Structured Review

Millipore ck2 is
<t>CK2</t> 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
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Images

1) 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

2) Product Images from "Molecular Understanding of Growth Inhibitory Effect from Irradiated to Bystander Tumor Cells in Mouse Fibrosarcoma Tumor Model"

Article Title: Molecular Understanding of Growth Inhibitory Effect from Irradiated to Bystander Tumor Cells in Mouse Fibrosarcoma Tumor Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0161662

Apoptosis and senescence in tumor sections from control and mixed cells. (A) Sections of tumor tissues were subjected to TUNEL assay. Blue colour shows nuclei of cells stained with DAPI. Green foci show TUNEL positive regions. (B) Tumor sections processed for SA-β-gal assay. The part of image is shown below in the respective inset. Greenish blue color indicates active SA-β-galactosidase.
Figure Legend Snippet: Apoptosis and senescence in tumor sections from control and mixed cells. (A) Sections of tumor tissues were subjected to TUNEL assay. Blue colour shows nuclei of cells stained with DAPI. Green foci show TUNEL positive regions. (B) Tumor sections processed for SA-β-gal assay. The part of image is shown below in the respective inset. Greenish blue color indicates active SA-β-galactosidase.

Techniques Used: TUNEL Assay, Staining, β-Gal Assay

3) Product Images from "Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2"

Article Title: Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2016.2694

Nuclear factor-erythroid 2-related factor 2 (Nrf2) activation mediated by epigallocatechin-3-gallate (EGCG) pre-treatment suppresses H 2 O 2 -induced senescence and the expression of acetyl-p53 and p21 in human mesenchymal stem cells (hMSCs). (A) Senescence-associated β-galactosidase (SAβ-gal) staining analysis of control, H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed/Nrf2-siRNA-transfected and EGCG-pre-treated/H 2 O 2 -treated/control-siRNA-transfected cells. hMSCs were transiently transfected for 48 h with either Nrf2 or control siRNA and treated with 100 μ M EGCG for 6 h followed by H 2 O 2 exposure (200 μ M, 2 h). Twenty-four hours after H 2 O 2 exposure, the cells were subjected to SAβ-gal staining (blue cytoplasmic stain). Scale bar, 200 μ m. (B) Quantification of SAβ-gal activity. (C) Western blot analysis and quantification of Nrf2 at 48 h after Nrf2 siRNA or control siRNA transfection. (D–F) Western blot analysis and quantification of acetyl-p53 and p21 protein levels in each group. The levels determined in four independent experiments are presented as the means ± SEM. * P
Figure Legend Snippet: Nuclear factor-erythroid 2-related factor 2 (Nrf2) activation mediated by epigallocatechin-3-gallate (EGCG) pre-treatment suppresses H 2 O 2 -induced senescence and the expression of acetyl-p53 and p21 in human mesenchymal stem cells (hMSCs). (A) Senescence-associated β-galactosidase (SAβ-gal) staining analysis of control, H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed, EGCG-pre-treated/H 2 O 2 -exposed/Nrf2-siRNA-transfected and EGCG-pre-treated/H 2 O 2 -treated/control-siRNA-transfected cells. hMSCs were transiently transfected for 48 h with either Nrf2 or control siRNA and treated with 100 μ M EGCG for 6 h followed by H 2 O 2 exposure (200 μ M, 2 h). Twenty-four hours after H 2 O 2 exposure, the cells were subjected to SAβ-gal staining (blue cytoplasmic stain). Scale bar, 200 μ m. (B) Quantification of SAβ-gal activity. (C) Western blot analysis and quantification of Nrf2 at 48 h after Nrf2 siRNA or control siRNA transfection. (D–F) Western blot analysis and quantification of acetyl-p53 and p21 protein levels in each group. The levels determined in four independent experiments are presented as the means ± SEM. * P

Techniques Used: Activation Assay, Expressing, Staining, Transfection, Activity Assay, Western Blot

Epigallocatechin-3-gallate (EGCG) pre-treatment reduces cellular senescence in H 2 O 2 -treated human mesenchymal stem cells (hMSCs). (A–D) Senescence-associated β-galactosidase (SAβ-gal) staining of control (Con) and hMSCs before and after H 2 O 2 exposure. hMSCs were treated with 50 or 100 μ M of EGCG for 6 h and then exposed to H 2 O 2 (200 μ M) for 2 h. Twenty-four hours after H 2 O 2 exposure, the cells were stained with SAβ-gal (blue cytoplasmic stain). Scale bar, 200 μ m. (E) Quantification of SAβ-gal activity. (F) Cell viability of hMSCs. MTT assays were performed 24 h after H 2 O 2 exposure. Changes in cell survival observed in three independent experiments are presented as the means ± SEM. * P
Figure Legend Snippet: Epigallocatechin-3-gallate (EGCG) pre-treatment reduces cellular senescence in H 2 O 2 -treated human mesenchymal stem cells (hMSCs). (A–D) Senescence-associated β-galactosidase (SAβ-gal) staining of control (Con) and hMSCs before and after H 2 O 2 exposure. hMSCs were treated with 50 or 100 μ M of EGCG for 6 h and then exposed to H 2 O 2 (200 μ M) for 2 h. Twenty-four hours after H 2 O 2 exposure, the cells were stained with SAβ-gal (blue cytoplasmic stain). Scale bar, 200 μ m. (E) Quantification of SAβ-gal activity. (F) Cell viability of hMSCs. MTT assays were performed 24 h after H 2 O 2 exposure. Changes in cell survival observed in three independent experiments are presented as the means ± SEM. * P

Techniques Used: Staining, Activity Assay, MTT Assay

4) Product Images from "Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line"

Article Title: Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-017-0172-3

The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or ( c ) cytokines (50 ng/ml) during 5 days and stained for SA-β-GAL. Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or ( e ) cytokines, as indicated. The values were normalized to GADPH and relative to control cells ( dotted lines ). Error bars represent SEM. (* p
Figure Legend Snippet: The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or ( c ) cytokines (50 ng/ml) during 5 days and stained for SA-β-GAL. Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or ( e ) cytokines, as indicated. The values were normalized to GADPH and relative to control cells ( dotted lines ). Error bars represent SEM. (* p

Techniques Used: Staining, Expressing

5) Product Images from "Preventive Effects of Epigallocatechin-3-O-Gallate against Replicative Senescence Associated with p53 Acetylation in Human Dermal Fibroblasts"

Article Title: Preventive Effects of Epigallocatechin-3-O-Gallate against Replicative Senescence Associated with p53 Acetylation in Human Dermal Fibroblasts

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2012/850684

SABG expression (a) of serially passaged HDFs in the absence or presence of 50 and 100 μ M EGCG. The quantitative result (b) showed that the HDFs at early passage (PN 5) that were not treated with EGCG did not stain blue, but the cells at late passage (PN 30 or ≥40) showed much blue staining of senescent cells. Serial passage-induced senescence in HDFs was partly prevented at 50 μ M EGCG, while 100 μ M EGCG treatment significantly reduced the number of senescent cells. All variables were tested in three independent cultures for each experiment, which was repeated twice independently ( n = 6). The results are reported as a mean ± SD and analyzed by a Tukey HSD test. * P
Figure Legend Snippet: SABG expression (a) of serially passaged HDFs in the absence or presence of 50 and 100 μ M EGCG. The quantitative result (b) showed that the HDFs at early passage (PN 5) that were not treated with EGCG did not stain blue, but the cells at late passage (PN 30 or ≥40) showed much blue staining of senescent cells. Serial passage-induced senescence in HDFs was partly prevented at 50 μ M EGCG, while 100 μ M EGCG treatment significantly reduced the number of senescent cells. All variables were tested in three independent cultures for each experiment, which was repeated twice independently ( n = 6). The results are reported as a mean ± SD and analyzed by a Tukey HSD test. * P

Techniques Used: Expressing, Staining

SABG expression of serially passaged RVSMCs (a) and HACs (b) in the absence or presence of 50 μ M EGCG. The quantitative results showed that the RVSMCs (c) and HACs (d) at early passage (PN 3 or 5) that were not treated with EGCG did not stain blue or only a few stained, but the cells at late passage (PN 20) showed much blue staining of senescent cells. Furthermore, both cells treated with EGCG showed the number of senescent cells comparable to those with 5 PN. All variables were tested in three independent cultures for each experiment, which was repeated twice independently ( n = 6). The results are reported as a mean ± SD and analyzed by a Tukey HSD test. * P
Figure Legend Snippet: SABG expression of serially passaged RVSMCs (a) and HACs (b) in the absence or presence of 50 μ M EGCG. The quantitative results showed that the RVSMCs (c) and HACs (d) at early passage (PN 3 or 5) that were not treated with EGCG did not stain blue or only a few stained, but the cells at late passage (PN 20) showed much blue staining of senescent cells. Furthermore, both cells treated with EGCG showed the number of senescent cells comparable to those with 5 PN. All variables were tested in three independent cultures for each experiment, which was repeated twice independently ( n = 6). The results are reported as a mean ± SD and analyzed by a Tukey HSD test. * P

Techniques Used: Expressing, Staining

6) Product Images from "Translational control of PML contributes to TNFα-induced apoptosis of MCF7 breast cancer cells and decreased angiogenesis in HUVECs"

Article Title: Translational control of PML contributes to TNFα-induced apoptosis of MCF7 breast cancer cells and decreased angiogenesis in HUVECs

