) or XL413+AZD8055 combination (n=8) for 14 days. Among the XL413-treated mice, a subset (n=10) was sacrificed directly 14 days post treatment, concomitantly to the combination-treated group. The rest of the XL413-treated mice (n=10) underwent XL413-drug withdrawal for 4 days. The absolute number of senescent cells per tumour nodule were visualized by SA-β-Gal staining performed on frozen sections and quantified for each treatment group (XL413 n=60 biologically independent nodules out of 10 mice; XL413 withdrawn n=63 biologically independent nodules out of 10 mice; XL413+AZD8055 n=57 biologically independent nodules out of 7 mice). Graphs show mean ± s.e.m. analysed with two-sided unpaired Student t -test. Data in c are representative of three independent biological experiments. Fig. 4f

    Techniques Used: CRISPR, Plasmid Preparation, Injection, Staining, Mouse Assay, Magnetic Resonance Imaging, Immunohistochemistry, Expressing, Knock-Out, Flow Cytometry

    27) Product Images from "Oxidative stress triggered by naturally occurring flavone apigenin results in senescence and chemotherapeutic effect in human colorectal cancer cells"

    Article Title: Oxidative stress triggered by naturally occurring flavone apigenin results in senescence and chemotherapeutic effect in human colorectal cancer cells

    Journal: Redox Biology

    doi: 10.1016/j.redox.2015.04.009

    Phase contrast micrographs of (A) untreated/control HCT-15 cells, and (B–H) treated HCT-15 cells with apigenin (conc. range from 1.5625 to 25 µM) stained with senescence Cells Histo-chemical staining kit after 5 day drug treatment regimen. Cells stained as blue/green represent senescent cells.
    Figure Legend Snippet: Phase contrast micrographs of (A) untreated/control HCT-15 cells, and (B–H) treated HCT-15 cells with apigenin (conc. range from 1.5625 to 25 µM) stained with senescence Cells Histo-chemical staining kit after 5 day drug treatment regimen. Cells stained as blue/green represent senescent cells.

    Techniques Used: Staining

    28) Product Images from "MicroRNA-101 targets EZH2, MCL-1 and FOS to suppress proliferation, invasion and stem cell-like phenotype of aggressive endometrial cancer cells"

    Article Title: MicroRNA-101 targets EZH2, MCL-1 and FOS to suppress proliferation, invasion and stem cell-like phenotype of aggressive endometrial cancer cells

    Journal: Oncotarget

    doi:

    MiR-101 is downregulated in aggressive EC cell lines and modulates cell proliferation (a) Relative miR-101 expression of four aggressive endometrial cancer cell lines and immortalized endometrial epithelial cell line EM were examined with the quantitative real-time RT-PCR (qRT-PCR) assay. The expression of GAPDH was used as a normalization control, and the results are presented as the fold-change in expression compared with EM. Effects of ectopic expression of miR-101 on the proliferation of SPAC-1-L cells (b) and HEC-50 cells (c) were assessed with cell counting kit-8 assay. Clone formation assays were performed in SPAC-1-L (d) and HEC-50 (e) cells transduced with pre-miR-101 (101) or pre-miRNA negative control (NC). (f) Representative images of TUNEL assay in SPAC-1-L cells at 72 hours after transfection. Arrows indicate TUNEL-positive cells. (g) The percentages of TUNEL-positive SPAC-1-L and HEC-50 cells. (h) SPAC-1-L and HEC-50 cells were transfected with 101 or NC for 72 hours, and the relative ratio of caspase-3/7 activities were determined. (i) SA-β-gal staining analysis in SPAC-1-L cells transfected with 101 or NC at 72 hours after transfection. Arrows indicate blue senescent cells positive for SA-β-gal staining. (j) The percentages of SA-β-gal-positive SPAC-1-L and HEC-50 cells. (k) Western blot analysis of p21, Bax, total PARP and cleaved PARP in SPAC-1-L and HEC-50 cells after transduction with 101 or NC. ** P
    Figure Legend Snippet: MiR-101 is downregulated in aggressive EC cell lines and modulates cell proliferation (a) Relative miR-101 expression of four aggressive endometrial cancer cell lines and immortalized endometrial epithelial cell line EM were examined with the quantitative real-time RT-PCR (qRT-PCR) assay. The expression of GAPDH was used as a normalization control, and the results are presented as the fold-change in expression compared with EM. Effects of ectopic expression of miR-101 on the proliferation of SPAC-1-L cells (b) and HEC-50 cells (c) were assessed with cell counting kit-8 assay. Clone formation assays were performed in SPAC-1-L (d) and HEC-50 (e) cells transduced with pre-miR-101 (101) or pre-miRNA negative control (NC). (f) Representative images of TUNEL assay in SPAC-1-L cells at 72 hours after transfection. Arrows indicate TUNEL-positive cells. (g) The percentages of TUNEL-positive SPAC-1-L and HEC-50 cells. (h) SPAC-1-L and HEC-50 cells were transfected with 101 or NC for 72 hours, and the relative ratio of caspase-3/7 activities were determined. (i) SA-β-gal staining analysis in SPAC-1-L cells transfected with 101 or NC at 72 hours after transfection. Arrows indicate blue senescent cells positive for SA-β-gal staining. (j) The percentages of SA-β-gal-positive SPAC-1-L and HEC-50 cells. (k) Western blot analysis of p21, Bax, total PARP and cleaved PARP in SPAC-1-L and HEC-50 cells after transduction with 101 or NC. ** P

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Counting, Transduction, Negative Control, TUNEL Assay, Transfection, Staining, Western Blot

    29) Product Images from "TNFα-senescence initiates a STAT-dependent positive feedback loop, leading to a sustained interferon signature, DNA damage, and cytokine secretion"

    Article Title: TNFα-senescence initiates a STAT-dependent positive feedback loop, leading to a sustained interferon signature, DNA damage, and cytokine secretion

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101328

    Persistent activation of STAT1/3 Cells were exposed to TNFα (20ng/ml) for 3 days, then washed to remove the residual TNFα and cultured for 3 days in the absence of exogenous TNFα (TNFα-PST). Parallel cultures were exposed to exogenous TNFα throughout the experiment. ( A ) Levels of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT3 proteins were quantified by immunoblot. ( B ) Secretion of IL-6/IFNγ was assessed in culture supernatants from cells treated with TNFα as indicated. ( C ) Immunodetection of ROS production and γH2AX foci in control or cells treated with TNFα or TNFα-post-stimulated (PST), as indicated. ( D ) Real-time gene expression of IRF1 and MX1 in cells exposed to TNFα or TNFα-PST for 3 days. Results were normalized to internal control TBP and are shown relative to untreated cells. ( E ) SA-β-gal activity in TNFα-treated cells for 3 days or in cells treated with TNFα (20ng/ml) for three days, then washed to remove residual TNFα and left untreated for another 3 days. (F) mRNA expression of p21 and p16 quantified by real-time PCR in cells exposed to TNFα or TNFα-PST for 3 days. Data in D and F represent mean value of ± sd from 2 independent experiments.
    Figure Legend Snippet: Persistent activation of STAT1/3 Cells were exposed to TNFα (20ng/ml) for 3 days, then washed to remove the residual TNFα and cultured for 3 days in the absence of exogenous TNFα (TNFα-PST). Parallel cultures were exposed to exogenous TNFα throughout the experiment. ( A ) Levels of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT3 proteins were quantified by immunoblot. ( B ) Secretion of IL-6/IFNγ was assessed in culture supernatants from cells treated with TNFα as indicated. ( C ) Immunodetection of ROS production and γH2AX foci in control or cells treated with TNFα or TNFα-post-stimulated (PST), as indicated. ( D ) Real-time gene expression of IRF1 and MX1 in cells exposed to TNFα or TNFα-PST for 3 days. Results were normalized to internal control TBP and are shown relative to untreated cells. ( E ) SA-β-gal activity in TNFα-treated cells for 3 days or in cells treated with TNFα (20ng/ml) for three days, then washed to remove residual TNFα and left untreated for another 3 days. (F) mRNA expression of p21 and p16 quantified by real-time PCR in cells exposed to TNFα or TNFα-PST for 3 days. Data in D and F represent mean value of ± sd from 2 independent experiments.

    Techniques Used: Activation Assay, Cell Culture, Immunodetection, Expressing, Activity Assay, Real-time Polymerase Chain Reaction

    Prolonged activation of JAK/ STAT signaling in TNFα-induced senescence ( A ) Immunoblot detection of p-Ser727-STAT1 and total STAT1 in cells exposed to TNFα 20ng/ml for the indicated times. ( B ) SA-β-gal activity in TNFα (20ng/ml)-treated or control cells for 3 or 6 days. ( C ) Immunoblot detection of p-Ser727-STAT1, total STAT1, p-Tyr705-STAT3, and total STAT3 in cells exposed to TNFα (5ng/ml) for the indicated intervals. ( D ) Immunoblot detection of pSTAT1, total STAT1, pSTAT3, and total STAT3 in cells stimulated with IL6 (10ng/ml) or IFNγ (1ng/ml) for the indicated intervals. ( E ) Secretion of IFNγ/IL6 quantified by ELISA in conditioned medium collected in the presence or absence of TNFα. ( F ) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, STAT1, and STAT3 in cells treated with conditioned medium (CM) (cell free-culture supernatants from control and cells stimulated with TNFα for 3 days transferred after 1:4 dilution with fresh culture medium) from TNFα-induced senescent cells or from non-senescent cells for the indicated times.
    Figure Legend Snippet: Prolonged activation of JAK/ STAT signaling in TNFα-induced senescence ( A ) Immunoblot detection of p-Ser727-STAT1 and total STAT1 in cells exposed to TNFα 20ng/ml for the indicated times. ( B ) SA-β-gal activity in TNFα (20ng/ml)-treated or control cells for 3 or 6 days. ( C ) Immunoblot detection of p-Ser727-STAT1, total STAT1, p-Tyr705-STAT3, and total STAT3 in cells exposed to TNFα (5ng/ml) for the indicated intervals. ( D ) Immunoblot detection of pSTAT1, total STAT1, pSTAT3, and total STAT3 in cells stimulated with IL6 (10ng/ml) or IFNγ (1ng/ml) for the indicated intervals. ( E ) Secretion of IFNγ/IL6 quantified by ELISA in conditioned medium collected in the presence or absence of TNFα. ( F ) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, STAT1, and STAT3 in cells treated with conditioned medium (CM) (cell free-culture supernatants from control and cells stimulated with TNFα for 3 days transferred after 1:4 dilution with fresh culture medium) from TNFα-induced senescent cells or from non-senescent cells for the indicated times.

