Journal: International Journal of Molecular Sciences

Article Title: A Simple Ionic-Gelation Method for Chitosan Nanoparticle Synthesis and Standardized Protocols for Biological Safety Assessment: Antibacterial Activity, Phytotoxicity, and Biocompatibility

doi: 10.3390/ijms27083673

Figure Lengend Snippet: Cell viability of HDF cells treated with Ch NPs at concentrations of 2.5–160 µg/mL for 24 h. Viability was determined using the MTT assay and expressed as mean ± SE (n = 3). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: The biocompatibility of Ch NPs was evaluated using primary HDF (ATCC ® PCS-201-012TM [ ], American Type Culture Collection, Manassas, VA, USA) and HaCaT cells (Cat. No. T0020001, AddexBio, San Diego, CA, USA).

Techniques: MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypothermically Stored Amnion Is Robust and Provides a Scaffold for Supporting Wound Healing by Retaining the Characteristics of Native Tissue

doi: 10.3390/ijms251910347

Figure Lengend Snippet: Fibroblast attachment and proliferation following seeding with human dermal fibroblasts (NHDF) and mouse fibroblasts (L929) onto hypothermically stored amniotic membranes. Cell number from AlamarBlue assessment showing metabolic activity of NHDFs ( A ) and L929s ( B ). ( C ) Representative immunofluorescence staining of seeded and non-seeded scaffolds at day 3 and 14; 40× magnification. Average ± standard deviation reported; ns denotes not significant; * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001; **** denotes p < 0.0001 compared to non-seeded controls. Blue: nuclei; green: TGF-β1; red: f-actin (phalloidin); arrow: epithelial layer; S: spongy layer; Scale bar: 25 µm.

Article Snippet: Primary normal adult human dermal fibroblasts (NHDFs, lot 22TL073035 [female, age 46, Black], Lonza Biosciences, Walkersville, MD, USA) and two lots of primary mouse fibroblasts (L929, lots 14A015/70058860, ATCC, Bethesda, MD, USA) were thawed and cultured as per the manufacturer’s recommendations.

Techniques: Activity Assay, Immunofluorescence, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Hypothermically Stored Amnion Is Robust and Provides a Scaffold for Supporting Wound Healing by Retaining the Characteristics of Native Tissue

doi: 10.3390/ijms251910347

Figure Lengend Snippet: Structural characterization of fibroblasts seeded onto hypothermically stored amniotic membranes. Representative scanning electron microscopy images of non-seeded and human fibroblasts seeded ( A ) and mouse fibroblast seeded ( B ) grafts. For SEM, representative images are at 100× (scale bar: 500 µm) and 500× (scale bar: 100 µm).

Article Snippet: Primary normal adult human dermal fibroblasts (NHDFs, lot 22TL073035 [female, age 46, Black], Lonza Biosciences, Walkersville, MD, USA) and two lots of primary mouse fibroblasts (L929, lots 14A015/70058860, ATCC, Bethesda, MD, USA) were thawed and cultured as per the manufacturer’s recommendations.

Techniques: Electron Microscopy

Journal: PLoS ONE

Article Title: Characterization of the Rat Oncostatin M Receptor Complex Which Resembles the Human, but Differs from the Murine Cytokine Receptor

doi: 10.1371/journal.pone.0043155

Figure Lengend Snippet: A , Rat dermal fibroblasts (RDF) and B , human dermal fibroblasts (HDF) were treated with 10 ng/ml hLIF, hOSM, mOSM or rOSM for 15 min. The phosphorylation levels of STAT1, STAT3, ERK1/2 as well as STAT5, p38 and Akt were detected via Western blot analysis. The blots were stripped and reprobed with antibodies recognizing the proteins irrespective of their activation status. Additionally an α-tubulin loading control was included. Phosphorylation intensities were quantified by chemiluminescence analysis and normalized to tubulin. Activation determined for rOSM was set to 100%. Shown are the means (n = 3) with standard error of mean (SEM). * p<0.05, ** p<0.01, *** p<0.001 untreated vs. cytokine-treated sample.

Article Snippet: Primary rat dermal fibroblasts were obtained from PELOBiotech (Martinsried, Germany).

Techniques: Phospho-proteomics, Western Blot, Activation Assay, Control

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: Cell lines 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: Cell lines 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: Cell lines 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: Cell lines 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