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2015.114

Identification of  PML  5′-UTR (-100- > -1) as an IRES activated by the TNF α– MNK1 axis. ( a ) A schematic representation of bi-cistronic pRF plasmids used in the transient transfection reporter assays. ( b ) The effects of  PML  5′-UTR (-100- > -1) on reporter activity. The relative ratio of Firefly luciferase/ Renilla  luciferase intensity is plotted. The results are mean±S.D. in triplicates ( n =3). ( c ) Determination of possible alternative splicing of the transcripts of  PML  5′-UTR (-100- > -1)-pRF. The cDNAs prepared from pRF or  PML  5′-UTR (-100- > -1)-pRF transfected HUVECs subjected to PCR using primer pairs as indicated in ( a ). The final PCR products were separated on a 0.8% agarose gel, stained by EtBr and images recorded. ( d ) Knockdown of  RLuc  in bicistronic system reduces FLuc driven by  EMCV  IRES and  PML  5′-UTR (-100- > -1) but not by  PML  5′-UTR (-140- > -1). ( e ) The reporter activity of  PML  5′-UTR (-100- > -1) is not derived from ribosomal read-through. HeLa cells were transiently transfected with engineered pRF and  β -gal plasmids as indicated. At 48 h post transfection, the Firefly luciferase and  Renilla  luciferase activity were measured and normalized to the transfection control,  β -gal. The luciferase activity of  PML  5′-UTR (-100- > -1)-pRF was set as 1. The results are mean ±S.D. in triplicates. ( f )  EMCV  IRES and  PML  5′-UTR (-100- > -1) harbors IRES activity as evidenced by transfection of  in vitro  transcribed mRNAs into HeLa cells. The relative activity of RLuc and FLuc was normalized to the corresponding mRNA levels 9 h post transfection. ( g ) TNF α  enhances  PML  5′-UTR (-100- > -1) activity in a MNK1-dependent manner. sh Ctrl  and sh MNK1  knockdown HeLa cells were transiently transfected with  PML  5′-UTR (-100- > -1)-pRF plasmid for 72 h, treated with TNF α  at the indicated times and cells harvested. The relative activity of Firefly luciferase over  Renilla  luciferase was plotted. The results are mean ±S.D. in triplicates ( n =3). ( h  and  i ) Ectopic expression of eIF4E inhibitor, 4E-BP or a mutant defective in phosphorylation by mTORC1, 4E-BP (5A), does not compromise TNF α -mediated PML protein accumulation or  PML  IRES activation. HeLa cells were transiently transfected with  PML  5′-UTR (-100- > -1)-pRF plasmid with the indicated plasmids for 48 h, treated with TNF α  for 20 h and cells harvested. ( h ) Endogenous PML protein in pEBG, pEBG-4E-BP and pEBG-4E-BP (5A) transfected cells were determined by western blottings. The relative PML protein expression was normalized by  β -actin levels and (PML)/( β -actin) in the absence of TNF α  was set as 1. The relative fold induction of PML protein abundance by TNF α  is shown in each sample. Both long (L.E.) and short (S.E.) exposures of PML western blot are shown. ( i ) The relative effect of TNF α  on (FLuc)/(RLuc) of  PML  5′-UTR (-100- > -1) reporter in transfected samples is plotted. The results are shown as mean±S.D. ( n =4)
Figure Legend Snippet: Identification of PML 5′-UTR (-100- > -1) as an IRES activated by the TNF α– MNK1 axis. ( a ) A schematic representation of bi-cistronic pRF plasmids used in the transient transfection reporter assays. ( b ) The effects of PML 5′-UTR (-100- > -1) on reporter activity. The relative ratio of Firefly luciferase/ Renilla luciferase intensity is plotted. The results are mean±S.D. in triplicates ( n =3). ( c ) Determination of possible alternative splicing of the transcripts of PML 5′-UTR (-100- > -1)-pRF. The cDNAs prepared from pRF or PML 5′-UTR (-100- > -1)-pRF transfected HUVECs subjected to PCR using primer pairs as indicated in ( a ). The final PCR products were separated on a 0.8% agarose gel, stained by EtBr and images recorded. ( d ) Knockdown of RLuc in bicistronic system reduces FLuc driven by EMCV IRES and PML 5′-UTR (-100- > -1) but not by PML 5′-UTR (-140- > -1). ( e ) The reporter activity of PML 5′-UTR (-100- > -1) is not derived from ribosomal read-through. HeLa cells were transiently transfected with engineered pRF and β -gal plasmids as indicated. At 48 h post transfection, the Firefly luciferase and Renilla luciferase activity were measured and normalized to the transfection control, β -gal. The luciferase activity of PML 5′-UTR (-100- > -1)-pRF was set as 1. The results are mean ±S.D. in triplicates. ( f ) EMCV IRES and PML 5′-UTR (-100- > -1) harbors IRES activity as evidenced by transfection of in vitro transcribed mRNAs into HeLa cells. The relative activity of RLuc and FLuc was normalized to the corresponding mRNA levels 9 h post transfection. ( g ) TNF α enhances PML 5′-UTR (-100- > -1) activity in a MNK1-dependent manner. sh Ctrl and sh MNK1 knockdown HeLa cells were transiently transfected with PML 5′-UTR (-100- > -1)-pRF plasmid for 72 h, treated with TNF α at the indicated times and cells harvested. The relative activity of Firefly luciferase over Renilla luciferase was plotted. The results are mean ±S.D. in triplicates ( n =3). ( h and i ) Ectopic expression of eIF4E inhibitor, 4E-BP or a mutant defective in phosphorylation by mTORC1, 4E-BP (5A), does not compromise TNF α -mediated PML protein accumulation or PML IRES activation. HeLa cells were transiently transfected with PML 5′-UTR (-100- > -1)-pRF plasmid with the indicated plasmids for 48 h, treated with TNF α for 20 h and cells harvested. ( h ) Endogenous PML protein in pEBG, pEBG-4E-BP and pEBG-4E-BP (5A) transfected cells were determined by western blottings. The relative PML protein expression was normalized by β -actin levels and (PML)/( β -actin) in the absence of TNF α was set as 1. The relative fold induction of PML protein abundance by TNF α is shown in each sample. Both long (L.E.) and short (S.E.) exposures of PML western blot are shown. ( i ) The relative effect of TNF α on (FLuc)/(RLuc) of PML 5′-UTR (-100- > -1) reporter in transfected samples is plotted. The results are shown as mean±S.D. ( n =4)

Techniques Used: Transfection, Activity Assay, Luciferase, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Derivative Assay, In Vitro, Plasmid Preparation, Expressing, Mutagenesis, Activation Assay, Western Blot

7) 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

8) 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

9) 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

10) 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

11) 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

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

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

13) 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

14) 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

15) Product Images from "Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors"

Article Title: Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors

Journal: Stem Cell Research & Therapy

doi: 10.1186/scrt452

β-galactosidase (β-gal) senescence evaluation of hASC-M, −T, −TS and -TE cells. Representative β-gal staining of cells. The percentage of senescent cells were calculated by analyzing eight different fields for each cell line. The histogram represents the mean value ± SD of four different experiments ** P
Figure Legend Snippet: β-galactosidase (β-gal) senescence evaluation of hASC-M, −T, −TS and -TE cells. Representative β-gal staining of cells. The percentage of senescent cells were calculated by analyzing eight different fields for each cell line. The histogram represents the mean value ± SD of four different experiments ** P

Techniques Used: Staining

16) 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

17) 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:

18) 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

19) 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

20) 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

21) 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

22) Product Images from "Polycomb Repressor Complex 1 Member, BMI1 Contributes to Urothelial Tumorigenesis through p16-Independent Mechanisms"

Article Title: Polycomb Repressor Complex 1 Member, BMI1 Contributes to Urothelial Tumorigenesis through p16-Independent Mechanisms

Journal: Translational Oncology

doi: 10.1016/j.tranon.2015.08.002

(A) Overexpression of BMI1 led to a bypass of senescence in NHUC. Filled bars show senescence-associated β-galactosidase expression in empty vector-transduced NHUC; and unfilled bars, BMI1-transduced cells. (B) Ectopic expression of BMI1 led to exponential growth of the cell population and apparent immortalization in cells from three independent donors. Filled symbol shows vector-transduced cells; and unfilled symbols, NHUC-BMI1. (C) Detection of telomerase activity in NHUC-BMI1 using quantitative real-time PCR-based method. HI represents heat-inactivated sample, No Taq is a no polymerase negative control, and positive control is a lysate of the control pellet from the TRAPEZE XL kit. (D) Overexpression of BMI1 repressed induction of differentiation in NHUC. Induction of uroplakin 2 (UPK2), a marker of urothelial differentiation, was repressed in NHUC-BMI1 compared with vector control cells after treatment with trogiltazone and EGFR inhibitor. Expression is normalized to NHUC-vector control treatment cells. (E) Quantitative real-time PCR confirmed that overexpression of BMI1 did not repress CDKN2A transcription in NHUC. Expression was quantified relative to pooled cultured NHUC.
Figure Legend Snippet: (A) Overexpression of BMI1 led to a bypass of senescence in NHUC. Filled bars show senescence-associated β-galactosidase expression in empty vector-transduced NHUC; and unfilled bars, BMI1-transduced cells. (B) Ectopic expression of BMI1 led to exponential growth of the cell population and apparent immortalization in cells from three independent donors. Filled symbol shows vector-transduced cells; and unfilled symbols, NHUC-BMI1. (C) Detection of telomerase activity in NHUC-BMI1 using quantitative real-time PCR-based method. HI represents heat-inactivated sample, No Taq is a no polymerase negative control, and positive control is a lysate of the control pellet from the TRAPEZE XL kit. (D) Overexpression of BMI1 repressed induction of differentiation in NHUC. Induction of uroplakin 2 (UPK2), a marker of urothelial differentiation, was repressed in NHUC-BMI1 compared with vector control cells after treatment with trogiltazone and EGFR inhibitor. Expression is normalized to NHUC-vector control treatment cells. (E) Quantitative real-time PCR confirmed that overexpression of BMI1 did not repress CDKN2A transcription in NHUC. Expression was quantified relative to pooled cultured NHUC.