    Techniques Used: Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    JAK2 inhibitor decreases TNFα-mediated inflammation, ROS levels, and interferon signature Cells were exposed to TNFα alone, TNFα in combination with AG490 (30μM), or AG490 alone for 3 days. ( A ) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT1 and STAT3. ( B ) Secretion of IL6 was estimated by ELISA in culture supernatants from cells treated with TNFα alone, or in combination with AG490, or AG490 alone for 3 days. ( C ) FACS analysis of ROS levels in cells stimulated either with TNFα, TNFα along with AG490, or AG490 alone for 3 days using DCFDA staining 2′,7′-dichlorofluorescein (DCF) positive cells were analyzed. Inhibition of STAT signals modulates senescence. ( D ) Cell cycle analysis using BrdU and 7- aminoactinomycin D (7-AAD) staning in cells exposed to TNFα, TNFα in combination with AG490, or AG490 alone for 3 days. ( E ) Percentage of SA-β-gal-positive cells. Quantification of SA-β-gal activity in cells stimulated with TNFα 20ng/ml, TNFα in combination with AG490, or AG490 alone for 3 days. The data represent means of 3 independent counts of 200 cells from 2 independent experiments. ( F ) Effect of AG490 on cell cycle regulatory proteins. Western analysis performed using cells treated with AG490 for 3 days and blotted against anti-p21, CDK2, and NDC80. Actin serves as loading control.
    Figure Legend Snippet: JAK2 inhibitor decreases TNFα-mediated inflammation, ROS levels, and interferon signature Cells were exposed to TNFα alone, TNFα in combination with AG490 (30μM), or AG490 alone for 3 days. ( A ) Immunoblot detection of p-Ser727-STAT1, p-Tyr705-STAT3, and total STAT1 and STAT3. ( B ) Secretion of IL6 was estimated by ELISA in culture supernatants from cells treated with TNFα alone, or in combination with AG490, or AG490 alone for 3 days. ( C ) FACS analysis of ROS levels in cells stimulated either with TNFα, TNFα along with AG490, or AG490 alone for 3 days using DCFDA staining 2′,7′-dichlorofluorescein (DCF) positive cells were analyzed. Inhibition of STAT signals modulates senescence. ( D ) Cell cycle analysis using BrdU and 7- aminoactinomycin D (7-AAD) staning in cells exposed to TNFα, TNFα in combination with AG490, or AG490 alone for 3 days. ( E ) Percentage of SA-β-gal-positive cells. Quantification of SA-β-gal activity in cells stimulated with TNFα 20ng/ml, TNFα in combination with AG490, or AG490 alone for 3 days. The data represent means of 3 independent counts of 200 cells from 2 independent experiments. ( F ) Effect of AG490 on cell cycle regulatory proteins. Western analysis performed using cells treated with AG490 for 3 days and blotted against anti-p21, CDK2, and NDC80. Actin serves as loading control.

    Techniques Used: Enzyme-linked Immunosorbent Assay, FACS, Staining, Inhibition, Cell Cycle Assay, Activity Assay, Western Blot

    TNFα induces senescence and DNA damage in HUVECs ( A ) Long-term growth curve of cells exposed to recombinant human TNFα (5ng/ml). Untreated cells were used as controls. Population doubling and doubling times were calculated based on cell density at confluence. Data represent mean values from 3 independent experiments. ( B ) The percentage of BrdU-positive cells was determined by FACS analysis in cells untreated or chronically treated with TNFα at the concentration indicated. ( C ) Western blot analysis of p21, p16, and actin in cells treated with TNFα 5ng/ml for the indicated times. ( D ) SA-β-gal activity in TNFα (5ng/ml)-treated or control cells for the indicated number of days. ( E ) Percentages of SA-β-gal-positive cells in control or TNFα-treated cultures. The data represent 2 independent counts of 200 cells from 3 independent experiments. ( F ) Intracellular ROS levels were monitored by 2′,7′-dichlorodihydrofluorescein diacetate staining followed by flow cytometry. Bar graph represents percentage of DCFDA-positive cells treated with TNFα or medium alone. ( G ) Immunofluorescence detection of γH2AX foci in controls or cells treated with TNFα (5ng/ml) for indicated days. Data in A , B , E , and F represent mean value ± standard deviation (s.d.) from n=3, 2, 3, and 2 independent experiments, respectively.
    Figure Legend Snippet: TNFα induces senescence and DNA damage in HUVECs ( A ) Long-term growth curve of cells exposed to recombinant human TNFα (5ng/ml). Untreated cells were used as controls. Population doubling and doubling times were calculated based on cell density at confluence. Data represent mean values from 3 independent experiments. ( B ) The percentage of BrdU-positive cells was determined by FACS analysis in cells untreated or chronically treated with TNFα at the concentration indicated. ( C ) Western blot analysis of p21, p16, and actin in cells treated with TNFα 5ng/ml for the indicated times. ( D ) SA-β-gal activity in TNFα (5ng/ml)-treated or control cells for the indicated number of days. ( E ) Percentages of SA-β-gal-positive cells in control or TNFα-treated cultures. The data represent 2 independent counts of 200 cells from 3 independent experiments. ( F ) Intracellular ROS levels were monitored by 2′,7′-dichlorodihydrofluorescein diacetate staining followed by flow cytometry. Bar graph represents percentage of DCFDA-positive cells treated with TNFα or medium alone. ( G ) Immunofluorescence detection of γH2AX foci in controls or cells treated with TNFα (5ng/ml) for indicated days. Data in A , B , E , and F represent mean value ± standard deviation (s.d.) from n=3, 2, 3, and 2 independent experiments, respectively.

    Techniques Used: Recombinant, FACS, Concentration Assay, Western Blot, Activity Assay, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Standard Deviation

    30) Product Images from "Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung"

    Article Title: Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10327

    Analysis of senescence markers in normal (cancer-naive) lung fibroblasts co-cultured with lung cancer cell lines derived from ADC, SCC and LCC patients A. Outline of the Transwell-based co-cultures. B. Representative phase contrast images of SA-βgal stainings of CCD-19Lu fibroblasts co-cultured with a panel of lung cancer cell lines. More images are shown in Supplementary Figure S2 . C. Average percentage of SA-βgal+ CCD-19Lu fibroblasts co-cultured with a panel of lung cancer cell lines. Bare Transwell inserts were used as negative control. All pair-wise comparisons were performed with respect to Bare. D. Average percentage of growth arrested CCD-19Lu fibroblasts co-cultured with a panel of lung cancer cell lines with 0% or 10% FBS. E. Average relative changes in growth arrested cells computed from data in (D) as in Figure 1G .
    Figure Legend Snippet: Analysis of senescence markers in normal (cancer-naive) lung fibroblasts co-cultured with lung cancer cell lines derived from ADC, SCC and LCC patients A. Outline of the Transwell-based co-cultures. B. Representative phase contrast images of SA-βgal stainings of CCD-19Lu fibroblasts co-cultured with a panel of lung cancer cell lines. More images are shown in Supplementary Figure S2 . C. Average percentage of SA-βgal+ CCD-19Lu fibroblasts co-cultured with a panel of lung cancer cell lines. Bare Transwell inserts were used as negative control. All pair-wise comparisons were performed with respect to Bare. D. Average percentage of growth arrested CCD-19Lu fibroblasts co-cultured with a panel of lung cancer cell lines with 0% or 10% FBS. E. Average relative changes in growth arrested cells computed from data in (D) as in Figure 1G .

    Techniques Used: Cell Culture, Derivative Assay, Negative Control

    Analysis of myofibroblast and senescence markers in primary lung fibroblasts from major NSCLC subtypes (ADC, SCC and LCC) A. Representative fluorescence images of α-SMA stainings of cultured CFs and TAFs from a randomly selected patient of each histologic subtype. Patient number is indicated in the bottom-left of each image. Scale bar here and thereafter, 50 μm. B. Average fold α-SMA fluorescence intensity per cell of TAFs with respect to paired CFs for each subtype (6 ADC, 8 SCC, 3 LCC). Data shown as mean ± SE. C. Representative phase contrast images of SA-βgal stainings of cultured CFs and TAFs from a randomly selected patient of each histologic subtype. SA-βgal+ fibroblasts appear in blue. More images are shown in Supplementary Figure S1 . D. Box-plot of the percentage of SA-βgal+ fibroblasts in CFs and TAFs for each subtype from two independent collections (10 ADC, 8 SCC, 4 LCC). E. Average percentage of growth arrested fibroblasts (G0/G1 of the cell cycle) in CFs and TAFs for each subtype (4 ADC, 4 SCC, 3 LCC) cultured with 0% and 10% FBS. F. Average relative change in arrested fibroblasts at 10% versus 0% FBS computed from the data in G. All pair-wise comparisons were performed with respect to CFs except in (E). Mann–Whitney rank sum test was used in (D). *, P
    Figure Legend Snippet: Analysis of myofibroblast and senescence markers in primary lung fibroblasts from major NSCLC subtypes (ADC, SCC and LCC) A. Representative fluorescence images of α-SMA stainings of cultured CFs and TAFs from a randomly selected patient of each histologic subtype. Patient number is indicated in the bottom-left of each image. Scale bar here and thereafter, 50 μm. B. Average fold α-SMA fluorescence intensity per cell of TAFs with respect to paired CFs for each subtype (6 ADC, 8 SCC, 3 LCC). Data shown as mean ± SE. C. Representative phase contrast images of SA-βgal stainings of cultured CFs and TAFs from a randomly selected patient of each histologic subtype. SA-βgal+ fibroblasts appear in blue. More images are shown in Supplementary Figure S1 . D. Box-plot of the percentage of SA-βgal+ fibroblasts in CFs and TAFs for each subtype from two independent collections (10 ADC, 8 SCC, 4 LCC). E. Average percentage of growth arrested fibroblasts (G0/G1 of the cell cycle) in CFs and TAFs for each subtype (4 ADC, 4 SCC, 3 LCC) cultured with 0% and 10% FBS. F. Average relative change in arrested fibroblasts at 10% versus 0% FBS computed from the data in G. All pair-wise comparisons were performed with respect to CFs except in (E). Mann–Whitney rank sum test was used in (D). *, P

