Journal: The American journal of pathology

Article Title: The antiviral cytokines IFN-α and IFN-β modulate parietal epithelial cells and promote podocyte loss: implications for IFN toxicity, viral glomerulonephritis, and glomerular regeneration.

doi: 10.1016/j.ajpath.2013.04.017

Figure Lengend Snippet: Figure 2 IFN-a arrests PEC growth by affecting the cell cycle. A and D: Human and murine renal PECs were cultured with 5000 U/mL human IFN-a and IFN-b for 24 hours and cell proliferation was assessed. Data are means SEM of three experiments, each analyzed in quadruplicate. *P < 0.05 versus control. B and E: In a similar experiment, cellular lysate was analyzed by Western blot for the expression of p21. b-Actin staining is shown as loading control. One representative blot of several blots from three independent experiments is displayed. C and F: Human and murine PECs stimulated for 24 hours with 5000 U/mL IFN-a and IFN-b were stained with annexin V and PI and analyzed by flow cytometry. Increase of PEC death was not detected on IFN-a or IFN-b stimulation.

Article Snippet: Protein Isolation and Western Blotting Protein was extracted from renal cells using RIPA buffer (Sigma, Munich, Germany) containing protease inhibitors (Roche, Mannheim, Germany) and processed for Western blotting as described.21 The membranes were incubated overnight at 4 C with goat antibody against mouse p21 (clone F-5; Santa Cruz, Heidelberg, Germany).

Techniques: Cell Culture, Control, Western Blot, Expressing, Staining, Cytometry

Journal: Disease Models & Mechanisms

Article Title: TRANSLATIONAL IMPACT

doi: 10.1242/dmm.002675

Figure Lengend Snippet: Fig. 5. Accumulation of p21 and p19ARF in K5TRF2 skin. (a) Representative image of a western blot analysis of p21 protein expression in wild-type and K5TRF2 ventral skin. (b) Quantification of p21 expression relative to β-actin expression is expressed as mean (values indicated) ± s.e.m. n=number of samples analyzed per group. Statistical analysis was performed by using the Student’s t-test with Welch’s correction for unequal variance. (c) Representative images of wild-type and K5TRF2 ventral skin stained for p21 (brown) and hematoxylin (blue). Black arrowheads indicate basal cells that are positive for p21 expression. Bar, 20 μm. (d) Percentage of p21-positive basal cells observed in ventral skin sections. All groups include at least three mice (M) and at least six microscopic fields (n) were analyzed per mouse; in total, over 800 basal cells were analyzed. The percentage of p21-positive cells is shown, as well as the number of p21- positive cells per total number of basal cells analyzed. (e) Representative image of western blot analysis of p19ARF protein expression in wild-type and K5TRF2 ventral skin. (f) Quantification of p19ARF expression relative to β-actin expression. (g) Representative image of ventral skin from wild-type and K5TRF2 mice stained for p19ARF (brown) and hematoxylin (blue). Black arrowheads indicate the p19ARF- positive basal cells. Bar, 20 μm. (h) Percentage of p19ARF-positive basal cells detected in the ventral skin. All groups include at least three mice (M) and at least six microscopic fields (n) were analyzed per mouse; in total, over 900 basal cells were analyzed. The percentage of p19ARF- positive cells is shown, as well as the number of p19ARF-positive cells per total number of basal cells analyzed. (b,d,f,h) All data expressed as mean (values indicated) ± s.e.m. n=number of samples analyzed per group. Statistical analysis was performed by using the Student’s t-test with Welch’s correction for unequal variance.

Article Snippet: Sections were then blocked with fetal bovine serum (FBS) and incubated with rabbit anti-MSHalpha (1:1200; M0939, Sigma), goat polyclonal anti-p21 (1:75; Santa Cruz C-19-G, sc397-G), rat monoclonal antibody anti-p19arf (1:15; Santa Cruz Clone 5-C3-1), HRP-conjugated Ki67 (pre-diluted 1:200; Master Diagnostica 000310QD) and rabbit polyclonal anti-C3A (1:200; RyD AF835).