Techniques Used: Over Expression, Expressing, Plasmid Preparation, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Marker, Cell Culture

23) Product Images from "Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth"

Article Title: Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017924

C/EBPs induce clusterin. A ) C/EBPβ is up-regulated in the αGSU. PTTG pituitary. Confocal image showing C/EBPβ co-localization with αGSU-positive, GH-positive and PRL-positive cells in WT and pre-tumorous αGSU. PTTG pituitary glands. (Hormones-green, cytoplsmic, C/EBPβ–red, intranuclear); B ) Western blot analysis of C/EBPβ and δ isoforms induced in LβT2 cells stably transfected with m Pttg ; C ) Effects of C/EBPs on the clusterin promoter in LβT2 and αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-m Clu reporter plasmid and 800 ng murine pCDNA3-C/EBPα, β or δ. The ratio of luciferase to co-trasfected β-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown.*, p
Figure Legend Snippet: C/EBPs induce clusterin. A ) C/EBPβ is up-regulated in the αGSU. PTTG pituitary. Confocal image showing C/EBPβ co-localization with αGSU-positive, GH-positive and PRL-positive cells in WT and pre-tumorous αGSU. PTTG pituitary glands. (Hormones-green, cytoplsmic, C/EBPβ–red, intranuclear); B ) Western blot analysis of C/EBPβ and δ isoforms induced in LβT2 cells stably transfected with m Pttg ; C ) Effects of C/EBPs on the clusterin promoter in LβT2 and αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-m Clu reporter plasmid and 800 ng murine pCDNA3-C/EBPα, β or δ. The ratio of luciferase to co-trasfected β-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown.*, p

Techniques Used: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Luciferase, Expressing

FOXL2 stimulates the clusterin promoter. A ) Effects of FOXL2 on the clusterin promoter in αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-m Clu reporter plasmid and indicated amounts of pcDNA3-His-mFoxl2. The ratio of luciferase to co-trasfected β-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown; **,p
Figure Legend Snippet: FOXL2 stimulates the clusterin promoter. A ) Effects of FOXL2 on the clusterin promoter in αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-m Clu reporter plasmid and indicated amounts of pcDNA3-His-mFoxl2. The ratio of luciferase to co-trasfected β-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown; **,p

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Expressing

Senescence markers in gonadotroph-derived LβT2 cells transfected with m Pttg . Western blot analysis of senescence markers in A ) LβT2 cells transiently transfected with m Pttg ; B ) in LβT2 cells stably transfected with m Pttg ; C ) Percent BrdU positive cells in two selected clones stably transfected with m Pttg . Duplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry; D ) Senescent morphology of LβT2 cells stably transfected with m Pttg . Brown dots depict incorporated BrdU; E ) Percent apoptotic cells stably transfected with m Pttg . Cells were fixed, and one thousand cells/field counted in three randomly chosen visual fields; F ) Percent SA-β-galactosidase positivity in cells stably transfected with Pttg was assessed in 6-well plates in triplicate. One thousand cells/field were counted in three fields/well. G ) SA-β-galactosidase enzymatic activity (blue) in cells stably transfected with m Pttg . *, p
Figure Legend Snippet: Senescence markers in gonadotroph-derived LβT2 cells transfected with m Pttg . Western blot analysis of senescence markers in A ) LβT2 cells transiently transfected with m Pttg ; B ) in LβT2 cells stably transfected with m Pttg ; C ) Percent BrdU positive cells in two selected clones stably transfected with m Pttg . Duplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry; D ) Senescent morphology of LβT2 cells stably transfected with m Pttg . Brown dots depict incorporated BrdU; E ) Percent apoptotic cells stably transfected with m Pttg . Cells were fixed, and one thousand cells/field counted in three randomly chosen visual fields; F ) Percent SA-β-galactosidase positivity in cells stably transfected with Pttg was assessed in 6-well plates in triplicate. One thousand cells/field were counted in three fields/well. G ) SA-β-galactosidase enzymatic activity (blue) in cells stably transfected with m Pttg . *, p

Techniques Used: Derivative Assay, Transfection, Western Blot, Stable Transfection, Clone Assay, Flow Cytometry, Cytometry, Activity Assay

24) Product Images from "Mesenchymal stem cells (MSCs) from scleroderma patients (SSc) preserve their immunomodulatory properties although senescent and normally induce T regulatory cells (Tregs) with a functional phenotype: implications for cellular-based therapy"

Article Title: Mesenchymal stem cells (MSCs) from scleroderma patients (SSc) preserve their immunomodulatory properties although senescent and normally induce T regulatory cells (Tregs) with a functional phenotype: implications for cellular-based therapy

Journal: Clinical and Experimental Immunology

doi: 10.1111/cei.12111

β-Galactosidase (β-Gal) activity. (a) Systemic sclerosis (SSc)–mesenchymal stem cells (MSCs) showed a significant increase in the percentage of β-Gal-positive cells in respect to healthy controls (HC) cells. β-Gal-stained cells showed flat and enlarged morphology. (b) Doxorubicin (5 μg/ml) treatment increased the percentage of β-Gal-positive cells in both HC and SSc cells. β-Gal-positive cells in SSc–MSCs were increased significantly when compared to HC–MSC. Percentage of β-Gal-positive cells were quantified and showed in histograms. The values are expressed as mean ± standard deviation (s.d.) (* P
Figure Legend Snippet: β-Galactosidase (β-Gal) activity. (a) Systemic sclerosis (SSc)–mesenchymal stem cells (MSCs) showed a significant increase in the percentage of β-Gal-positive cells in respect to healthy controls (HC) cells. β-Gal-stained cells showed flat and enlarged morphology. (b) Doxorubicin (5 μg/ml) treatment increased the percentage of β-Gal-positive cells in both HC and SSc cells. β-Gal-positive cells in SSc–MSCs were increased significantly when compared to HC–MSC. Percentage of β-Gal-positive cells were quantified and showed in histograms. The values are expressed as mean ± standard deviation (s.d.) (* P

Techniques Used: Activity Assay, Staining, Standard Deviation

25) Product Images from "AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells"

Article Title: AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-020-1565-6

Distinct effects of AICAR, NAM, and concomitant AICAR+NAM treatment on senescence-associated changes of MSCs and total cellular reactive oxygen species (ROS). MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. a Phase-contrast images of MSCs (P10) (scale bar = 500 μm), SA-β-gal expression, visualized using light microscopy (scale bar = 100 μm), and fluorescent micrograph (scale bar = 50 μm) of the Acridine Orange stained MSCs at P10 of the four groups. b Left panel: the surface area of the MSCs (P10), calculated using ImageJ software, indicates that cells treated with AICAR alone or AICAR+NAM displayed a significantly lower cross-sectional surface area compared to the NAM-treated cells and the untreated group. Middle panel: prevalence of the SA-β-gal-positive cells, calculated as the number of blue cells per the total number of cells counted. Our data show that treatment with AICAR and NAM reduces the expression of SA-β-gal. Right panel: prevalence of senescent cells determined by the number of green fluorescence-emitting cells per the total number of cells counted. Untreated cells displayed the highest frequency of cells emitting green fluorescence and the least frequency of red fluorescence-emitting cells, compared to the treatment groups. Each bar indicates mean ± SD. c Total cellular ROS was measured at P5 and P10 by staining with DCFDA, followed by flow cytometry analysis ( n = 3 independent experiments). d Comparison between total cellular ROS of the study groups at P10 ( n = 3 independent experiments). Each bar indicates mean ± SD (* p
Figure Legend Snippet: Distinct effects of AICAR, NAM, and concomitant AICAR+NAM treatment on senescence-associated changes of MSCs and total cellular reactive oxygen species (ROS). MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. a Phase-contrast images of MSCs (P10) (scale bar = 500 μm), SA-β-gal expression, visualized using light microscopy (scale bar = 100 μm), and fluorescent micrograph (scale bar = 50 μm) of the Acridine Orange stained MSCs at P10 of the four groups. b Left panel: the surface area of the MSCs (P10), calculated using ImageJ software, indicates that cells treated with AICAR alone or AICAR+NAM displayed a significantly lower cross-sectional surface area compared to the NAM-treated cells and the untreated group. Middle panel: prevalence of the SA-β-gal-positive cells, calculated as the number of blue cells per the total number of cells counted. Our data show that treatment with AICAR and NAM reduces the expression of SA-β-gal. Right panel: prevalence of senescent cells determined by the number of green fluorescence-emitting cells per the total number of cells counted. Untreated cells displayed the highest frequency of cells emitting green fluorescence and the least frequency of red fluorescence-emitting cells, compared to the treatment groups. Each bar indicates mean ± SD. c Total cellular ROS was measured at P5 and P10 by staining with DCFDA, followed by flow cytometry analysis ( n = 3 independent experiments). d Comparison between total cellular ROS of the study groups at P10 ( n = 3 independent experiments). Each bar indicates mean ± SD (* p

Techniques Used: Expressing, Light Microscopy, Staining, Software, Fluorescence, Flow Cytometry

26) 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

27) 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

28) 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

29) Product Images from "Targeting LINC00673 expression triggers cellular senescence in lung cancer"

Article Title: Targeting LINC00673 expression triggers cellular senescence in lung cancer

Journal: RNA Biology

doi: 10.1080/15476286.2018.1553481

LINC00673 is a cytoplasmic lncRNA with a short half-life. (A) Cellular fractionation of A549 cells and subsequent RT-qPCR (represented as nuclear/cytosolic (log2) ratios) indicated an enrichment of LINC00673 in the cytoplasmic fraction. Data represent the mean ± SEM ( n = 3). (B) RNA FISH using custom Stellaris (LGC Biosearch Technologies) probes for LINC00673 revealed a cytoplasmic localization. Yellow arrows indicate lncRNA signal, and the scale bars represent 20 μm. BF: brightfield. (C) RNA stability was determined by actinomycin D (10 µg/ml) treatment of A549 cells for up to 24 h and the RNA levels were measured by RT-qPCR. LINC00673 expression was detected with two different primer pairs spanning exon 2–3 (E2-3) and located within exon 4 (E4), respectively. Data represent the mean ± SEM ( n = 3), t 1/2 : half-life, CI: confidence interval. Data was fitted to a non-linear least squares regression curve (one phase decay) with GraphPad Prism 5.
Figure Legend Snippet: LINC00673 is a cytoplasmic lncRNA with a short half-life. (A) Cellular fractionation of A549 cells and subsequent RT-qPCR (represented as nuclear/cytosolic (log2) ratios) indicated an enrichment of LINC00673 in the cytoplasmic fraction. Data represent the mean ± SEM ( n = 3). (B) RNA FISH using custom Stellaris (LGC Biosearch Technologies) probes for LINC00673 revealed a cytoplasmic localization. Yellow arrows indicate lncRNA signal, and the scale bars represent 20 μm. BF: brightfield. (C) RNA stability was determined by actinomycin D (10 µg/ml) treatment of A549 cells for up to 24 h and the RNA levels were measured by RT-qPCR. LINC00673 expression was detected with two different primer pairs spanning exon 2–3 (E2-3) and located within exon 4 (E4), respectively. Data represent the mean ± SEM ( n = 3), t 1/2 : half-life, CI: confidence interval. Data was fitted to a non-linear least squares regression curve (one phase decay) with GraphPad Prism 5.