    Techniques Used: Fluorescence, Cell Culture, MANN-WHITNEY

    Effect of oxidative stress and exogenous TGF-β1 on fibroblast senescence induction by LCC cells A. Average percentage of SA-βgal+ CCD-19Lu fibroblasts co-cultured with H460 in the presence of increasing doses of the antioxidant NAC or vehicle. B, C. Average percentage of SA-βgal+ CCD-19Lu fibroblasts in response to direct or indirect oxidative stress elicited by (B) 2h treatment of H 2 O 2 followed by 4 days of recovery or (C) 9 day treatment with bleomycin (BLM). D. Average percentage of SA-βgal+ CCD-19Lu fibroblasts daily treated with TGF-β1-continuously or intermitently for 4h/day as in [ 8 ]- for 2 weeks. E. Average percentage of SA-βgal+ CCD-19Lu fibroblasts co-cultured with H460 in the presence 5 μM of the TGF-β pathway inhibitor SB505124 for 9 days. All results correspond to two replicates from at least three independent experiments. All pair-wise comparisons were performed with respect to Bare or vehicle.
    Figure Legend Snippet: Effect of oxidative stress and exogenous TGF-β1 on fibroblast senescence induction by LCC cells A. Average percentage of SA-βgal+ CCD-19Lu fibroblasts co-cultured with H460 in the presence of increasing doses of the antioxidant NAC or vehicle. B, C. Average percentage of SA-βgal+ CCD-19Lu fibroblasts in response to direct or indirect oxidative stress elicited by (B) 2h treatment of H 2 O 2 followed by 4 days of recovery or (C) 9 day treatment with bleomycin (BLM). D. Average percentage of SA-βgal+ CCD-19Lu fibroblasts daily treated with TGF-β1-continuously or intermitently for 4h/day as in [ 8 ]- for 2 weeks. E. Average percentage of SA-βgal+ CCD-19Lu fibroblasts co-cultured with H460 in the presence 5 μM of the TGF-β pathway inhibitor SB505124 for 9 days. All results correspond to two replicates from at least three independent experiments. All pair-wise comparisons were performed with respect to Bare or vehicle.

    Techniques Used: Cell Culture

    31) Product Images from "Comparison of Oxidative Stress Effects on Senescence Patterning of Human Adult and Perinatal Tissue-Derived Stem Cells in Short and Long-term Cultures"

    Article Title: Comparison of Oxidative Stress Effects on Senescence Patterning of Human Adult and Perinatal Tissue-Derived Stem Cells in Short and Long-term Cultures

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.27181

    hASC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hASC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hASCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.
    Figure Legend Snippet: hASC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hASC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hASCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.

    Techniques Used: Activity Assay, Concentration Assay, Staining, Light Microscopy, Imaging

    hWJ-MSC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hWJ-MSC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hWJ-MSCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.
    Figure Legend Snippet: hWJ-MSC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hWJ-MSC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hWJ-MSCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.

    Techniques Used: Activity Assay, Concentration Assay, Staining, Light Microscopy, Imaging

    32) Product Images from "Comparison of Oxidative Stress Effects on Senescence Patterning of Human Adult and Perinatal Tissue-Derived Stem Cells in Short and Long-term Cultures"

    Article Title: Comparison of Oxidative Stress Effects on Senescence Patterning of Human Adult and Perinatal Tissue-Derived Stem Cells in Short and Long-term Cultures

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.27181

    hASC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hASC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hASCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.
    Figure Legend Snippet: hASC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hASC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hASCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.

    Techniques Used: Activity Assay, Concentration Assay, Staining, Light Microscopy, Imaging

    hWJ-MSC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hWJ-MSC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hWJ-MSCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.
    Figure Legend Snippet: hWJ-MSC SA β-Gal activity after H 2 O 2 treatment. Effect of H 2 O 2 treatment expressed in term of ratio between the percentage of blue cells in treated sample and the relative percentage of blue cells in untreated sample after 1 h (Panel A) and 2 h (Panel B) of treatment. In each panel the effect of every H 2 O 2 concentration was presented after 24, 48 and 72 h from the end of the stimulus . Data were obtained in triplicate from one subject and expressed as ratio (n=3) ± SD. Control value is 1 (not shown) and treatment was arbitrarily considered effective when ratio was > 3. In Panels (C) and (D) the effects of different H 2 O 2 concentrations on hWJ-MSC SA β-Gal activity tested at 48 h from the end of the treatment are shown. In Panel (C), a graph representative of three (obtained from three different subjects) showing the percentage of blue cells in untreated (CTR) and treated cells after 1 and 2 h of H 2 O 2 treatment. Data were expressed as percentage of blue cells ± SD. Panel (D): representative SA β-Gal staining images of hWJ-MSCs untreated (CTR) and treated with H 2 O 2 at final concentrations of 100 μM, 150 μM and 200 μM after 2-h treatment. Cells were analyzed under a light microscope (at 200× magnification) and cell images were detected under bright field illumination with the Leica MC170 HD Imaging System. Blue staining indicates senescent cells and scale bars correspond to 100 μm.

    Techniques Used: Activity Assay, Concentration Assay, Staining, Light Microscopy, Imaging

    33) Product Images from "p27Kip1 deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process"

    Article Title: p27Kip1 deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-541

    Effects of 9cRA on cell proliferation and senescence in DLP of p27 deficient mice . Fig. 2A: Cell proliferation, as determined by Ki-67 antibody in DLP of 9-month-old p27+/+ mice. Note several Ki-67-positive cells (brawn-stained, arrows) in glandular structures which are covered by a single layer of epithelial cells. The slide is counterstained by hematoxylin × 200. 2B: Ki-67 positive cells (arrows) in anterior (large left) and DLP (two right glands) of 9-month-old p27-/- mice. Note the thick connective tissue surrounding glandular structures. The slide is counterstained by hematoxylin × 200. 2C: Ki-67 positive cells in PIN of MNU-treated mice. The number of Ki-67 positive cells is higher (arrow), as compared to those in Fig. 2B. The slide is counterstained by hematoxylin × 200. 2D: Treatment of p27-/- mice with 9cRA for 6 months reduced the number of Ki-67 positive cells in PIN (arrow). Note the inflammation associated cells in PINs' lumen. The slide is counterstained by hematoxylin × 200. 2E: Senescent cells (blue stained) determined by SA-β-Gal staining in glandular and ductal structures of DLP of 9-month-old p27+/- mice. Senescent cells are also detectable among myoepithelial and stroma cells (arrows). The slide is counterstained by nuclear fast red × 200. 2F: Treatment of animals with 9cRA increased senescent cells (blue stained) in DLP and AP of a p27+/- mice. The slide is counterstained by nuclear fast red × 200. 2G: 9cRA increased senescent cells in low grade PIN (arrows) as compared to non-PIN areas of a 10-month-old p27+/- animal. The slide is counterstained by nuclear fast red × 200. 2H: 9cRA also increased senescent cells in high grade PIN as shown in the right glandular structure. The slide is counterstained by nuclear fast red × 200.
    Figure Legend Snippet: Effects of 9cRA on cell proliferation and senescence in DLP of p27 deficient mice . Fig. 2A: Cell proliferation, as determined by Ki-67 antibody in DLP of 9-month-old p27+/+ mice. Note several Ki-67-positive cells (brawn-stained, arrows) in glandular structures which are covered by a single layer of epithelial cells. The slide is counterstained by hematoxylin × 200. 2B: Ki-67 positive cells (arrows) in anterior (large left) and DLP (two right glands) of 9-month-old p27-/- mice. Note the thick connective tissue surrounding glandular structures. The slide is counterstained by hematoxylin × 200. 2C: Ki-67 positive cells in PIN of MNU-treated mice. The number of Ki-67 positive cells is higher (arrow), as compared to those in Fig. 2B. The slide is counterstained by hematoxylin × 200. 2D: Treatment of p27-/- mice with 9cRA for 6 months reduced the number of Ki-67 positive cells in PIN (arrow). Note the inflammation associated cells in PINs' lumen. The slide is counterstained by hematoxylin × 200. 2E: Senescent cells (blue stained) determined by SA-β-Gal staining in glandular and ductal structures of DLP of 9-month-old p27+/- mice. Senescent cells are also detectable among myoepithelial and stroma cells (arrows). The slide is counterstained by nuclear fast red × 200. 2F: Treatment of animals with 9cRA increased senescent cells (blue stained) in DLP and AP of a p27+/- mice. The slide is counterstained by nuclear fast red × 200. 2G: 9cRA increased senescent cells in low grade PIN (arrows) as compared to non-PIN areas of a 10-month-old p27+/- animal. The slide is counterstained by nuclear fast red × 200. 2H: 9cRA also increased senescent cells in high grade PIN as shown in the right glandular structure. The slide is counterstained by nuclear fast red × 200.

