bj human foreskin fibroblast cultures  (ATCC)

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 induces cellular senescence in fibroblasts

doi: 10.1007/s12079-016-0359-1

Figure Lengend Snippet: CCN2 induces cellular senescence in normal human BJ fibroblasts. a Immunoblots of purified CCN1 and CCN2 protein probed with either anti-CCN1 or anti-CCN2 antibodies. b Human BJ fibroblasts treated with BSA, purified recombinant CCN1, or CCN2 (2.5 μg/ml each) were grown for indicated days and cell numbers counted using a haemocytometer. c After indicated treatment for 3 days, cells were subjected to immunostaining for Ki-67 and counterstained with DAPI. Cells positive for Ki-67 were counted in ten random fields and expressed as percentages of total cell number. d Cells were stained for SA-β-gal activity; positive cells were counted in ten random fields and expressed as percentages of total cell number. e Phase-contrast microscopy showing enlarged and flattened senescence morphology in cells treated with CCN2. f Total RNA was isolated from BJ cells treated with either BSA or CCN2 for 4 days and mRNAs for IL6, IL8, MMP1, and COL1a1 were quantified by qPCR (n = 4 per time point). g BJ cells were infected with recombinant adenoviral vectors (Ad-LacZ and Ad-CCN2) at a multiplicity of infection of 10 for 2 h. Subsequently, cell numbers were counted at indicated days. h Gene expression at day 4 after adenoviral infection was quantified using qPCR (n = 3 per time point). Experiments were done in triplicates and data presented as means ± s.d. *p < 0.05, **p < 0.01

Article Snippet: * p < 0.05, ** p < 0.01 Cell culture Human BJ foreskin fibroblasts (ATCC# CRL-2522) were maintained at 37 °C in 5 % CO 2 in Eagle’s minimum essential medium (EMEM) containing 10 % fetal bovine serum (FBS; Hyclone), 1 % penicillin/streptomycin, non-essential amino acids and sodium pyruvate.

Techniques: Western Blot, Purification, Recombinant, Immunostaining, Staining, Activity Assay, Microscopy, Isolation, Infection, Gene Expression

Journal: Journal of Cell Communication and Signaling

Article Title: CCN2 induces cellular senescence in fibroblasts

doi: 10.1007/s12079-016-0359-1

Figure Lengend Snippet: CCN2-induced senescence requires p53 and p16INK4a. a Cell lysates from BJ fibroblasts treated with either BSA or CCN2 were electrophoresed and immunoblotted to show increases in p53, p16INK4a, and hypophosphorylated pRb. b Cells were infected with either empty lentivirus (LV) or lentivirus driving expression of shRNAs against p53; cells were then treated with BSA or CCN2 for indicated times before cell numbers counted. c Cells were infected with either LV or lentivirus driving expression of shRNAs against p16INK4a; cells were then treated with BSA or CCN2 for indicated times and cell numbers counted. Knockdown effects of shp53 and shp16 were confirmed using Western blotting. Experiments were done in triplicates and data presented as means ± s.d

Article Snippet: * p < 0.05, ** p < 0.01 Cell culture Human BJ foreskin fibroblasts (ATCC# CRL-2522) were maintained at 37 °C in 5 % CO 2 in Eagle’s minimum essential medium (EMEM) containing 10 % fetal bovine serum (FBS; Hyclone), 1 % penicillin/streptomycin, non-essential amino acids and sodium pyruvate.

Techniques: Infection, Expressing, Knockdown, Western Blot