Techniques Used: Cell Fractionation, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Expressing

LINC00673 acts by regulating p53 translation. (A) A549 cells were adapted to SILAC medium and total protein lysates were prepared at 48 h following siPOOL-mediated knockdown of LINC00673 . The lysates were analyzed by mass spectrometry and proteins that displayed an absolute fold change > 1.5 as compared to the siPOOL control are listed ( n = 2, t test with P
Figure Legend Snippet: LINC00673 acts by regulating p53 translation. (A) A549 cells were adapted to SILAC medium and total protein lysates were prepared at 48 h following siPOOL-mediated knockdown of LINC00673 . The lysates were analyzed by mass spectrometry and proteins that displayed an absolute fold change > 1.5 as compared to the siPOOL control are listed ( n = 2, t test with P

Techniques Used: Mass Spectrometry

LINC00673 depletion induces a senescence-like phenotype. (A) SA-β-Gal accumulated in A549 cells 4 days after LINC00673 knockdown with siPOOLs (3 nM). As a positive control for staining, cells were treated with 200 nM doxorubicin. At least 200 cells were counted per condition ( n = 4). The scale bar represents 100 µm. (B) SA-β-Gal staining of IMR-90 cells as in A ( n = 3). (C) Cell cycle and E2F1-regulated genes were determined by RT-qPCR at 48 h after LINC00673 knockdown with siPOOLs (3 nM) in A549 cells. GAPDH was used as reference gene ( n = 3). (D) SASP gene expression in A549 cells was determined by RT-qPCR as in C ( n = 3). (E) Western Blot analysis 48 h after LINC00673 knockdown with siPOOLs (3 nM) in A549 cells. Specific antibodies were used for indicated proteins. One representative Western Blot is shown ( n = 3). All data is shown as mean + SEM and the statistical significance was determined per two-sided unpaired Student t test, with *, P
Figure Legend Snippet: LINC00673 depletion induces a senescence-like phenotype. (A) SA-β-Gal accumulated in A549 cells 4 days after LINC00673 knockdown with siPOOLs (3 nM). As a positive control for staining, cells were treated with 200 nM doxorubicin. At least 200 cells were counted per condition ( n = 4). The scale bar represents 100 µm. (B) SA-β-Gal staining of IMR-90 cells as in A ( n = 3). (C) Cell cycle and E2F1-regulated genes were determined by RT-qPCR at 48 h after LINC00673 knockdown with siPOOLs (3 nM) in A549 cells. GAPDH was used as reference gene ( n = 3). (D) SASP gene expression in A549 cells was determined by RT-qPCR as in C ( n = 3). (E) Western Blot analysis 48 h after LINC00673 knockdown with siPOOLs (3 nM) in A549 cells. Specific antibodies were used for indicated proteins. One representative Western Blot is shown ( n = 3). All data is shown as mean + SEM and the statistical significance was determined per two-sided unpaired Student t test, with *, P

Techniques Used: Positive Control, Staining, Quantitative RT-PCR, Expressing, Western Blot

LINC00673 reduces cell proliferation and induces a cell cycle arrest. (A) CellTiter-Glo Luminescent Cell Viability Assay (Promega) was performed 48 h after LINC00673 knockdown in A549 cells with GapmeRs (60 nM; n = 3) and siPOOLs (10 nM; n = 4), respectively. (B) Caspase-Glo 3/7 luminescent assay (Promega) was performed 24 h after LINC00673 knockdown in A549 cells (final concentration of GapmeRs and siPOOLs as in A; n = 6 for GapmeR-mediated knockdown and n = 4 for siPOOL-mediated knockdown experiments). Treatment with actinomycin D (ActD; 5 µg/ml) served as a positive control for apoptosis induction. (C) BrdU Cell Proliferation ELISA Kit (Roche) was used to quantify cell proliferation 24 h and 48 h after LINC00673 knockdown (final concentrations as in A; n = 3 for GapmeR-mediated knockdown in A549 and Calu-3; n = 5 for siPOOL-mediated knockdown in A549 and n = 3 for Calu-3). (D) BrdU Cell Proliferation ELISA Kit was used to quantify cell proliferation 48 h after siPOOL-mediated knockdown (final concentration of 1 nM) of LINC00673 in IMR-90 and WI-38 cells ( n = 3). (E) Cell cycle profiles of A549 cells were analyzed by FACS after 48 h and 72 h of LINC00673 knockdown. Quantification of cells with the cell cycle function of FlowJo (V10) and the cell cycle profiles of one representative experiment are shown ( n = 3). (F) IMR-90 cells were serum starved for 24 h and subsequently transfected with 0.3 nM siPOOLs. Cells were released by adding complete medium supplemented with 10% FBS at 72 h after knockdown and BrdU incorporation was measured 8 h and 36 h following block release ( n = 4). In A-F the mean + SEM is shown and the statistical significance was determined per two-sided unpaired Student t test, with *, P
Figure Legend Snippet: LINC00673 reduces cell proliferation and induces a cell cycle arrest. (A) CellTiter-Glo Luminescent Cell Viability Assay (Promega) was performed 48 h after LINC00673 knockdown in A549 cells with GapmeRs (60 nM; n = 3) and siPOOLs (10 nM; n = 4), respectively. (B) Caspase-Glo 3/7 luminescent assay (Promega) was performed 24 h after LINC00673 knockdown in A549 cells (final concentration of GapmeRs and siPOOLs as in A; n = 6 for GapmeR-mediated knockdown and n = 4 for siPOOL-mediated knockdown experiments). Treatment with actinomycin D (ActD; 5 µg/ml) served as a positive control for apoptosis induction. (C) BrdU Cell Proliferation ELISA Kit (Roche) was used to quantify cell proliferation 24 h and 48 h after LINC00673 knockdown (final concentrations as in A; n = 3 for GapmeR-mediated knockdown in A549 and Calu-3; n = 5 for siPOOL-mediated knockdown in A549 and n = 3 for Calu-3). (D) BrdU Cell Proliferation ELISA Kit was used to quantify cell proliferation 48 h after siPOOL-mediated knockdown (final concentration of 1 nM) of LINC00673 in IMR-90 and WI-38 cells ( n = 3). (E) Cell cycle profiles of A549 cells were analyzed by FACS after 48 h and 72 h of LINC00673 knockdown. Quantification of cells with the cell cycle function of FlowJo (V10) and the cell cycle profiles of one representative experiment are shown ( n = 3). (F) IMR-90 cells were serum starved for 24 h and subsequently transfected with 0.3 nM siPOOLs. Cells were released by adding complete medium supplemented with 10% FBS at 72 h after knockdown and BrdU incorporation was measured 8 h and 36 h following block release ( n = 4). In A-F the mean + SEM is shown and the statistical significance was determined per two-sided unpaired Student t test, with *, P

Techniques Used: Cell Viability Assay, Luminescence Assay, Concentration Assay, Positive Control, Enzyme-linked Immunosorbent Assay, FACS, Transfection, BrdU Incorporation Assay, Blocking Assay

LINC00673 -mediated senescence is p53-dependent. (A) BrdU Cell Proliferation ELISA Kit was used to quantify cell proliferation 48 h after knockdown of p53, pRb and LINC00673 in A549 ( n = 4). The reactions contained siPOOL negative control (12 nM) or a mix of 1 nM siPOOL p53, pRb and 10 nM siPOOL LINC00673 supplemented with siPOOL negative control for a final concentration of 12 nM. (B) BrdU incorporation in IMR-90 cells was measured as in A. The reactions contained siPOOL negative control (0.9 nM) or a mix of 0.3 nM siPOOL p53, pRb and LINC00673 supplemented with siPOOL negative control for a final concentration of 0.9 nM. (C) Relative RNA levels were determined by RT-qPCR at 48 h after knockdown in A549 cells ( n = 3). The reactions contained siPOOL negative control (5 nM) or a mix of 1 nM siPOOL p53, pRb and 3 nM siPOOL LINC00673 supplemented with siPOOL negative control for a final concentration of 5 nM. GAPDH was used as reference gene. One representative Western Blot is shown ( n = 3). (D) RT-qPCR as in C using IMR-90 cells ( n = 4). The siPOOL concentrations were as described in B. One representative Western Blot is shown ( n = 4). (E) SA-β-Gal accumulated in A549 cells 4 days after knockdown. The reactions contained siPOOL negative control (4 nM) or a mix of 3 nM siPOOL LINC00673 and 1 nM siPOOL p53 or pRb supplemented with siPOOL negative control for a final concentration of 4 nM ( n = 4, scale bar = 100 µm). In A-F, the mean + SEM is shown. The statistical significance was determined per two-sided unpaired Student t test, with *, P
Figure Legend Snippet: LINC00673 -mediated senescence is p53-dependent. (A) BrdU Cell Proliferation ELISA Kit was used to quantify cell proliferation 48 h after knockdown of p53, pRb and LINC00673 in A549 ( n = 4). The reactions contained siPOOL negative control (12 nM) or a mix of 1 nM siPOOL p53, pRb and 10 nM siPOOL LINC00673 supplemented with siPOOL negative control for a final concentration of 12 nM. (B) BrdU incorporation in IMR-90 cells was measured as in A. The reactions contained siPOOL negative control (0.9 nM) or a mix of 0.3 nM siPOOL p53, pRb and LINC00673 supplemented with siPOOL negative control for a final concentration of 0.9 nM. (C) Relative RNA levels were determined by RT-qPCR at 48 h after knockdown in A549 cells ( n = 3). The reactions contained siPOOL negative control (5 nM) or a mix of 1 nM siPOOL p53, pRb and 3 nM siPOOL LINC00673 supplemented with siPOOL negative control for a final concentration of 5 nM. GAPDH was used as reference gene. One representative Western Blot is shown ( n = 3). (D) RT-qPCR as in C using IMR-90 cells ( n = 4). The siPOOL concentrations were as described in B. One representative Western Blot is shown ( n = 4). (E) SA-β-Gal accumulated in A549 cells 4 days after knockdown. The reactions contained siPOOL negative control (4 nM) or a mix of 3 nM siPOOL LINC00673 and 1 nM siPOOL p53 or pRb supplemented with siPOOL negative control for a final concentration of 4 nM ( n = 4, scale bar = 100 µm). In A-F, the mean + SEM is shown. The statistical significance was determined per two-sided unpaired Student t test, with *, P

Techniques Used: Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay, BrdU Incorporation Assay, Quantitative RT-PCR, Western Blot

30) Product Images from "Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer"

Article Title: Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer

Journal: American Journal of Cancer Research

doi:

HSP70-2 shRNA treatment initiates the onset of apoptosis and inhibits EMT in tumor cells. A. Western blot data show upregulation of pro-apoptotic molecules and downregulation of anti-apoptotic molecules. B. Representative images of IHC of serial sections
Figure Legend Snippet: HSP70-2 shRNA treatment initiates the onset of apoptosis and inhibits EMT in tumor cells. A. Western blot data show upregulation of pro-apoptotic molecules and downregulation of anti-apoptotic molecules. B. Representative images of IHC of serial sections

Techniques Used: shRNA, Western Blot, Immunohistochemistry

HSP70-2 ablation retards tumor growth in-vivo human xenograft mouse model. A. Representative images show the difference in tumor dissected and compared on scale shows reduced tumor size when treated with shRNA3 as compared to NC shRNA treated mice. B.
Figure Legend Snippet: HSP70-2 ablation retards tumor growth in-vivo human xenograft mouse model. A. Representative images show the difference in tumor dissected and compared on scale shows reduced tumor size when treated with shRNA3 as compared to NC shRNA treated mice. B.