    Techniques Used: Mouse Assay, Staining

    34) Product Images from "APE1 deficiency promotes cellular senescence and premature aging features"

    Article Title: APE1 deficiency promotes cellular senescence and premature aging features

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky326

    Senescence results in altered expression of BER proteins in primary human BJ fibroblasts. ( A ) Representative images of SA-β-gal staining at P3, P30 and P50 in primary BJ fibroblasts. ( B ) Quantitation of the percentage of cells that has undergone senescence (SA-β-gal positive) at the indicated passage number. ( C ) Expression of several BER proteins at P3, P30 and P50 as determined by western blot analysis. Increased p16 INK4a expression is consistent with a senescent phenotype. ( D ) Quantitation of expression of the indicated BER protein (after normalization to the β-actin loading control) relative to the P3 group. In panels B and D, results are averages and standard deviations of three biological replicates. * P -value
    Figure Legend Snippet: Senescence results in altered expression of BER proteins in primary human BJ fibroblasts. ( A ) Representative images of SA-β-gal staining at P3, P30 and P50 in primary BJ fibroblasts. ( B ) Quantitation of the percentage of cells that has undergone senescence (SA-β-gal positive) at the indicated passage number. ( C ) Expression of several BER proteins at P3, P30 and P50 as determined by western blot analysis. Increased p16 INK4a expression is consistent with a senescent phenotype. ( D ) Quantitation of expression of the indicated BER protein (after normalization to the β-actin loading control) relative to the P3 group. In panels B and D, results are averages and standard deviations of three biological replicates. * P -value

    Techniques Used: Expressing, Staining, Quantitation Assay, Western Blot

    Effect of Apex1 deletion at post-natal day 7/12 on mouse growth and organ development. ( A ) Tamoxifen treatment strategy for post-natal day 7/12 (D7/D12) mice. ( B ) Expression of Apex1 protein in the indicated tissue/organ of tamoxifen-treated wildtype (Apex1 +/+ ) or Apex1-floxed (Apex1 −/− ) animals ( n = 4 mice per group). Shown is a representative western blot using Apex1 or β-tubulin (loading control) antibody. ( C ) Comparison of overall size of Apex1 −/− and Apex1 +/+ animals ( n = 4 mice per group) at D28. ( D ) Comparison of tissues/organs, including heart, digestive track, liver, kidney, spleen and brain of Apex1 −/− and Apex1 +/+ animals ( n = 4 mice per group) after sacrifice at D28. (E) SA-β-gal staining and immunohistochemistry for Ki67 in the colon and skin of Apex1 −/− and Apex1 +/+ mice ( n = 4 mice per group).
    Figure Legend Snippet: Effect of Apex1 deletion at post-natal day 7/12 on mouse growth and organ development. ( A ) Tamoxifen treatment strategy for post-natal day 7/12 (D7/D12) mice. ( B ) Expression of Apex1 protein in the indicated tissue/organ of tamoxifen-treated wildtype (Apex1 +/+ ) or Apex1-floxed (Apex1 −/− ) animals ( n = 4 mice per group). Shown is a representative western blot using Apex1 or β-tubulin (loading control) antibody. ( C ) Comparison of overall size of Apex1 −/− and Apex1 +/+ animals ( n = 4 mice per group) at D28. ( D ) Comparison of tissues/organs, including heart, digestive track, liver, kidney, spleen and brain of Apex1 −/− and Apex1 +/+ animals ( n = 4 mice per group) after sacrifice at D28. (E) SA-β-gal staining and immunohistochemistry for Ki67 in the colon and skin of Apex1 −/− and Apex1 +/+ mice ( n = 4 mice per group).

    Techniques Used: Mouse Assay, Expressing, Western Blot, Staining, Immunohistochemistry

    Effect of Apex1 deletion at post-weaning week 6 on animal health. ( A ) The protocol for tamoxifen treatment of post-weaning week 6 (W6) mice. ( B ) Expression of Apex1 protein in the indicated tissue/organ of tamoxifen-treated wild-type (Apex1 +/+ ) or Apex1-floxed (Apex1 −/− ) animals ( n = 6 mice per group). Shown is a representative western blot using Apex1 or β-tubulin (loading control) antibody. ( C ) Comparison of the tissues/organs of Apex1 −/− and Apex1 +/+ mice ( n = 6 mice per group). ( D ) Comparison of the overall size of the Apex1 −/− and Apex1 +/+ animals ( n = 6 mice per group). Analysis of animals and associated tissue/organs was carried out at W40. ( E ) Comparison of skin wound healing after incision of the back of Apex1 +/+ and Apex1 −/− animals ( n = 6 mice per group). Representative images of wound at day 0 and 10 days post-incision are shown. SA-β-gal staining and Ki67 immunohistochemistry in the colon ( F ) and skin ( G ) of Apex1 −/− and Apex1 +/+ mice (n = 6 mice per group). The expression of p16 INK4a , p21 WAF1 , γ-H2AX and Apex1 was determined in extracts from the colon ( H ) or skin ( I ) of Apex1 −/− and Apex1 +/+ mice ( n = 6 mice per group) by western blot analysis. ( J ) The amount of abasic sites in skin and colon of Apex1 −/− and Apex1 +/+ animals ( n = 6 mice per group). Results are the average and standard deviation of three biological replicates with four technical replicates each. ** P -value
    Figure Legend Snippet: Effect of Apex1 deletion at post-weaning week 6 on animal health. ( A ) The protocol for tamoxifen treatment of post-weaning week 6 (W6) mice. ( B ) Expression of Apex1 protein in the indicated tissue/organ of tamoxifen-treated wild-type (Apex1 +/+ ) or Apex1-floxed (Apex1 −/− ) animals ( n = 6 mice per group). Shown is a representative western blot using Apex1 or β-tubulin (loading control) antibody. ( C ) Comparison of the tissues/organs of Apex1 −/− and Apex1 +/+ mice ( n = 6 mice per group). ( D ) Comparison of the overall size of the Apex1 −/− and Apex1 +/+ animals ( n = 6 mice per group). Analysis of animals and associated tissue/organs was carried out at W40. ( E ) Comparison of skin wound healing after incision of the back of Apex1 +/+ and Apex1 −/− animals ( n = 6 mice per group). Representative images of wound at day 0 and 10 days post-incision are shown. SA-β-gal staining and Ki67 immunohistochemistry in the colon ( F ) and skin ( G ) of Apex1 −/− and Apex1 +/+ mice (n = 6 mice per group). The expression of p16 INK4a , p21 WAF1 , γ-H2AX and Apex1 was determined in extracts from the colon ( H ) or skin ( I ) of Apex1 −/− and Apex1 +/+ mice ( n = 6 mice per group) by western blot analysis. ( J ) The amount of abasic sites in skin and colon of Apex1 −/− and Apex1 +/+ animals ( n = 6 mice per group). Results are the average and standard deviation of three biological replicates with four technical replicates each. ** P -value

    Techniques Used: Mouse Assay, Expressing, Western Blot, Staining, Immunohistochemistry, Standard Deviation

    APE1 knock-down results in increased cellular senescence in hTERT-negative fibroblasts. ( A ) Representative images of SA-β-gal staining in BJ (at P2–5), GM05565 (at P3–8) and GM00969 (at P10–15) fibroblasts that were infected at the indicated passage number with an APE1-specific shRNA [TRCN0000007959 (#59) or TRCN0000007961 (#61)] or the scramble control. Sample analysis was conducted 48–72 hr post-infection. ( B ) Quantitation of the percentage of cells that have undergone senescence in early passage fibroblasts following APE1 knock-down or scramble control. ( C ) The expression of APE1, p16 INK4a and p21 WAF1 in the indicated scramble control (Scr) or APE1-knock-down (shAPE1) cell population, as determined by western blot analysis. Following normalization of the indicated protein to β-actin, the relative expression level (specified below each) was obtained by comparing knock-down to its corresponding scramble control. ( D ) Quantitation of the percentage of cells that have undergone senescence (SA-β-gal) in early passage primary BJ, GM05565 and GM00969 fibroblasts upon shRNA knock-down of POLB or XRCC1. ( E ) Quantitation of the percentage of cells that have undergone apoptosis in hTERT-negative (primary BJ) or hTERT-positive (BJ-5ta) cells following APE1 knock-down (shRNA, #59), in comparison to the scramble control. Results are averages and standard deviations of three biological replicates (panel B and D) or two biological replicates with three technical replicates each (panel E). * P -value
    Figure Legend Snippet: APE1 knock-down results in increased cellular senescence in hTERT-negative fibroblasts. ( A ) Representative images of SA-β-gal staining in BJ (at P2–5), GM05565 (at P3–8) and GM00969 (at P10–15) fibroblasts that were infected at the indicated passage number with an APE1-specific shRNA [TRCN0000007959 (#59) or TRCN0000007961 (#61)] or the scramble control. Sample analysis was conducted 48–72 hr post-infection. ( B ) Quantitation of the percentage of cells that have undergone senescence in early passage fibroblasts following APE1 knock-down or scramble control. ( C ) The expression of APE1, p16 INK4a and p21 WAF1 in the indicated scramble control (Scr) or APE1-knock-down (shAPE1) cell population, as determined by western blot analysis. Following normalization of the indicated protein to β-actin, the relative expression level (specified below each) was obtained by comparing knock-down to its corresponding scramble control. ( D ) Quantitation of the percentage of cells that have undergone senescence (SA-β-gal) in early passage primary BJ, GM05565 and GM00969 fibroblasts upon shRNA knock-down of POLB or XRCC1. ( E ) Quantitation of the percentage of cells that have undergone apoptosis in hTERT-negative (primary BJ) or hTERT-positive (BJ-5ta) cells following APE1 knock-down (shRNA, #59), in comparison to the scramble control. Results are averages and standard deviations of three biological replicates (panel B and D) or two biological replicates with three technical replicates each (panel E). * P -value

    Techniques Used: Staining, Infection, shRNA, Quantitation Assay, Expressing, Western Blot

