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