Techniques Used: In Vivo, shRNA, Mouse Assay

HSP70-2 shRNA treatment arrests cell growth. A. Flow cytometric analysis shows cell cycle analysis of NC shRNA, shRNA3 and shRNA4 transfected cells stained with PI (M1: G0-G1, M2: S and M3: G2-M phase). Bar diagram depicts cumulative percentage accumulation
Figure Legend Snippet: HSP70-2 shRNA treatment arrests cell growth. A. Flow cytometric analysis shows cell cycle analysis of NC shRNA, shRNA3 and shRNA4 transfected cells stained with PI (M1: G0-G1, M2: S and M3: G2-M phase). Bar diagram depicts cumulative percentage accumulation

Techniques Used: shRNA, Flow Cytometry, Cell Cycle Assay, Transfection, Staining

HSP70-2 ablation initiates apoptosis. A. Representative SEM images show no phenotypic changes in cells treated with NC shRNA. Initiation of membrane blebbing and apoptotic bodies formation is observed in cells treated with shRNA3 and shRNA4 after 12 hours
Figure Legend Snippet: HSP70-2 ablation initiates apoptosis. A. Representative SEM images show no phenotypic changes in cells treated with NC shRNA. Initiation of membrane blebbing and apoptotic bodies formation is observed in cells treated with shRNA3 and shRNA4 after 12 hours

Techniques Used: shRNA

Ablation of HSP70-2 alters the malignant properties of ovarian cancer cell, A10. A. Bar diagram depicts qPCR data of all four HSP70-2 shRNA targets. B. Western blotting shows reduced HSP70-2 protein expression in shRNA3 and shRNA4 transfected cells as
Figure Legend Snippet: Ablation of HSP70-2 alters the malignant properties of ovarian cancer cell, A10. A. Bar diagram depicts qPCR data of all four HSP70-2 shRNA targets. B. Western blotting shows reduced HSP70-2 protein expression in shRNA3 and shRNA4 transfected cells as

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

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 "Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts"

Article Title: Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-11-59

Expression of senescence associated (SA) β-galactosidase with different experimental treatments . Senescent HDFs, passage 30 (A); cells with H 2 O 2 -induced oxidative stress (B); HDFs at young age, passage 4 (C) and γ-tocotrienol-treated HDFs (D). Cells with blue staining indicated positive for β-galactosidase activity. Micrographs are shown at ×200 magnification.
Figure Legend Snippet: Expression of senescence associated (SA) β-galactosidase with different experimental treatments . Senescent HDFs, passage 30 (A); cells with H 2 O 2 -induced oxidative stress (B); HDFs at young age, passage 4 (C) and γ-tocotrienol-treated HDFs (D). Cells with blue staining indicated positive for β-galactosidase activity. Micrographs are shown at ×200 magnification.

Techniques Used: Expressing, Staining, Activity Assay

33) Product Images from "Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7"

Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7

Journal: Journal of Virology

doi: 10.1128/JVI.75.16.7583-7591.2001

HPV-16 E7 destabilizes p107 by a proteasome-dependent mechanism. (A) HPV-16 E7 decreases steady-state levels of p107. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding HA-tagged p107, wild-type HPV-16 E7 (16E7), or the pRB-binding-deficient mutant 16E7 ΔD21-C24. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for p107 using an HA antibody (upper panel) and cotransfected GFP control (lower panel). Quantitation of p107 levels, normalized for GFP expression, is shown underneath these panels. (B) HPV-16 E7 decreases the half-life of p107. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding HA-tagged p107 and HPV-16 E7 (16E7) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were harvested and then processed for immunoblot analysis for p107 using an HA antibody (upper panel) or cotransfected GFP control (lower panel). Quantitation of p107 levels, normalized for GFP expression, is shown underneath these panels. (C) Inhibition of the 26S proteasome blocks HPV-16 E7-mediated p107 degradation. SAOS2 cells were transfected with the indicated combinations of plasmids encoding HA-tagged p107, HPV-16 E7 (16E7), and HPV-16 E7 mutants 16E7 ΔD21-C24 (pRB binding and degradation deficient), 16E7 ΔP6-E10 (pRB binding competent and pRB degradation deficient), and 16E7 C91S (pRB binding and degradation competent) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (−) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for p107 using an HA antibody (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of p107 and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (D) Differential destabilization of pRB and p107 by pRB-binding-site E7 point mutants. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB or HA-tagged p107, HPV-16 E7 (16E7), and HPV-16 E7 mutants 16E7 C24G and 16E7 E26G (pRB binding and degradation deficient, partially p107 binding competent) or control vector (CMV). C, untransfected controls. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (left upper panel), p107 using an HA antibody (right upper panel), HPV-16 E7 (middle panels), and cotransfected GFP control (lower panels). Quantitation of pRB and p107 levels, normalized for GFP expression, is shown underneath these panels.
Figure Legend Snippet: HPV-16 E7 destabilizes p107 by a proteasome-dependent mechanism. (A) HPV-16 E7 decreases steady-state levels of p107. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding HA-tagged p107, wild-type HPV-16 E7 (16E7), or the pRB-binding-deficient mutant 16E7 ΔD21-C24. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for p107 using an HA antibody (upper panel) and cotransfected GFP control (lower panel). Quantitation of p107 levels, normalized for GFP expression, is shown underneath these panels. (B) HPV-16 E7 decreases the half-life of p107. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding HA-tagged p107 and HPV-16 E7 (16E7) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were harvested and then processed for immunoblot analysis for p107 using an HA antibody (upper panel) or cotransfected GFP control (lower panel). Quantitation of p107 levels, normalized for GFP expression, is shown underneath these panels. (C) Inhibition of the 26S proteasome blocks HPV-16 E7-mediated p107 degradation. SAOS2 cells were transfected with the indicated combinations of plasmids encoding HA-tagged p107, HPV-16 E7 (16E7), and HPV-16 E7 mutants 16E7 ΔD21-C24 (pRB binding and degradation deficient), 16E7 ΔP6-E10 (pRB binding competent and pRB degradation deficient), and 16E7 C91S (pRB binding and degradation competent) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (−) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for p107 using an HA antibody (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of p107 and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (D) Differential destabilization of pRB and p107 by pRB-binding-site E7 point mutants. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB or HA-tagged p107, HPV-16 E7 (16E7), and HPV-16 E7 mutants 16E7 C24G and 16E7 E26G (pRB binding and degradation deficient, partially p107 binding competent) or control vector (CMV). C, untransfected controls. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (left upper panel), p107 using an HA antibody (right upper panel), HPV-16 E7 (middle panels), and cotransfected GFP control (lower panels). Quantitation of pRB and p107 levels, normalized for GFP expression, is shown underneath these panels.

Techniques Used: Transfection, Binding Assay, Mutagenesis, SDS Page, Quantitation Assay, Expressing, Plasmid Preparation, Inhibition

HPV-16 E7 degradation and E7-mediated pRB degradation are 26S proteasome-dependent but separable processes. (A) The human osteosarcoma cell line SAOS2 was transiently transfected with the indicated combinations of plasmids encoding wild-type pRB and wild-type HPV-16 E7 (16E7). Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB levels, normalized for GFP expression, is shown underneath those panels (in arbitrary units based on densitometry). (B) HPV-16 E7, adenovirus E1A, and SV40 T antigen oncoproteins differ in their ability to destabilize pRB. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7, adenovirus (Ad) E1A, and SV40 T antigen (TAg) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were processed for immunoblot analysis for pRB (upper panel) and cotransfected GFP control (lower panel). Quantitation of pRB (RB) levels, normalized for GFP expression, is shown underneath these panels. (C) Effect of MG132 and Lactacystin on HPV-16 E7 and pRB steady-state levels. SAOS2 cells were transfected with expression plasmids encoding pRB alone or in combination with 16E7. At 40 h posttransfection, cells were treated with 10 or 40 μM MG132 or Lactacystin or mock treated with dimethyl sulfoxide (DMSO) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (D) Effect of proteasome inhibition on various HPV E7 proteins. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7 (16E7 and 16E7-HA), a pRB-binding- and pRB degradation-deficient 16E7 mutant (16E7 ΔD21-C24), pRB-binding-competent and pRB degradation-deficient versions of E7 (16E7 ΔP6-E10 and 1aE7-HA), and a pRB-binding- and pRB degradation-competent 16E7 mutant (16E7 C91S) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (D) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 or HPV-16 E7 tagged with HA (middle panel), and cotransfected GFP control or HPV-1a E7 tagged with HA (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (E) E7 degradation and E7-mediated pRB degradation are not linked. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7 (16E7), a lysineless mutant of 16E7 (16E7 K60,97R), an amino-terminal FLAG-tagged version of 16E7 (FLAG-16E7), and a carboxyl-terminal FLAG-tagged version of 16E7 (16E7-FLAG) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (D) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (F) HPV-16 E7 stability correlates with E7-mediated pRB degradation. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding pRB and amino- and carboxyl-terminal FLAG-tagged HPV-16 E7. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and E7 levels, normalized for GFP expression, is shown underneath these panels.
Figure Legend Snippet: HPV-16 E7 degradation and E7-mediated pRB degradation are 26S proteasome-dependent but separable processes. (A) The human osteosarcoma cell line SAOS2 was transiently transfected with the indicated combinations of plasmids encoding wild-type pRB and wild-type HPV-16 E7 (16E7). Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB levels, normalized for GFP expression, is shown underneath those panels (in arbitrary units based on densitometry). (B) HPV-16 E7, adenovirus E1A, and SV40 T antigen oncoproteins differ in their ability to destabilize pRB. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7, adenovirus (Ad) E1A, and SV40 T antigen (TAg) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were processed for immunoblot analysis for pRB (upper panel) and cotransfected GFP control (lower panel). Quantitation of pRB (RB) levels, normalized for GFP expression, is shown underneath these panels. (C) Effect of MG132 and Lactacystin on HPV-16 E7 and pRB steady-state levels. SAOS2 cells were transfected with expression plasmids encoding pRB alone or in combination with 16E7. At 40 h posttransfection, cells were treated with 10 or 40 μM MG132 or Lactacystin or mock treated with dimethyl sulfoxide (DMSO) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (D) Effect of proteasome inhibition on various HPV E7 proteins. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7 (16E7 and 16E7-HA), a pRB-binding- and pRB degradation-deficient 16E7 mutant (16E7 ΔD21-C24), pRB-binding-competent and pRB degradation-deficient versions of E7 (16E7 ΔP6-E10 and 1aE7-HA), and a pRB-binding- and pRB degradation-competent 16E7 mutant (16E7 C91S) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (D) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 or HPV-16 E7 tagged with HA (middle panel), and cotransfected GFP control or HPV-1a E7 tagged with HA (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (E) E7 degradation and E7-mediated pRB degradation are not linked. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB, HPV-16 E7 (16E7), a lysineless mutant of 16E7 (16E7 K60,97R), an amino-terminal FLAG-tagged version of 16E7 (FLAG-16E7), and a carboxyl-terminal FLAG-tagged version of 16E7 (16E7-FLAG) or control vector (CMV). Cells were treated with 40 μM MG132 (+) or mock treated with DMSO (D) for 4 h. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and HPV-16 E7 levels, normalized for GFP expression, is shown underneath these panels. (F) HPV-16 E7 stability correlates with E7-mediated pRB degradation. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding pRB and amino- and carboxyl-terminal FLAG-tagged HPV-16 E7. Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for pRB (upper panel), HPV-16 E7 (middle panel), and cotransfected GFP control (lower panel). Quantitation of pRB and E7 levels, normalized for GFP expression, is shown underneath these panels.