    BER protein expression correlates with senescence in patient samples. ( A ) Knock-down of APE1 (shAPE1; #59) in 10 primary fibroblast cultures from the excess skin removed during plastic surgery results in increased senescence relative to the scramble control. Results from a single run of each human fibroblast culture is shown below the relevant sample. ( B ) The results (% senescence, i.e. SA-β-gal positive) from each sample in panel A are plotted for the scramble control and comparative APE1 knock-down cells. Averages and standard deviations are indicated, with *** P -value
    Figure Legend Snippet: BER protein expression correlates with senescence in patient samples. ( A ) Knock-down of APE1 (shAPE1; #59) in 10 primary fibroblast cultures from the excess skin removed during plastic surgery results in increased senescence relative to the scramble control. Results from a single run of each human fibroblast culture is shown below the relevant sample. ( B ) The results (% senescence, i.e. SA-β-gal positive) from each sample in panel A are plotted for the scramble control and comparative APE1 knock-down cells. Averages and standard deviations are indicated, with *** P -value

    Techniques Used: Expressing

    35) Product Images from "Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis"

    Article Title: Hyaluronan synthase 2 regulates fibroblast senescence in pulmonary fibrosis

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2016.03.004

    Depletion of HAS2 expression induces fibroblast senescence. Fibroblasts transfected with HAS2 siRNA and control siRNA. (A). Cell numbers were counted at indicated time after transfection. Data shown is the mean of triplicates ± s. d. of one representative
    Figure Legend Snippet: Depletion of HAS2 expression induces fibroblast senescence. Fibroblasts transfected with HAS2 siRNA and control siRNA. (A). Cell numbers were counted at indicated time after transfection. Data shown is the mean of triplicates ± s. d. of one representative

    Techniques Used: Expressing, Transfection

    HAS2 depletion induced fibroblast senescence is associated with increased p27-CDK2-SKP2 expression. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western
    Figure Legend Snippet: HAS2 depletion induced fibroblast senescence is associated with increased p27-CDK2-SKP2 expression. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western

    Techniques Used: Expressing, Transfection, Western Blot

    Stress responses mediate HAS2 deficiency-induced senescence. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western blots. The expression of proteins
    Figure Legend Snippet: Stress responses mediate HAS2 deficiency-induced senescence. Fibroblasts were transfected with HAS2 siRNA or control siRNA. Cell lysates were harvested at various time points after transfection and subjected to Western blots. The expression of proteins

    Techniques Used: Transfection, Western Blot, Expressing

    36) Product Images from "Galactose‐modified duocarmycin prodrugs as senolytics, et al. Galactose‐modified duocarmycin prodrugs as senolytics"

    Article Title: Galactose‐modified duocarmycin prodrugs as senolytics, et al. Galactose‐modified duocarmycin prodrugs as senolytics

    Journal: Aging Cell

    doi: 10.1111/acel.13133

    Galactose‐modified duocarmycin prodrugs preferentially target senescent cells with high SA‐β‐galactosidase activity. (a) Representative pictures of fluorescent SA‐β‐galactosidase staining in IMR90 ER:RAS cells treated with prodrug A or vehicle. Scale bar, 100 µm. (b) Single‐cell intensities value for DDAO galactoside in a representative well of a 96‐well plate seeded with IMR90 ER:RAS cells treated with DMSO or 4OHT. Grey dotted line indicates quantification cut‐off. Cells were considered positives for SA‐β‐gal when their cell intensity was > 165. (c) Quantification of SA‐β‐galactosidase activity in IMR90 ER:RAS cells treated with prodrug A or vehicle. Statistical significance was calculated using two‐way ANOVA. (d‐g) The senolytic properties of prodrug A depend on the lysosomal β‐galactosidase (GLB1). (d) Experimental set‐up. (e) Representative pictures of cytochemical SA‐β‐Gal staining in IMR90 ER:RAS infected with different shRNAs against GLB1 or an empty vector and (f) quantification ( n = 3). Statistical significance was calculated using one‐way ANOVA. (g) Quantification of cell survival of senescent and control IMR90 ER:RAS infected with different shRNAs targeting GLB1 or an empty vector and treated with ABT‐263 or prodrug A for 3 days ( n = 3). Statistical significance was calculated using two‐tailed, Student's t test. All error bars represent mean ± SD ; n represents independent experiments; ns, not significant; * p
    Figure Legend Snippet: Galactose‐modified duocarmycin prodrugs preferentially target senescent cells with high SA‐β‐galactosidase activity. (a) Representative pictures of fluorescent SA‐β‐galactosidase staining in IMR90 ER:RAS cells treated with prodrug A or vehicle. Scale bar, 100 µm. (b) Single‐cell intensities value for DDAO galactoside in a representative well of a 96‐well plate seeded with IMR90 ER:RAS cells treated with DMSO or 4OHT. Grey dotted line indicates quantification cut‐off. Cells were considered positives for SA‐β‐gal when their cell intensity was > 165. (c) Quantification of SA‐β‐galactosidase activity in IMR90 ER:RAS cells treated with prodrug A or vehicle. Statistical significance was calculated using two‐way ANOVA. (d‐g) The senolytic properties of prodrug A depend on the lysosomal β‐galactosidase (GLB1). (d) Experimental set‐up. (e) Representative pictures of cytochemical SA‐β‐Gal staining in IMR90 ER:RAS infected with different shRNAs against GLB1 or an empty vector and (f) quantification ( n = 3). Statistical significance was calculated using one‐way ANOVA. (g) Quantification of cell survival of senescent and control IMR90 ER:RAS infected with different shRNAs targeting GLB1 or an empty vector and treated with ABT‐263 or prodrug A for 3 days ( n = 3). Statistical significance was calculated using two‐tailed, Student's t test. All error bars represent mean ± SD ; n represents independent experiments; ns, not significant; * p

    Techniques Used: Modification, Activity Assay, Staining, Infection, Plasmid Preparation, Two Tailed Test

    Prodrug A reduces the numbers of senescent cells accumulating after whole‐body irradiation. (a) Experimental design of the whole‐body irradiation‐induced senescence experiment. Mice ( n = 4/5 per group) were irradiated with 6 Gray to induce senescence. Two months later, mice were treated with vehicle, prodrug A (JHB75B) or ABT‐263 for 4 consecutive days, before being culled for analysis. (b, c) Representative pictures of lung cryosections (b) and quantification of the lung area positive for SA‐β‐Gal staining (c). (d) Expression levels of Cdkn1a , Il6 and Cxcl1 in lungs of nonirradiated mice or irradiated mice treated with prodrug A, ABT‐263 or vehicle. Statistical significance was calculated using unpaired Student's t test. Data represent mean ± SD ; n represents number of mice; ns, not significant; * p
    Figure Legend Snippet: Prodrug A reduces the numbers of senescent cells accumulating after whole‐body irradiation. (a) Experimental design of the whole‐body irradiation‐induced senescence experiment. Mice ( n = 4/5 per group) were irradiated with 6 Gray to induce senescence. Two months later, mice were treated with vehicle, prodrug A (JHB75B) or ABT‐263 for 4 consecutive days, before being culled for analysis. (b, c) Representative pictures of lung cryosections (b) and quantification of the lung area positive for SA‐β‐Gal staining (c). (d) Expression levels of Cdkn1a , Il6 and Cxcl1 in lungs of nonirradiated mice or irradiated mice treated with prodrug A, ABT‐263 or vehicle. Statistical significance was calculated using unpaired Student's t test. Data represent mean ± SD ; n represents number of mice; ns, not significant; * p

    Techniques Used: Irradiation, Mouse Assay, Staining, Expressing

    37) Product Images from "Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition"

    Article Title: Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2012.10

    CK2 inhibitor affects p53 target genes associated with cell cycle regulation and apoptosis. ( a ) CK2-I increases p21 expression in a p53-dependent manner. Representative blot is shown from three independent experiments with identical results. Blots were re-probed with c23 to establish equivalent loading. ( b ) CK2-I increases mRNA levels of pro-apoptotic molecules Noxa and GADD45 β in a p53-dependent manner. Total RNA was isolated from cells treated with different combinations of TNF α and CK2-I, and the mRNA levels for Noxa, GADD45 β and constitutive enzyme GAPDH were determined by RT-PCR. ( c ) Pifithrin- α reverses CK2-I-mediated G2/M phase arrest in A172 cells. FACS analysis was performed on A172 cells treated with different combinations of TNF α , CK2-Is and Pifithrin- α . Inset indicates percentage of cells in G1, S and G2/M phase of the cell cycle. C, T, Pf and A denote control, TNF α , Pifithrin- α and Apigenin, respectively
    Figure Legend Snippet: CK2 inhibitor affects p53 target genes associated with cell cycle regulation and apoptosis. ( a ) CK2-I increases p21 expression in a p53-dependent manner. Representative blot is shown from three independent experiments with identical results. Blots were re-probed with c23 to establish equivalent loading. ( b ) CK2-I increases mRNA levels of pro-apoptotic molecules Noxa and GADD45 β in a p53-dependent manner. Total RNA was isolated from cells treated with different combinations of TNF α and CK2-I, and the mRNA levels for Noxa, GADD45 β and constitutive enzyme GAPDH were determined by RT-PCR. ( c ) Pifithrin- α reverses CK2-I-mediated G2/M phase arrest in A172 cells. FACS analysis was performed on A172 cells treated with different combinations of TNF α , CK2-Is and Pifithrin- α . Inset indicates percentage of cells in G1, S and G2/M phase of the cell cycle. C, T, Pf and A denote control, TNF α , Pifithrin- α and Apigenin, respectively