Techniques: Western Blot, Expressing, Staining

Journal: Cell Cycle

Article Title: A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

doi: 10.4161/cc.23515

Figure Lengend Snippet: Figure 1. Identification of senescence-inducing kinase siRNAs. ( A ) Schematic overview of the high-content senescence screen. ( B ) Scatterplot analysis of data from the kinome HCS screen in p53+ cells. Cells were stained for Ki67, p53, p21 CIP1 and Hoechst at day 3, and multiparametric analysis was performed to determine siRNAs displaying decreased proliferation (< 0.5 StDev Ki67%) and an increased nuclear size (> 2.0 StDev Hoechst mean area). High, medium and low cell number indicators are assigned as the third parameter. ( C ) Senescence scores per gene are the sum of the senescence scores of its siRNA pool and the four individual siRNAs, omitting siRNAs conferring less than 50% specific gene knockdown. Scoring details are depicted in . References to studies establishing bona fide tumor suppressor functions for human ( Hs ) or mouse ( Mm ) genes are indicated.

Article Snippet: Primary antibodies were rabbit anti-p53 (Cell Signaling 928; 1:1500), goat anti-p21 (R&D Systems AF1047; 1:20.000) and mouse-anti-Ki67 (BD PharMingen 55600; 1:1500).

Techniques: Staining, Knockdown

Journal: Cell Cycle

Article Title: A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

doi: 10.4161/cc.23515

Figure Lengend Snippet: Figure 2. Delineation of senescence signatures. ( A ) hTERT-RPE1 p53 shRNA cells were transfected with kinome siRNAs in the absence of doxycycline and immunostained for p53 and p21 CIP1 following 72 h culture. Percentages of cells expressing p53, p21 CIP1 above threshold levels were calculated, and averages from four 384 wells are shown. ( B ) hTERT-RPE1 p53 shRNA cells were transfected with kinome siRNAs in the absence or presence of doxycycline and immunostained for Ki67. Percentages of cells expressing Ki67 protein above threshold levels were calculated, and averages from four 384 wells are shown. * indicates p ≤ 0,05 and ** indicates p ≤ 0,01. ( C ) Senescence scores per gene are the sum of the senescence scores of the siRNA pool plus four individual siRNAs, omitting siRNAs conferring less than 50% knockdown. Detailed scoring information is depicted in . ( D ) Quantitative p16 INK4A mRNA expression analyses during senescence induction. hTERT-RPE1 p53 shRNA cells were transfected with pooled siRNAs in the absence of doxycycline. RNA expression was quantitated using TaqMan analyses after 3 days of transfection. ( E ) Schematic model summarizing kinome screen data, using data depicted in ( A ) and ( D ). In an incipient tumor, modeled by hTERT-RPE1 cells, loss of selected tumor suppressors activates p53- and/or p16 INK4A -dependent senescence, and overt DNA damage. In a premalignant tumor, senescence may serve as a cell-intrinsic tumor suppressor mechanism to subvert oncogenic transformation. Loss of p16 INK4A and/or p53 promotes malignancy.

Article Snippet: Primary antibodies were rabbit anti-p53 (Cell Signaling 928; 1:1500), goat anti-p21 (R&D Systems AF1047; 1:20.000) and mouse-anti-Ki67 (BD PharMingen 55600; 1:1500).

Techniques: shRNA, Transfection, Expressing, Knockdown, RNA Expression, Transformation Assay

Journal: Cancer Research

Article Title: TheRgrOncogene Induces Tumorigenesis in Transgenic Mice

doi: 10.1158/0008-5472.can-03-3389

Figure Lengend Snippet: Fig. 5. Specific role of the lack of p15INK4b in MSV-RGR/KO-p15INK4b fibrosarco- mas. A. Table shows p21, p16, and p53 protein expression levels detected by immuno- histochemistry in seven MSV-RGR/KO-p15INK4b fibrosarcomas. Protein expression: , 5–50% positive cells; , 50% positive cells; and, , 50% positive cells with high levels of expression per cell. B. Representative pictures show the expression of p16, p21, and p53 in two different MSV-RGR/KO-p15INK4b fibrosarcomas. Sample 6609T shows normal expression of both p16 and p53 and moderated overexpression of p21. Sample 6866TA shows overexpression of the three analyzed proteins.

Article Snippet: Antibodies used were as follows: rabbit antimouse p16 (M-156; 1:35 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit antimouse p21 (C-19; 1:25 dilution; Santa Cruz Biotechnology), and rabbit antimouse p53 (CM5p; 1:100 dilution; Novocastra, Newcastle upon Tyne, United Kingdom).

Techniques: Expressing, Immunohistochemistry, Over Expression