Techniques Used: Transfection, SDS Page, Quantitation Assay, Expressing, Plasmid Preparation, Inhibition, Binding Assay, Mutagenesis

HPV-16 E7 destabilizes p130. (A) Decreased p130 steady-state levels in HPV-16 E7-expressing pools of HFKs. Two independent, matched populations of HFKs infected with control LXSN (LX) and HPV-16 E7-expressing LXSN (LX-16E7) vectors are shown. Pools of cells were obtained after G418 selection. Extracts (100 μg) were prepared and subjected to SDS-PAGE and p130 immunoblot analysis. Quantitation of p130 levels is shown underneath the blot. (B) HPV-16 E7 decreases p130 steady-state levels in the SAOS2 cell transient assay. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding p130 and HPV-16 E7 (16E7). Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for p130 (upper panel) and cotransfected GFP control (lower panel). Quantitation of p130 levels, normalized for GFP expression, is shown underneath these panels. Untransfected cells are shown as controls. (C) HPV-16 E7 decreases the half-life of p130. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding p130 and HPV-16 E7 (16E7) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were harvested and then processed for immunoblot analysis for p130 (upper panel) or cotransfected GFP control (lower panel). Quantitation of p130 levels, normalized for GFP expression, is shown underneath these panels.
Figure Legend Snippet: HPV-16 E7 destabilizes p130. (A) Decreased p130 steady-state levels in HPV-16 E7-expressing pools of HFKs. Two independent, matched populations of HFKs infected with control LXSN (LX) and HPV-16 E7-expressing LXSN (LX-16E7) vectors are shown. Pools of cells were obtained after G418 selection. Extracts (100 μg) were prepared and subjected to SDS-PAGE and p130 immunoblot analysis. Quantitation of p130 levels is shown underneath the blot. (B) HPV-16 E7 decreases p130 steady-state levels in the SAOS2 cell transient assay. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding p130 and HPV-16 E7 (16E7). Samples (100 μg) were subjected to SDS-PAGE and immunoblot analysis for p130 (upper panel) and cotransfected GFP control (lower panel). Quantitation of p130 levels, normalized for GFP expression, is shown underneath these panels. Untransfected cells are shown as controls. (C) HPV-16 E7 decreases the half-life of p130. SAOS2 cells were transiently transfected with the indicated combinations of plasmids encoding p130 and HPV-16 E7 (16E7) or control plasmid (CMV). At 24 h posttransfection, protein synthesis was blocked by treatment with cycloheximide (Chx). At the indicated times, samples (100 μg) were harvested and then processed for immunoblot analysis for p130 (upper panel) or cotransfected GFP control (lower panel). Quantitation of p130 levels, normalized for GFP expression, is shown underneath these panels.

Techniques Used: Expressing, Infection, Selection, SDS Page, Quantitation Assay, Transfection, Plasmid Preparation

Abrogation of pRB functions by HPV-16 E7-mediated degradation. (A) HPV-16 E7 inactivates pRB in an SAOS2 flat-cell assay. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB alone (panel a) or in combination with carboxyl-terminal HA-tagged HPV-16 E7 (16E7) (panel b), HPV-16 E7 mutants 16E7 ΔD21-C24, 16E7 C24G, 16E7 E26G, and 16E7 ΔP6-E10 (panels c, d, e, and f, respectively), and HPV-1a E7 (panel g). At 10 days posttransfection, cells were stained for senescence-associated β-galactosidase (β-gal) activity. (B) Quantitation of the results from panel A. The results represent averages and standard deviations from two independent experiments. RB, pRB.
Figure Legend Snippet: Abrogation of pRB functions by HPV-16 E7-mediated degradation. (A) HPV-16 E7 inactivates pRB in an SAOS2 flat-cell assay. SAOS2 cells were transfected with the indicated combinations of plasmids encoding pRB alone (panel a) or in combination with carboxyl-terminal HA-tagged HPV-16 E7 (16E7) (panel b), HPV-16 E7 mutants 16E7 ΔD21-C24, 16E7 C24G, 16E7 E26G, and 16E7 ΔP6-E10 (panels c, d, e, and f, respectively), and HPV-1a E7 (panel g). At 10 days posttransfection, cells were stained for senescence-associated β-galactosidase (β-gal) activity. (B) Quantitation of the results from panel A. The results represent averages and standard deviations from two independent experiments. RB, pRB.

Techniques Used: Transfection, Staining, Activity Assay, Quantitation Assay

34) Product Images from "Sequence-specific Recruitment of Heterochromatin Protein 1 via Interaction with Kr?ppel-like Factor 11, a Human Transcription Factor Involved in Tumor Suppression and Metabolic Diseases *"

Article Title: Sequence-specific Recruitment of Heterochromatin Protein 1 via Interaction with Kr?ppel-like Factor 11, a Human Transcription Factor Involved in Tumor Suppression and Metabolic Diseases *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.342634

HP1 recruitment is necessary for KLF11-mediated apoptosis and senescence. A and B, HP1 plays a role in the ability of KLF11 to induce apoptosis., Apoptosis was measured by both, nuclear morphology via Hoechst 33342 staining ( A ) and caspase 3 cleavage assay ( B ). As observed by Hoescht staining ( A ), KLF11 WT increases apoptosis. KLF11ΔHP1 is defective in inducing this effect, with an apoptotic index similar to EV. The depicted results are the mean ± S.E. from three independent experiments. In caspase 3 cleavage assays ( B ), KLF11 WT increased the amount of caspase 3 cleavage, as shown by Western blot ( upper ), whereas KLF11ΔHP1 had reduced levels similar to control. Pro-caspase 3 was used as a control ( lower ). The levels of caspase 3 cleavage are shown for both, 48- and 72-h post-serum starvation. C , KLF11ΔHP1 demonstrates an increase in adherent cell number compared with KLF WT. Assessment of adherent cell count is shown after normalization to EV. Although KLF11 WT results in a significant decrease in cell numbers, this effect is blunted with KLF11ΔHP1. The graph represents the mean ± S.E. of results from three independent experiments. D and E, loss of HP1 recruitment eliminates the ability of KLF11 to increase senescence. To measure cells undergoing senescence, senescence-associated β-galactosidase staining was performed in primary fibroblasts. KLF11 increased the percentage of cells undergoing senescence. However, KLF11ΔHP1 lost this ability, behaving similar to control. The graph ( D ) depicts the mean ± S.E. from three independent experiments. A representative picture of senescence-associated β-galactosidase staining for each experimental group is shown ( E ). F, KLF11ΔHP1 is no longer able to repress the telomerase promoter. Activity of the hTERT promoter, as measured by the luciferase reporter assay, was significantly repressed by KLF11 WT, however, KLF11ΔHP1 loses this repression. Graphical depiction of the results is the mean ± S.E. from at least three independent experiments performed in triplicate. * denotes p
Figure Legend Snippet: HP1 recruitment is necessary for KLF11-mediated apoptosis and senescence. A and B, HP1 plays a role in the ability of KLF11 to induce apoptosis., Apoptosis was measured by both, nuclear morphology via Hoechst 33342 staining ( A ) and caspase 3 cleavage assay ( B ). As observed by Hoescht staining ( A ), KLF11 WT increases apoptosis. KLF11ΔHP1 is defective in inducing this effect, with an apoptotic index similar to EV. The depicted results are the mean ± S.E. from three independent experiments. In caspase 3 cleavage assays ( B ), KLF11 WT increased the amount of caspase 3 cleavage, as shown by Western blot ( upper ), whereas KLF11ΔHP1 had reduced levels similar to control. Pro-caspase 3 was used as a control ( lower ). The levels of caspase 3 cleavage are shown for both, 48- and 72-h post-serum starvation. C , KLF11ΔHP1 demonstrates an increase in adherent cell number compared with KLF WT. Assessment of adherent cell count is shown after normalization to EV. Although KLF11 WT results in a significant decrease in cell numbers, this effect is blunted with KLF11ΔHP1. The graph represents the mean ± S.E. of results from three independent experiments. D and E, loss of HP1 recruitment eliminates the ability of KLF11 to increase senescence. To measure cells undergoing senescence, senescence-associated β-galactosidase staining was performed in primary fibroblasts. KLF11 increased the percentage of cells undergoing senescence. However, KLF11ΔHP1 lost this ability, behaving similar to control. The graph ( D ) depicts the mean ± S.E. from three independent experiments. A representative picture of senescence-associated β-galactosidase staining for each experimental group is shown ( E ). F, KLF11ΔHP1 is no longer able to repress the telomerase promoter. Activity of the hTERT promoter, as measured by the luciferase reporter assay, was significantly repressed by KLF11 WT, however, KLF11ΔHP1 loses this repression. Graphical depiction of the results is the mean ± S.E. from at least three independent experiments performed in triplicate. * denotes p