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, FACS

    CK2 inhibition-induced p53 activation decreases telomerase activity and induces senescence but has no effect on NF- κ B activity. ( a ) CK2-Is decrease telomerase activity in glioma cells in a p53-dependent manner. Glioma cells were treated with TNF α in the presence or absence of CK2-Is and/or Pifithrin- α ; and T elo TAGGG Telomerase PCR ELISA was performed. Values represent the means±S.E.M. from three independent experiments. ( b ) CK2-I-induced senescence in glioma cells is p53-dependent as evident from senescence-specific β -gal staining. Images were taken at 20 × magnification. The graph represents fold increase in β -gal-positive cells upon treatment with different combinations of Apigenin, TNF α and Pifithrin- α . ( c ) Pifithrin- α does not affect the ability of DRB to abrogate TNF α -induced NF- κ B activation. The graph represents fold change in NF- κ B luciferase activity over control, in cells treated with different combinations of TNF α , DRB and Pifithrin- α . Values in ( a – c ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from control ( P
    Figure Legend Snippet: CK2 inhibition-induced p53 activation decreases telomerase activity and induces senescence but has no effect on NF- κ B activity. ( a ) CK2-Is decrease telomerase activity in glioma cells in a p53-dependent manner. Glioma cells were treated with TNF α in the presence or absence of CK2-Is and/or Pifithrin- α ; and T elo TAGGG Telomerase PCR ELISA was performed. Values represent the means±S.E.M. from three independent experiments. ( b ) CK2-I-induced senescence in glioma cells is p53-dependent as evident from senescence-specific β -gal staining. Images were taken at 20 × magnification. The graph represents fold increase in β -gal-positive cells upon treatment with different combinations of Apigenin, TNF α and Pifithrin- α . ( c ) Pifithrin- α does not affect the ability of DRB to abrogate TNF α -induced NF- κ B activation. The graph represents fold change in NF- κ B luciferase activity over control, in cells treated with different combinations of TNF α , DRB and Pifithrin- α . Values in ( a – c ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from control ( P

    Techniques Used: Inhibition, Activation Assay, Activity Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Staining, Luciferase

    CK2 inhibition triggers p53 expression and activation to induce glioma cell death. ( a ) Western blot indicated phosphorylated, acetylated and total p53 levels in cells treated with CK2-Is in the presence and absence of TNF α . A representative blot is shown from three independent experiments with identical results. c23 was used as loading control. ( b ) Increased binding of p53 to its DNA-binding sequence in cells treated with CK2-Is, either alone or in combination with TNF α , is abrogated by p53 inhibitor, Pifithrin- α , The graphs represent relative binding activity of p53 under different conditions, as compared with untreated controls. Values represent means ±S.E.M. from three individual experiments. ( c ) CK2-I-mediated increase in p53 transcriptional activity is abrogated upon p53 inhibition. The graphs represent fold change in p53 luciferase reporter activity over control in cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 12 h. ( d ) siRNA-mediated knockdown of CK2 α / β induces p53 transcriptional activity in p53 wild-type but not in mutant cell lines. The graph represents fold change in p53 luciferase reporter activity over NS-siRNA-transfected control. ( e ) CK2-I-mediated sensitization of glioma cells to TNF α -induced death is reversed by Pifithrin- α and ( f ) p53 siRNA. The graphs represent percentage viable glioma cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 24 h, as determined by MTS assay. (Inset) p53 siRNA abrogates CK2-I-induced p53 levels as determined by western blot analysis. ( g ) CK2-I reduced viability of p53 null cells lines only in presence of TNF α . The graph represents percentage viable SAOS2 and H1299 cells treated with different combinations of CK2-Is and TNF α . Values in ( b – g ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from untreated control, # denotes significant change from CK2-I-treated cells either alone or in presence of TNF α ( P
    Figure Legend Snippet: CK2 inhibition triggers p53 expression and activation to induce glioma cell death. ( a ) Western blot indicated phosphorylated, acetylated and total p53 levels in cells treated with CK2-Is in the presence and absence of TNF α . A representative blot is shown from three independent experiments with identical results. c23 was used as loading control. ( b ) Increased binding of p53 to its DNA-binding sequence in cells treated with CK2-Is, either alone or in combination with TNF α , is abrogated by p53 inhibitor, Pifithrin- α , The graphs represent relative binding activity of p53 under different conditions, as compared with untreated controls. Values represent means ±S.E.M. from three individual experiments. ( c ) CK2-I-mediated increase in p53 transcriptional activity is abrogated upon p53 inhibition. The graphs represent fold change in p53 luciferase reporter activity over control in cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 12 h. ( d ) siRNA-mediated knockdown of CK2 α / β induces p53 transcriptional activity in p53 wild-type but not in mutant cell lines. The graph represents fold change in p53 luciferase reporter activity over NS-siRNA-transfected control. ( e ) CK2-I-mediated sensitization of glioma cells to TNF α -induced death is reversed by Pifithrin- α and ( f ) p53 siRNA. The graphs represent percentage viable glioma cells treated with different combinations of CK2-Is, TNF α and Pifithrin- α for 24 h, as determined by MTS assay. (Inset) p53 siRNA abrogates CK2-I-induced p53 levels as determined by western blot analysis. ( g ) CK2-I reduced viability of p53 null cells lines only in presence of TNF α . The graph represents percentage viable SAOS2 and H1299 cells treated with different combinations of CK2-Is and TNF α . Values in ( b – g ) represent the means ± S.E.M. from three independent experiments. * Denotes significant change from untreated control, # denotes significant change from CK2-I-treated cells either alone or in presence of TNF α ( P

    Techniques Used: Inhibition, Expressing, Activation Assay, Western Blot, Binding Assay, Sequencing, Activity Assay, Luciferase, Mutagenesis, Transfection, MTS Assay

    38) Product Images from "Effect of Advanced Glycation End Products on Oxidative Stress and Senescence of Trabecular Meshwork Cells"

    Article Title: Effect of Advanced Glycation End Products on Oxidative Stress and Senescence of Trabecular Meshwork Cells

    Journal: Korean Journal of Ophthalmology : KJO

    doi: 10.3341/kjo.2012.26.2.123

    Effect of advanced glycation end products (AGE) on the senescence of trabecular meshwork cells. AGE significantly increased the percentage of senescence-associated β-galactosidase (SA-β-gal) (+) cells in a dose-dependent manner ( * p
    Figure Legend Snippet: Effect of advanced glycation end products (AGE) on the senescence of trabecular meshwork cells. AGE significantly increased the percentage of senescence-associated β-galactosidase (SA-β-gal) (+) cells in a dose-dependent manner ( * p

    Techniques Used:

    39) Product Images from "Branched-Chain Amino Acids Enhance Premature Senescence through Mammalian Target of Rapamycin Complex I-Mediated Upregulation of p21 Protein"

    Article Title: Branched-Chain Amino Acids Enhance Premature Senescence through Mammalian Target of Rapamycin Complex I-Mediated Upregulation of p21 Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080411

    DNA damage-inducing drugs cause premature senescence. (A) HepG2 cells were cultured in RPMI medium with 0.1% DMSO or 10 µM etoposide for 0, 12, 24, 36 and 48 hours. (B) U2OS cells were cultured in RPMI medium with 0.1% DMSO, 2 µM etoposide, or 2 µM bleomycin for 0, 3, 5 and 7 days. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods . The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results ( P values) are shown.
    Figure Legend Snippet: DNA damage-inducing drugs cause premature senescence. (A) HepG2 cells were cultured in RPMI medium with 0.1% DMSO or 10 µM etoposide for 0, 12, 24, 36 and 48 hours. (B) U2OS cells were cultured in RPMI medium with 0.1% DMSO, 2 µM etoposide, or 2 µM bleomycin for 0, 3, 5 and 7 days. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods . The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results ( P values) are shown.

    Techniques Used: Cell Culture, Activity Assay, Staining

    BCAAs upregulate p21 protein level mediated through the mTORC1 pathway. (A) HepG2 cells cultured in RPMI medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 1 or 2 days. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. (B) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 2 days. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated.
    Figure Legend Snippet: BCAAs upregulate p21 protein level mediated through the mTORC1 pathway. (A) HepG2 cells cultured in RPMI medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 1 or 2 days. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. (B) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 2 days. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated.

    Techniques Used: Cell Culture, SDS Page

    Cells cultured in BCAA_3 medium have higher activities of mTOR and higher protein levels of p21. (A) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide for 48 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. The intensities of the bands corresponding to phosphorylated S6K at Thr389 and S6K were quantified by ImageJ, and the ratio of the phosphorylated S6K at Thr389 to S6K was shown as mTORC1 activities. (B) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide for 48 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. The intensities of the bands corresponding to p21 and α-tubulin were quantified by ImageJ, and the ratio of p21 to α-tubulin was shown. (C) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 48 hours. The mRNA expressions of p21 and GAPDH were examined by RT-PCR using specific primers against p21 and GAPDH . The intensities of the bands corresponding to p21 and GAPDH were quantified by ImageJ, and the ratio of p21 to GAPDH was shown.
    Figure Legend Snippet: Cells cultured in BCAA_3 medium have higher activities of mTOR and higher protein levels of p21. (A) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide for 48 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. The intensities of the bands corresponding to phosphorylated S6K at Thr389 and S6K were quantified by ImageJ, and the ratio of the phosphorylated S6K at Thr389 to S6K was shown as mTORC1 activities. (B) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide for 48 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated. The intensities of the bands corresponding to p21 and α-tubulin were quantified by ImageJ, and the ratio of p21 to α-tubulin was shown. (C) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 48 hours. The mRNA expressions of p21 and GAPDH were examined by RT-PCR using specific primers against p21 and GAPDH . The intensities of the bands corresponding to p21 and GAPDH were quantified by ImageJ, and the ratio of p21 to GAPDH was shown.

