Journal: Cell Cycle

Article Title: Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

doi: 10.1080/15384101.2016.1189041

Figure Lengend Snippet: Doxorubicin induced premature senescence and expression of Sirtuin isoforms. (A) U2OS cells were treated with doxorubicin (Dox, 1 µM, 2 h), grown in fresh culture medium for 120 h and assayed for SA-βgal (blue). Untreated cells served as control. (B) Bar diagram showing percentage SA-βgal positive cells in control and doxorubicin treated U2OS cells at 120 h (*P < 0.05). (C) Immunoblots showing expression of senescence-associated markers viz., p53, p21, PAI-1 and Lamin B1 in control and senescent cells at 120 h. (D) Time kinetics showing cell cycle distribution of control and doxorubicin treated cells. (E) Bar diagram showing relative fold change in expression levels of various Sirtuin transcripts in control and senescent U2OS cells at 120 h. The transcript levels of SIRT1-SIRT7 were quantified by real-time PCR and normalized to GAPDH mRNA (*P < 0.05). (F) Immunoblots showing expression of various Sirtuin isoforms in control vs. senescent cells at 120 h. (G) Bar diagram showing the fold increase in expression levels of Sirtuin proteins in senescent cells in comparison to control cells. Note the presence of 2 distinct isoforms for both SIRT2 (39 and 43 kDa) and SIRT7 (45 and 47.5 kDa) on the immunoblot. To calculate the total fold change, the corresponding increase in both the isoforms were normalized first to GAPDH and the values obtained were averaged together to represent the total pool of either SIRT2 or SIRT7 in the bar graph (*P < 0.05).

Article Snippet: U2OS (p53 +/+ ) and Saos-2 cells (p53 −/− ) were a kind gift by Dr. Renu Wadhwa (AIST, Japan) and primary dermal fibroblasts (p53 wild type) of human origin were purchased from HiMedia, India.

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

Journal: Cell Cycle

Article Title: Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

doi: 10.1080/15384101.2016.1189041

Figure Lengend Snippet: SIRT2 expression in various modes of senescent conditions viz., stress-, replicative- and oncogene- induced. (A) SA-βgal staining of U2OS cells at 120 h following treatment with DNA damaging agents such as doxorubicin (Dox, 1 µM, 2 h), camptothecin (Campto, 1 µM, 2 h), etoposide (Etopo, 1 µM, 72 h) or oxidative stress agent, H2O2 (200 µM, 2 h). (B) Immunoblots showing expression of SIRT2 and p21 in U2OS control and senescent cells (120 h) induced by various agents. (C) Bar diagram showing fold change in expression level of SIRT2 in stress-induced senescence by different agents (*P < 0.05). (D) SA-βgal activity in human primary adult dermal fibroblasts undergoing replicative senescence. (E) Immunoblots showing expression of SIRT2 and Lamin B1 in young (passage 4, 17) and senescent fibroblasts (passage 57). The values below the blots indicate the relative fold change in total SIRT2 and Lamin B1 levels. (F) SA-βgal staining in untransfected U2OS cells and transfected with either vector alone or mutant RAS (HRAS-V12). (G) Immunoblots showing expression of RAS, SIRT2 and Lamin B1 in the untransfected cells (U2OS) and those transfected with either vector or HRAS-V12. (H) Bar graph showing relative change in total SIRT2 levels in oncogene-induced senescent cells compared to vector control cells (*P < 0.05).

Article Snippet: U2OS (p53 +/+ ) and Saos-2 cells (p53 −/− ) were a kind gift by Dr. Renu Wadhwa (AIST, Japan) and primary dermal fibroblasts (p53 wild type) of human origin were purchased from HiMedia, India.

Techniques: Expressing, Staining, Western Blot, Control, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis

Journal: Cell Cycle

Article Title: Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

doi: 10.1080/15384101.2016.1189041

Figure Lengend Snippet: p53 regulates expression of SIRT2. (A) SA-βgal staining in p53 null cells (Saos-2) treated with doxorubicin (0.5 µM, 1 h or 50 nM, 48 h) at 120 h. (B) Immunoblots showing expression of total SIRT2 in control and senescent Saos-2 cells at 120 h. Values below the blot indicate fold change in total level of SIRT2 normalized to GAPDH. (C) U2OS cells transfected with either p53 shRNA or non-target shRNA (NT shRNA) were treated with doxorubicin (1 µM, 2 h) and SA-βgal staining was performed at 120 h. (D) Immunoblots showing expression of p53, SIRT2 and p21 in U2OS cells with or without p53 depletion (p53 shRNA) treated with varying doses of doxorubicin. (E) Quantification of fold change in expression of SIRT2 in NT shRNA and p53 depleted cells (*P < 0.05). (F) SA-βgal staining of U2OS cells treated with doxorubicin, and nutlin either alone or in combination. (G) Immunoblot analysis of p53, SIRT2 and p21 in U2OS cells treated with doxorubicin (0.2 µM, 2 h) and nutlin (5 µM) either alone or in combination. (H) Quantification of fold change of total SIRT2 in doxorubicin and nutlin treated U2OS cells either alone or together.

Article Snippet: U2OS (p53 +/+ ) and Saos-2 cells (p53 −/− ) were a kind gift by Dr. Renu Wadhwa (AIST, Japan) and primary dermal fibroblasts (p53 wild type) of human origin were purchased from HiMedia, India.

Techniques: Expressing, Staining, Western Blot, Control, Transfection, shRNA

Journal: Cell Cycle

Article Title: Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

doi: 10.1080/15384101.2016.1189041

Figure Lengend Snippet: SIRT2 promoter has a p53 binding site. (A) Schematic representation of chromosome 19 showing location of SIRT2 gene (39370265-39390629 bp) and p53 binding site at its promoter region [+348 bp to +368 bp from transcription start site (TSS)]. Positions of primers used in ChIP assay are indicated by the arrows (+239 bp to +487 bp from TSS). (B, C) ChIP assay was performed in control and senescent U2OS cells using a p53 specific antibody or mouse IgG as control, followed by quantitative PCR with primers specific for SIRT2 and p21 promoter regions. Bar diagram indicates the fold enrichment of p53 binding at SIRT2/p21 promoter in control and doxorubicin induced senescent cells (*P < 0.05). (D) U2OS control and doxorubicin induced senescent cells were transfected with either SEAP plasmid alone (Vector) or with SEAP plasmid containg SIRT2 promoter with intact p53 binding site (SIRT2) or with deletion of p53 binding region (SIRT2_p53 del). Later at 24 h post transfection, SEAP assay was performed using the culture-conditioned medium from both control and senescent cells. Bar diagram indicates the SEAP activity (plotted as fold change over control) in control and senescent U2OS cells (* P < 0.05).

Article Snippet: U2OS (p53 +/+ ) and Saos-2 cells (p53 −/− ) were a kind gift by Dr. Renu Wadhwa (AIST, Japan) and primary dermal fibroblasts (p53 wild type) of human origin were purchased from HiMedia, India.

Techniques: Binding Assay, Control, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, SEAP Assay, Activity Assay