Techniques Used: Staining, Cleavage Assay, Western Blot, Cell Counting, Activity Assay, Luciferase, Reporter Assay

35) Product Images from "Zerumbone Exhibits Antiphotoaging and Dermatoprotective Properties in Ultraviolet A-Irradiated Human Skin Fibroblast Cells via the Activation of Nrf2/ARE Defensive Pathway"

Article Title: Zerumbone Exhibits Antiphotoaging and Dermatoprotective Properties in Ultraviolet A-Irradiated Human Skin Fibroblast Cells via the Activation of Nrf2/ARE Defensive Pathway

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2019/4098674

Effect of ZER on the nuclear translocation, activation of Nrf2, and its associated proteins in UVA-irradiated HSF cells: ZER pretreated (2-8 μ M for 2 h) HSF cells were irradiated in the presence (a, b) or presence (c, d) of 3 J/cm 2 UVA (for the indicated time) were subjected to western blot for the measurement of total Nrf2 and Keap-1 protein expressions. The effect of ZER on this pattern was represented as fold change of Nrf2/Keap-1 ratio over the control values. This ratio determines the dissociation pattern of Nrf2 from Keap-1 in the cytoplasm. (e, f) HSF cells were treated with 8 μ M of ZER at different time points (0, 0.5, 1, 2, or 4 h), and the expression of cytosolic, nuclear Nrf2 protein fractions was determined using western blot method. β -Actin and histone proteins were used as internal controls for cytosolic and nuclear Nrf2, respectively. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗ p
Figure Legend Snippet: Effect of ZER on the nuclear translocation, activation of Nrf2, and its associated proteins in UVA-irradiated HSF cells: ZER pretreated (2-8 μ M for 2 h) HSF cells were irradiated in the presence (a, b) or presence (c, d) of 3 J/cm 2 UVA (for the indicated time) were subjected to western blot for the measurement of total Nrf2 and Keap-1 protein expressions. The effect of ZER on this pattern was represented as fold change of Nrf2/Keap-1 ratio over the control values. This ratio determines the dissociation pattern of Nrf2 from Keap-1 in the cytoplasm. (e, f) HSF cells were treated with 8 μ M of ZER at different time points (0, 0.5, 1, 2, or 4 h), and the expression of cytosolic, nuclear Nrf2 protein fractions was determined using western blot method. β -Actin and histone proteins were used as internal controls for cytosolic and nuclear Nrf2, respectively. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗ p

Techniques Used: Translocation Assay, Activation Assay, Irradiation, Western Blot, Expressing

Effect of ZER on ARE promoter activation and subsequent expression of HO-1 and γ -GCLC proteins in HSF cells. (a) Cells were treated with ZER (8 μ M for 2 h) and subcellular localization of Nrf2 was determined using immunostaining method. (b) HSF cells were cotransfected with pGL3-ARE and treated with various concentrations of ZER (2-8 μ M for 2 h) to measure the percentage of ARE promoter activity. Data was presented as fold over increase in the percentage of ARE promoter activity. (c, d) The effect of ZER treatment (8 μ M) on the expression of total Nrf2 and antioxidant proteins (HO-1 and γ -GCLC) at different time points (0, 1, 2, 4, 8, 12, 16, or 24 h) was measured using western blot method against β -actin as an internal control. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗ p
Figure Legend Snippet: Effect of ZER on ARE promoter activation and subsequent expression of HO-1 and γ -GCLC proteins in HSF cells. (a) Cells were treated with ZER (8 μ M for 2 h) and subcellular localization of Nrf2 was determined using immunostaining method. (b) HSF cells were cotransfected with pGL3-ARE and treated with various concentrations of ZER (2-8 μ M for 2 h) to measure the percentage of ARE promoter activity. Data was presented as fold over increase in the percentage of ARE promoter activity. (c, d) The effect of ZER treatment (8 μ M) on the expression of total Nrf2 and antioxidant proteins (HO-1 and γ -GCLC) at different time points (0, 1, 2, 4, 8, 12, 16, or 24 h) was measured using western blot method against β -actin as an internal control. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗ p

Techniques Used: Activation Assay, Expressing, Immunostaining, Activity Assay, Western Blot

Effect of ZER on intracellular ROS levels and SA- β -gal activity in UVA-irradiated HSF cells, (a) Cells were pretreated with ZER (0, 2, 4, or 8 μ M for 24 h) followed by irradiated with UVA in the absence or presence of 3 J/cm 2 UVA. The accumulation of UVA-induced ROS and SA- β -gal-positive cells was measured using fluorescence microscopy (200x magnification) as described. (b) The data was represented as fold change over control intracellular ROS levels. (c) The data was reported as fold increase over control SA- β -gal-positive cells. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗∗ p
Figure Legend Snippet: Effect of ZER on intracellular ROS levels and SA- β -gal activity in UVA-irradiated HSF cells, (a) Cells were pretreated with ZER (0, 2, 4, or 8 μ M for 24 h) followed by irradiated with UVA in the absence or presence of 3 J/cm 2 UVA. The accumulation of UVA-induced ROS and SA- β -gal-positive cells was measured using fluorescence microscopy (200x magnification) as described. (b) The data was represented as fold change over control intracellular ROS levels. (c) The data was reported as fold increase over control SA- β -gal-positive cells. Results from three or more experiments were presented as mean ± SD, and the statistical significance was considered as ∗∗∗ p

Techniques Used: Activity Assay, Irradiation, Fluorescence, Microscopy

36) Product Images from "Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties"

Article Title: Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects “stemness” properties

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0705-0

Impact of long-term expansion of DPSCs and aBMMSCs using three different culture media (CCM, StemMacs and StemPro) on β-galactosidase activity. a, b Optical microscopy photographs of DPSCs and aBMMSCs, respectively (sale bars: 100 μm). c Percentage of SA-β-gal-positive cells (DPSCs and aBMMSCs) at early, middle and late passages of each expansion medium. Values are mean (± SD) of DPSCs ( n = 6 donors, experiments repeated three times in duplicates) and aBMMSCs ( n = 4 donors, experiments repeated three times in duplicates). Asterisks indicate statistically significant differences (* p
Figure Legend Snippet: Impact of long-term expansion of DPSCs and aBMMSCs using three different culture media (CCM, StemMacs and StemPro) on β-galactosidase activity. a, b Optical microscopy photographs of DPSCs and aBMMSCs, respectively (sale bars: 100 μm). c Percentage of SA-β-gal-positive cells (DPSCs and aBMMSCs) at early, middle and late passages of each expansion medium. Values are mean (± SD) of DPSCs ( n = 6 donors, experiments repeated three times in duplicates) and aBMMSCs ( n = 4 donors, experiments repeated three times in duplicates). Asterisks indicate statistically significant differences (* p

Techniques Used: Activity Assay, Microscopy

37) 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

38) 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

39) Product Images from "Xela DS2 and Xela VS2: two novel skin epithelial-like cell lines from adult African clawed frog (Xenopus laevis) and their response to an extracellular viral dsRNA analogue"

Article Title: Xela DS2 and Xela VS2: two novel skin epithelial-like cell lines from adult African clawed frog (Xenopus laevis) and their response to an extracellular viral dsRNA analogue

Journal: bioRxiv

doi: 10.1101/2020.05.08.084723

Senescence associated β-galactosidase activity of Xela DS2 and Xela VS2 cells from early and established cultures. Xela DS2 and Xela VS2 cells were tested for senescence associated β-galactosidase activity appear blue. Photomicrographs of Xela BMW3 cells at passage 11 (A), Xela DS2 cells at passage 9 (C), Xela VS2 cells at passage 13 (E), Xela BMW3 cells at passage 20 (B), Xela DS2 cells at passage 74 (D), and Xela VS2 cells at passage 75 (F). Xela BMW3 cells were used as a positive control for β-galactosidase activity. The percentages of β-galactosidase positive cells are indicated in the top-right hand corner of each image. Scale bars represent a distance of 50 μM.
Figure Legend Snippet: Senescence associated β-galactosidase activity of Xela DS2 and Xela VS2 cells from early and established cultures. Xela DS2 and Xela VS2 cells were tested for senescence associated β-galactosidase activity appear blue. Photomicrographs of Xela BMW3 cells at passage 11 (A), Xela DS2 cells at passage 9 (C), Xela VS2 cells at passage 13 (E), Xela BMW3 cells at passage 20 (B), Xela DS2 cells at passage 74 (D), and Xela VS2 cells at passage 75 (F). Xela BMW3 cells were used as a positive control for β-galactosidase activity. The percentages of β-galactosidase positive cells are indicated in the top-right hand corner of each image. Scale bars represent a distance of 50 μM.

Techniques Used: Activity Assay, Positive Control

40) Product Images from "The UbL-UBA Ubiquilin4 protein functions as a tumor suppressor in gastric cancer by p53-dependent and p53-independent regulation of p21"

Article Title: The UbL-UBA Ubiquilin4 protein functions as a tumor suppressor in gastric cancer by p53-dependent and p53-independent regulation of p21

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-018-0141-4

Effects of Ubqln4 in GC cells are mediated by p21. MKN45 cells were infected with Ubqln4 lentivirus alone or together with p21-sh2, p21-sh5, or GFP-sh lentivirus. Cells infected with empty vector lentivirus served as controls. Cells were screened in 2 μg/ml puromycin for 5 days. a MTT assay, b colony-formation assays, and c SA-β-Gal staining assays were conducted in the indicated cell lines as described in Figs. 2 and 3 . Student’s t -test, ** P
Figure Legend Snippet: Effects of Ubqln4 in GC cells are mediated by p21. MKN45 cells were infected with Ubqln4 lentivirus alone or together with p21-sh2, p21-sh5, or GFP-sh lentivirus. Cells infected with empty vector lentivirus served as controls. Cells were screened in 2 μg/ml puromycin for 5 days. a MTT assay, b colony-formation assays, and c SA-β-Gal staining assays were conducted in the indicated cell lines as described in Figs. 2 and 3 . Student’s t -test, ** P