    Techniques Used: Cell Culture, SDS Page, Reverse Transcription Polymerase Chain Reaction

    Cells cultured in BCAA_3 medium have higher activities to induce premature senescence. (A) HepG2 cells cultured in BCAA_1, 3, 5 and BCAA_5 with 100 nM rapamycin were treated with 10 µM etoposide for 2 days, and observed with microscope after SA-β-Gal staining assay. (B, C) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide (B) or 2 µM bleomycin (C) for 2 days. (D) U2OS cells cultured in RPMI-based medium with or without 100 nM rapamycin as indicated were treated with 2 µM etoposide for 7 days. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods . The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results ( P values) are shown.
    Figure Legend Snippet: Cells cultured in BCAA_3 medium have higher activities to induce premature senescence. (A) HepG2 cells cultured in BCAA_1, 3, 5 and BCAA_5 with 100 nM rapamycin were treated with 10 µM etoposide for 2 days, and observed with microscope after SA-β-Gal staining assay. (B, C) HepG2 cells cultured in BCAA medium with or without 100 nM rapamycin as indicated were treated with 10 µM etoposide (B) or 2 µM bleomycin (C) for 2 days. (D) U2OS cells cultured in RPMI-based medium with or without 100 nM rapamycin as indicated were treated with 2 µM etoposide for 7 days. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods . The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results ( P values) are shown.

    Techniques Used: Cell Culture, Microscopy, Staining, Activity Assay

    BCAAs enhance the execution of premature senescence induced by DNA damage-inducing drugs. (A) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 48 hours, and observed with microscope after SA-β-Gal staining assay. (B) HepG2 cells were cultured in BCAA as described in A. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods . The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results ( P values) are shown. (C) U2OS cells cultured in BCAA medium were treated with or without 2 µM etoposide and 100 nM rapamycin as indicated for 7 days, and observed with microscope after SA-β-Gal staining assay. (D) U2OS cells were cultured in BCAA medium as described in C. The assay of SA-β-Gal activity was carried out as described in B. (E) U2OS cells cultured in BCAA medium were treated with or without 100 nM rapamycin as indicated for 24 hours and cells were harvested at each time point. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated.
    Figure Legend Snippet: BCAAs enhance the execution of premature senescence induced by DNA damage-inducing drugs. (A) HepG2 cells cultured in BCAA medium were treated with or without 10 µM etoposide and 100 nM rapamycin as indicated for 48 hours, and observed with microscope after SA-β-Gal staining assay. (B) HepG2 cells were cultured in BCAA as described in A. For the assay of SA-β-Gal activity, cells stained with blue color were counted as described in Materials and Methods . The data (mean ± S.D.) were obtained from at least three independent experiments. Significant test results ( P values) are shown. (C) U2OS cells cultured in BCAA medium were treated with or without 2 µM etoposide and 100 nM rapamycin as indicated for 7 days, and observed with microscope after SA-β-Gal staining assay. (D) U2OS cells were cultured in BCAA medium as described in C. The assay of SA-β-Gal activity was carried out as described in B. (E) U2OS cells cultured in BCAA medium were treated with or without 100 nM rapamycin as indicated for 24 hours and cells were harvested at each time point. Cell lysates were subjected to SDS-PAGE and immunoblotted with the antibodies as indicated.

    Techniques Used: Cell Culture, Microscopy, Staining, Activity Assay, SDS Page

    40) Product Images from "The canonical NF-?B pathway differentially protects normal and human tumor cells from ROS-induced DNA damage"

    Article Title: The canonical NF-?B pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2012.06.010

    p53 knock-down delays the premature senescence of control and H 2 O 2 -treated MRC-5 and MRC-5 canonical NF-κB compromised cells. (A) Two days post infection with a high titer RS-Puro-p53 KD retrovirus, cells were analyzed for p53 and β-actin expression by immunoblotting. (B) Untreated and H 2 O 2 -treated MRC-5 carrying an empty vector or IκBαSR and their corresponding p53 KD cells were stained for SA-β-Gal expression. Cells were allowed to recover for 4 days post-H 2 O 2 treatment. Images shown are representative of three independent experiments. (C) Comparisons of the percentages of SA-β-Gal positive cells quantified from 3 independent experiments. Results were analyzed by paired two-way ANOVA, followed by Tukey post-hoc comparisons with all values presented as mean ± SEM. Symbols above specific connected bars represent the following statistically significant differences: *p
    Figure Legend Snippet: p53 knock-down delays the premature senescence of control and H 2 O 2 -treated MRC-5 and MRC-5 canonical NF-κB compromised cells. (A) Two days post infection with a high titer RS-Puro-p53 KD retrovirus, cells were analyzed for p53 and β-actin expression by immunoblotting. (B) Untreated and H 2 O 2 -treated MRC-5 carrying an empty vector or IκBαSR and their corresponding p53 KD cells were stained for SA-β-Gal expression. Cells were allowed to recover for 4 days post-H 2 O 2 treatment. Images shown are representative of three independent experiments. (C) Comparisons of the percentages of SA-β-Gal positive cells quantified from 3 independent experiments. Results were analyzed by paired two-way ANOVA, followed by Tukey post-hoc comparisons with all values presented as mean ± SEM. Symbols above specific connected bars represent the following statistically significant differences: *p

    Techniques Used: Infection, Expressing, Plasmid Preparation, Staining

    Suppressing canonical NF-κB signaling accelerates the senescence response of MRC-5 HDFs in response to H 2 O 2 treatment. SA-β-Gal staining was performed on the indicated proliferating cell populations with and without prior exposure to increasing H 2 O 2 doses as described in Materials and Methods. Representative examples of SA-β-Gal staining observed in 3 independent experiments are shown in Panel A-C. (A) MRC-5 vector and MRC-5 IκBαSR cells, (B) MRC-5 GL2 and IKKβ KD cells. (C) MRC-5 vector and IκBαSR cells with and without exposure to H 2 O 2 as in Panel A but in conjunction with NAC to suppress ROS accumulation. (D) Comparisons of the percentages of SA-β-Gal positive cells in different populations of MRC-5 cells quantified from 3 independent experiments. Black bars in the left and right sides of Panel D show the percentages of SA-β-Gal stained MRC-5 vector control, MRC-5 IκBαSR and MRC-5 GL2 cells with and without prior H 2 O 2 treatment. Gray bars on the left side of Panel D quantify the effects of NAC on MRC-5 vector control and IκBαSR cells with and without H 2 O 2 exposure; and the gray bars in the right side of panel D show the percentages of senescent MRC-5 IKKβ KD cells with and without prior H 2 O 2 exposure. Symbols directly above individual bars refer to statistical comparisons between that specific bar’s H 2 O 2 dose and the same cells without H 2 O 2 exposure. All data were analyzed by paired two-way ANOVA, followed by Tukey post-hoc comparisons and are presented as means ± SEM (*p
    Figure Legend Snippet: Suppressing canonical NF-κB signaling accelerates the senescence response of MRC-5 HDFs in response to H 2 O 2 treatment. SA-β-Gal staining was performed on the indicated proliferating cell populations with and without prior exposure to increasing H 2 O 2 doses as described in Materials and Methods. Representative examples of SA-β-Gal staining observed in 3 independent experiments are shown in Panel A-C. (A) MRC-5 vector and MRC-5 IκBαSR cells, (B) MRC-5 GL2 and IKKβ KD cells. (C) MRC-5 vector and IκBαSR cells with and without exposure to H 2 O 2 as in Panel A but in conjunction with NAC to suppress ROS accumulation. (D) Comparisons of the percentages of SA-β-Gal positive cells in different populations of MRC-5 cells quantified from 3 independent experiments. Black bars in the left and right sides of Panel D show the percentages of SA-β-Gal stained MRC-5 vector control, MRC-5 IκBαSR and MRC-5 GL2 cells with and without prior H 2 O 2 treatment. Gray bars on the left side of Panel D quantify the effects of NAC on MRC-5 vector control and IκBαSR cells with and without H 2 O 2 exposure; and the gray bars in the right side of panel D show the percentages of senescent MRC-5 IKKβ KD cells with and without prior H 2 O 2 exposure. Symbols directly above individual bars refer to statistical comparisons between that specific bar’s H 2 O 2 dose and the same cells without H 2 O 2 exposure. All data were analyzed by paired two-way ANOVA, followed by Tukey post-hoc comparisons and are presented as means ± SEM (*p

    Techniques Used: Staining, Plasmid Preparation

    Related Articles

    Irradiation:

    Article Title: A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination
    Article Snippet: .. We irradiated (5 Gy) siRNA-transfected U2OS cells and incubated them in medium containing 100 ng ml−1 nocodazole (Sigma) for 24 h. We fixed cells in 4% (v/v) formaldehyde and permeabilized them in cold (−20 °C) 90% (v/v) methanol. .. We seeded siRNA-U2OS cells (3 × 103 per 200 μl) into octuplicate microtiter wells, incubated them overnight, and then treated them with CPT for 48 h. We measured viability as described , .

    Transfection:

    Article Title: Increased activity of chondrocyte translational apparatus accompanies osteoarthritis
    Article Snippet: .. Transfections with mTOR siRNA (nt2381; Sigma) were performed using Lipofectamine RNAiMax according to the manufacturer's protocol. .. MISSION siRNA Universal Negative Control was used as a control.

    Article Title: Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression
    Article Snippet: .. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously ( ). .. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich.

    Article Title: Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development
    Article Snippet: .. Having defined an important role for zebrafish Wdr43 in ribosome biogenesis, we further examined its function in cultured human HeLa cells, which we transfected with human WDR43 small interfering RNA (siRNA) (Sigma MISSION esiRNA) to silence WDR43 expression. .. GFP esiRNA was used as a negative control for these studies.

    Incubation:

    Article Title: A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination
    Article Snippet: .. We irradiated (5 Gy) siRNA-transfected U2OS cells and incubated them in medium containing 100 ng ml−1 nocodazole (Sigma) for 24 h. We fixed cells in 4% (v/v) formaldehyde and permeabilized them in cold (−20 °C) 90% (v/v) methanol. .. We seeded siRNA-U2OS cells (3 × 103 per 200 μl) into octuplicate microtiter wells, incubated them overnight, and then treated them with CPT for 48 h. We measured viability as described , .