Techniques Used: Infection, Plasmid Preparation, MTT Assay, Staining

Ubqln4 induces cellular senescence and cell cycle arrest. a Induction of cellular senescence by Ubqln4 overexpression. Senescence-associated β-galactosidase (SA-β-Gal) staining was performed in MKN45 and BGC-823 stable cell lines overexpressing Ubqln4 and the respective controls. Data represents mean ± SD of triplicate assays. Student’s t -test, *** P
Figure Legend Snippet: Ubqln4 induces cellular senescence and cell cycle arrest. a Induction of cellular senescence by Ubqln4 overexpression. Senescence-associated β-galactosidase (SA-β-Gal) staining was performed in MKN45 and BGC-823 stable cell lines overexpressing Ubqln4 and the respective controls. Data represents mean ± SD of triplicate assays. Student’s t -test, *** P

Techniques Used: Over Expression, Staining, Stable Transfection

Related Articles

Staining:

Article Title: Isolation of a Stable Subpopulation of Mobilized Dental Pulp Stem Cells (MDPSCs) with High Proliferation, Migration, and Regeneration Potential Is Independent of Age
Article Snippet: .. Senescence associated (SA)-β-gal (Senescence Cells Histochemical Staining Kit, Sigma-Aldrich, St. Louis, MO, USA) staining assay and real-time RT-PCR analysis of senescence related genes (p16, p21, Interleukin-1β (IL-1β ), Interleukin-6 (IL-6 ), Interleukin-8 (IL-8 ) and Growth related oncogene-α (GROα ) were performed in the 6th and 20th passages of young MDPSCs and DPSCs in comparison with aged MDPSCs and DPSCs as described previously . .. To analyze the karyotype of aged MDPSCs at the 7th passage, chromosomes prepared from cells were stained with quinacrine mustard (Sigma-Aldrich) and Hoechst 33258 (Sigma-Aldrich) dissolved in a McIlvane's buffered solution (pH 7.0) and examined under a fluorescence microscope.

Article Title: Astragaloside IV inhibits astrocyte senescence: implication in Parkinson’s disease
Article Snippet: .. Astrocyte senescence was evaluated using β-galactosidase-based Senescence Cells Staining Kit (CS0030-1KT, Sigma-Aldrich, USA) according to the manufacturer’s instructions. .. Astrocytes stained blue at pH 6 were observed by a light microscope.

Article Title: Oxidative stress triggered by naturally occurring flavone apigenin results in senescence and chemotherapeutic effect in human colorectal cancer cells
Article Snippet: .. Chemicals and reagents Apigenin, Senescence Cells Histochemical Staining Kit, Griess reagent were purchased from Sigma Chemicals Co., USA. .. Dulbecco's modified Eagle's medium (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with l -glutamine, fetal bovine serum (FBS), penicillin, streptomycin, Dulbecco's phosphate-buffered saline (D -PBS) and Hank's balanced salt solution (HBSS) were all procured from Gibco (Invitrogen), USA.

Article Title: Rapamycin prevents the intervertebral disc degeneration via inhibiting differentiation and senescence of annulus fibrosus cells
Article Snippet: .. SA-β-gal staining by histochemical staining kit The cell senescence were detected with β-galactosidase positively stained cells using a senescent cell histochemical staining kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol. ..

Article Title: Rapamycin prevents the intervertebral disc degeneration via inhibiting differentiation and senescence of annulus fibrosus cells
Article Snippet: .. The cell senescence were detected with β-galactosidase positively stained cells using a senescent cell histochemical staining kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol. ..

Article Title: Derivation of Cell-Engineered Nanovesicles from Human Induced Pluripotent Stem Cells and Their Protective Effect on the Senescence of Dermal Fibroblasts
Article Snippet: .. SA-β-Gal Staining SA-β-Gal staining was conducted using senescent cells histochemical staining kit (Sigma-Aldrich). .. HDFs were plated in 96-well plate, and incubated in growth medium for 24 h. Following treatment with the indicated concentration of iPSC-CENVs for 48 h, cells were further incubated in serum-free DMEM/F12 for another 48 h. After washing with PBS, cells were fixed with 4% paraformaldehyde, and SA-β-Gal was stained by treatment with 100 µL staining mixture.

Article Title: Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast
Article Snippet: .. Senescent cell assay Senescence-associated β-galactosidase (SA-β-Gal) activity, which is increased through aging, was measured by the Senescent Cells Histochemical Staining Kit (Sigma, USA) ( ; ). .. Skin fibroblast was cultured in 35-mm culture dishes (1×105 / well) with DMEM media with each HLI, HFI and HTI for 72 h. After fixing for 7 min with 0.2% glutaraldehyde and 2% formaldehyde, the staining solution (5mM potassium ferricyanide, 5mM potassium ferrocyanide, X-gal solution) was added to the media and incubated at 37℃ without CO2 for 24 h. The senescent skin fibroblast, dyed in blue, was examined under a microscope (×200) (Olympus, Japan)

Activity Assay:

Article Title: Oxidative stress triggered by naturally occurring flavone apigenin results in senescence and chemotherapeutic effect in human colorectal cancer cells
Article Snippet: .. SA-β-Gal activity assay (senescence estimation) The SA-β-gal activity assay was performed by using the Senescence Cells Histochemical Staining Kit (Sigma Chemicals Co., USA) as described by the manufacturer. ..

Article Title: Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast
Article Snippet: .. Senescent cell assay Senescence-associated β-galactosidase (SA-β-Gal) activity, which is increased through aging, was measured by the Senescent Cells Histochemical Staining Kit (Sigma, USA) ( ; ). .. Skin fibroblast was cultured in 35-mm culture dishes (1×105 / well) with DMEM media with each HLI, HFI and HTI for 72 h. After fixing for 7 min with 0.2% glutaraldehyde and 2% formaldehyde, the staining solution (5mM potassium ferricyanide, 5mM potassium ferrocyanide, X-gal solution) was added to the media and incubated at 37℃ without CO2 for 24 h. The senescent skin fibroblast, dyed in blue, was examined under a microscope (×200) (Olympus, Japan)

Quantitative RT-PCR:

Article Title: Isolation of a Stable Subpopulation of Mobilized Dental Pulp Stem Cells (MDPSCs) with High Proliferation, Migration, and Regeneration Potential Is Independent of Age
Article Snippet: .. Senescence associated (SA)-β-gal (Senescence Cells Histochemical Staining Kit, Sigma-Aldrich, St. Louis, MO, USA) staining assay and real-time RT-PCR analysis of senescence related genes (p16, p21, Interleukin-1β (IL-1β ), Interleukin-6 (IL-6 ), Interleukin-8 (IL-8 ) and Growth related oncogene-α (GROα ) were performed in the 6th and 20th passages of young MDPSCs and DPSCs in comparison with aged MDPSCs and DPSCs as described previously . .. To analyze the karyotype of aged MDPSCs at the 7th passage, chromosomes prepared from cells were stained with quinacrine mustard (Sigma-Aldrich) and Hoechst 33258 (Sigma-Aldrich) dissolved in a McIlvane's buffered solution (pH 7.0) and examined under a fluorescence microscope.

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  • 90
    Millipore senescence assay senescence associated β galactosidase activity
    Apoptosis and senescence in tumor sections from control and mixed cells. (A) Sections of tumor tissues were subjected to TUNEL assay. Blue colour shows nuclei of cells stained with DAPI. Green foci show TUNEL positive regions. (B) Tumor sections processed for SA-β-gal assay. The part of image is shown below in the respective inset. Greenish blue color indicates active <t>SA-β-galactosidase.</t>
    Senescence Assay Senescence Associated β Galactosidase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/senescence assay senescence associated β galactosidase activity/product/Millipore
    Average 90 stars, based on 77 article reviews
    Price from $9.99 to $1999.99
    senescence assay senescence associated β galactosidase activity - by Bioz Stars, 2020-08
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    91
    Millipore senescence associated β galactosidase sa βgal assay senescence associated β galactosidase
    The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or ( c ) cytokines (50 ng/ml) during 5 days and stained for <t>SA-β-GAL.</t> Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or ( e ) cytokines, as indicated. The values were normalized to GADPH and relative to control cells ( dotted lines ). Error bars represent SEM. (* p
    Senescence Associated β Galactosidase Sa βgal Assay Senescence Associated β Galactosidase, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/senescence associated β galactosidase sa βgal assay senescence associated β galactosidase/product/Millipore
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    senescence associated β galactosidase sa βgal assay senescence associated β galactosidase - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

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    Apoptosis and senescence in tumor sections from control and mixed cells. (A) Sections of tumor tissues were subjected to TUNEL assay. Blue colour shows nuclei of cells stained with DAPI. Green foci show TUNEL positive regions. (B) Tumor sections processed for SA-β-gal assay. The part of image is shown below in the respective inset. Greenish blue color indicates active SA-β-galactosidase.

    Journal: PLoS ONE

    Article Title: Molecular Understanding of Growth Inhibitory Effect from Irradiated to Bystander Tumor Cells in Mouse Fibrosarcoma Tumor Model

    doi: 10.1371/journal.pone.0161662

    Figure Lengend Snippet: Apoptosis and senescence in tumor sections from control and mixed cells. (A) Sections of tumor tissues were subjected to TUNEL assay. Blue colour shows nuclei of cells stained with DAPI. Green foci show TUNEL positive regions. (B) Tumor sections processed for SA-β-gal assay. The part of image is shown below in the respective inset. Greenish blue color indicates active SA-β-galactosidase.

    Article Snippet: Senescence assay Senescence associated β-galactosidase activity was assessed in tumor sections using cellular senescence assay kit (Millipore) as per the instructions provided by the manufacturer.

    Techniques: TUNEL Assay, Staining, β-Gal Assay

    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:

    The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or ( c ) cytokines (50 ng/ml) during 5 days and stained for SA-β-GAL. Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or ( e ) cytokines, as indicated. The values were normalized to GADPH and relative to control cells ( dotted lines ). Error bars represent SEM. (* p

    Journal: Cell Communication and Signaling : CCS

    Article Title: Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

    doi: 10.1186/s12964-017-0172-3

    Figure Lengend Snippet: The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or ( c ) cytokines (50 ng/ml) during 5 days and stained for SA-β-GAL. Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or ( e ) cytokines, as indicated. The values were normalized to GADPH and relative to control cells ( dotted lines ). Error bars represent SEM. (* p

    Article Snippet: Senescence-associated β-galactosidase (SA-βGAL) assay Senescence-associated β-galactosidase (SA-βGAL) activity was evaluated in senescent- and young fibroblasts after 2 days of culture by using the cellular senescence assay kit (KAA002, Millipore).

    Techniques: Staining, Expressing