    Article Title: Delivery of siRNA in vitro and in vivo using PEI-capped porous silicon nanoparticles to silence MRP1 and inhibit proliferation in glioblastoma
    Article Snippet: .. To cap the siRNA loaded pSiNPs with PEI, the pellet was then resuspended in 0.05% PEI of 25 kDa (Sigma-Aldrich, 408727) diluted in EtOH and incubated for 20 min. pSiNPs were centrifuged, and the pellet was washed with EtOH for three times to wash off excess PEI. .. To measure the loading efficiency of siRNA into pSiNPs, siRNA concentration in the supernatant was measured at 260 nm by using a Nanodrop spectrophotometer (ND2000, ThermoFisher) and the amount of siRNA was then back calculated.

    esiRNA:

    Article Title: Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development
    Article Snippet: .. Having defined an important role for zebrafish Wdr43 in ribosome biogenesis, we further examined its function in cultured human HeLa cells, which we transfected with human WDR43 small interfering RNA (siRNA) (Sigma MISSION esiRNA) to silence WDR43 expression. .. GFP esiRNA was used as a negative control for these studies.

    Negative Control:

    Article Title: Fidgetin-like 2: a microtubule-based regulator of wound healing
    Article Snippet: .. The hydrolyzed TMOS (100 µl) was added to 900 µl of 20 µM of siRNA (mouse FL2 (Sigma-Aldrich, SASI_Mm02_00354635) or the negative control) solution containing 10 mM phosphate, pH 7.4. ..

    Cell Culture:

    Article Title: Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development
    Article Snippet: .. Having defined an important role for zebrafish Wdr43 in ribosome biogenesis, we further examined its function in cultured human HeLa cells, which we transfected with human WDR43 small interfering RNA (siRNA) (Sigma MISSION esiRNA) to silence WDR43 expression. .. GFP esiRNA was used as a negative control for these studies.

    Ubiquitin Assay:

    Article Title: Rescue of ?F508-CFTR by the SGK1/Nedd4-2 Signaling Pathway *
    Article Snippet: .. For the CFTR ubiquitination assay, drug- or siRNA-treated cells were lysed in radioimmune precipitation assay buffer supplemented with 10 μ m MG132, a proteasome inhibitor (Sigma-Aldrich). .. After immunoprecipitation with anti-CFTR C-terminal antibody, the immunoprecipitated proteins were eluted, separated on 6% SDS-PAGE gels, and transferred to polyvinylidene difluoride membranes.

    Small Interfering RNA:

    Article Title: Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development
    Article Snippet: .. Having defined an important role for zebrafish Wdr43 in ribosome biogenesis, we further examined its function in cultured human HeLa cells, which we transfected with human WDR43 small interfering RNA (siRNA) (Sigma MISSION esiRNA) to silence WDR43 expression. .. GFP esiRNA was used as a negative control for these studies.

    Quantitative RT-PCR:

    Article Title: Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression
    Article Snippet: .. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously ( ). .. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich.

    Expressing:

    Article Title: Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development
    Article Snippet: .. Having defined an important role for zebrafish Wdr43 in ribosome biogenesis, we further examined its function in cultured human HeLa cells, which we transfected with human WDR43 small interfering RNA (siRNA) (Sigma MISSION esiRNA) to silence WDR43 expression. .. GFP esiRNA was used as a negative control for these studies.

    Western Blot:

    Article Title: Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression
    Article Snippet: .. Cancer cell lines were transfected with scrambled-siRNA or METTL14-siRNA/ALKBH5-siRNA (Sigma-Aldrich) for 48 or 72 hours before they were subjected to qRT-PCR or Western blot analysis, as described previously ( ). .. Antibodies against ALKBH5 (#HPA 007196), β-actin (#A3854), GAPDH (#G9295), and METTL14 (#HPA038002) were purchased from Sigma-Aldrich.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Millipore microglial sa β gal activity
    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase <t>(SA-β-gal)</t> activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p
    Microglial Sa β Gal Activity, supplied by Millipore, 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/microglial sa β gal activity/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    microglial sa β gal activity - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    99
    Millipore senescence associated β galactosidase
    MEK inhibition causes a decrease in thymidine uptake in SKMEL-28 (V600E BRAF) cells. A, SKMEL-28 and BT-474 cells were treated with PD0325901 for 48 h and thymidine uptake was measured. Thymidine uptake was inhibited by > 90% in SKMEL-28 (V600E BRAF) cells but minimally affected by PD0325901 treatment in BT-474 cells. B, on washout of drug, thymidine uptake was restored to pretreatment levels in SKMEL-28 cells. C and D, treatment of SKMEL-28 cells with PD0325901 (25 nmol/L) resulted in an increase in the fraction of cells staining positive for <t>SA-β-Gal.</t> The photomicrograph in D shows representative fields from control and PD0325901-treated cells at the 48-h time point.
    Senescence Associated β Galactosidase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/senescence associated β galactosidase/product/Millipore
    Average 99 stars, based on 76 article reviews
    Price from $9.99 to $1999.99
    senescence associated β galactosidase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    91
    Millipore sa β gal activity
    A high concentration of FCS induces mOPC senescence. ( A ) Morphology of mOPCs cultured in low FCS, high FCS, and reversion medium. (Scale bar, 10 μm.) ( B – D ) Data from cells cultured as in A . ( B ) <t>SA-β-gal</t> staining of mOPCs. (Scale
    Sa β Gal Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sa β gal activity/product/Millipore
    Average 91 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    sa β gal activity - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    Image Search Results


    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Microglia change from a reactive to an age-like phenotype with the time in culture

    doi: 10.3389/fncel.2014.00152

    Figure Lengend Snippet: Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

    Article Snippet: Microglial SA-β-gal activity was determined using the Cellular senescence assay kit (Millipore), according to the manufacturer instructions.

    Techniques: Activity Assay, Expressing, In Vitro, Staining, Real-time Polymerase Chain Reaction

    MEK inhibition causes a decrease in thymidine uptake in SKMEL-28 (V600E BRAF) cells. A, SKMEL-28 and BT-474 cells were treated with PD0325901 for 48 h and thymidine uptake was measured. Thymidine uptake was inhibited by > 90% in SKMEL-28 (V600E BRAF) cells but minimally affected by PD0325901 treatment in BT-474 cells. B, on washout of drug, thymidine uptake was restored to pretreatment levels in SKMEL-28 cells. C and D, treatment of SKMEL-28 cells with PD0325901 (25 nmol/L) resulted in an increase in the fraction of cells staining positive for SA-β-Gal. The photomicrograph in D shows representative fields from control and PD0325901-treated cells at the 48-h time point.

    Journal: Cancer research

    Article Title: 3?-Deoxy-3?-[18F]Fluorothymidine Positron Emission Tomography Is a Sensitive Method for Imaging the Response of BRAF-Dependent Tumors to MEK Inhibition

    doi: 10.1158/0008-5472.CAN-07-2976

    Figure Lengend Snippet: MEK inhibition causes a decrease in thymidine uptake in SKMEL-28 (V600E BRAF) cells. A, SKMEL-28 and BT-474 cells were treated with PD0325901 for 48 h and thymidine uptake was measured. Thymidine uptake was inhibited by > 90% in SKMEL-28 (V600E BRAF) cells but minimally affected by PD0325901 treatment in BT-474 cells. B, on washout of drug, thymidine uptake was restored to pretreatment levels in SKMEL-28 cells. C and D, treatment of SKMEL-28 cells with PD0325901 (25 nmol/L) resulted in an increase in the fraction of cells staining positive for SA-β-Gal. The photomicrograph in D shows representative fields from control and PD0325901-treated cells at the 48-h time point.

    Article Snippet: To determine the percentage of senescent cells, cells were treated with PD0325901 for the durations specified and then fixed with formaldehyde solution and then assayed for senescence-associated β-galactosidase (SA-β-Gal) activity using the Senescence Detection kit (Calbiochem) according to the manufacturer’s instructions.

    Techniques: Inhibition, Staining

    FL118 induces p53/p21-dependent senescence and p53/p21-independent apoptosis. A, FL118 effects on SA-β-gal positivity. HCT8 cells without treatment or treated with 10 nmol/L FL118 for 3 days followed by another 10-day culture in drug-free medium

    Journal: Cancer research

    Article Title: FL118 Induces p53-Dependent Senescence in Colorectal Cancer Cells by Promoting Degradation of MdmX

    doi: 10.1158/0008-5472.CAN-14-0683

    Figure Lengend Snippet: FL118 induces p53/p21-dependent senescence and p53/p21-independent apoptosis. A, FL118 effects on SA-β-gal positivity. HCT8 cells without treatment or treated with 10 nmol/L FL118 for 3 days followed by another 10-day culture in drug-free medium

    Article Snippet: Then the cells were fixed and stained for senescence-associated β-galactosidase (SA-β-gal) activity with a commercial kit performed according to manufacturer’s instructions (Calbiochem; catalog no. QIA117).

    Techniques:

    A high concentration of FCS induces mOPC senescence. ( A ) Morphology of mOPCs cultured in low FCS, high FCS, and reversion medium. (Scale bar, 10 μm.) ( B – D ) Data from cells cultured as in A . ( B ) SA-β-gal staining of mOPCs. (Scale

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

    Article Title: Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells

    doi: 10.1073/pnas.0911446107

    Figure Lengend Snippet: A high concentration of FCS induces mOPC senescence. ( A ) Morphology of mOPCs cultured in low FCS, high FCS, and reversion medium. (Scale bar, 10 μm.) ( B – D ) Data from cells cultured as in A . ( B ) SA-β-gal staining of mOPCs. (Scale

    Article Snippet: The SA-β-gal activity in cultured cells and brain sections was determined using a Senescence Detection Kit (Calbiochem) following the manufacturer's instructions.

    Techniques: Concentration Assay, Cell Culture, Staining