Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 1. Effect of in vitro (A and B) and in vivo (C and D) aging on fibroblast motility (A and C) and proliferation (B and D). A and B, Hs68 were passaged and tested at different passages with PDR back-calculated. Cell migration assays and cell proliferation assays were performed in the absence (E and open bars) and presence (G and black bars) of EGF (1 nM) as described under “Experimental Procedures.” The data are the mean 6 S.E. of more than three independent studies, each performed in triplicate. Statistical analysis was performed by Student’s t test as compared with P5 (PDR42) of Hs68: *, p , 0.05, **, p , 0.01 C, fibroblasts from fetus male (‚, basal; Œ, with EGF), 1-month-old male (CRL-1489) (M, basal; f, with EGF), 17-year-old male (CRL-7315) (L, basal; l, with EGF), and 83-year-old male (CRL-7815) (E, basal; G, with EGF) were assessed by cell migration assay. Fibroblasts from 1-month-old male (CRL-1489) and 83-year-old male (CRL-7815) were passaged and assessed at indicated passages. Basal and EGF-induced cell motility were measured as described under “Experimental Procedures.” The data are the mean of more than three independent studies, each performed in triplicate. Statistical analysis was preformed by Student’s t test as compared with P8 (PDR24) of CRL-1489 or P3 (PDR12) of CRL-7815: *, p , 0.05, **, p , 0.01 D, fibroblasts from different aged donor were obtained. Basal (clear bars) and EGF-induced (black bars) thymidine incorporation were measured as described under “Experimental Procedures.” The data are the mean 6 S.E. of more than three independent studies performed in triplicate. Statistical analysis was performed by Student’s t test as compared with fetal cells: *, p , 0.05, **, p , 0.01

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: In Vitro, In Vivo, Migration, Cell Migration Assay

Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 4. Ligand-induced internalization of EGFR in in vitro aged Hs68 fibroblasts (A) and fibroblasts from male donors (B). Internalized and surface-bound EGF were determined using 125I-EGF as described under “Experimental Procedures.” The endocytic rate con- stants were calculated by the time course of loss of surface-bound EGF and accumulation of internalized EGF. The data are the mean 6 S.E. of at least two experiments at each point except for CRL-7815. Statistical analysis was performed by Student’s t test as compared with early passage of cells: **, p , 0.01.

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: In Vitro

Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 8. Expression of exogenously encoded EGFR in near senescent Hs68 fibroblasts (C and D) and effects on cell motility (A) and mitogenesis (B). Eukaryotic expression plasmids for EGFR or GFP (control) were introduced into P18 (PDR3) of Hs68 using electropo- ration technology (Gene Pulser, Bio-Rad). The cells were incubated for 48 h in DME containing 0.1% dialyzed FBS before analyses as described under “Experimen- tal Procedures.” A, cell migration assay; B, thymidine incorporation; C, immuno- blot analysis with anti-phosphotyrosine antibody phosphotyrosine (PY-20, Trans- duction Laboratories); D, immunoblot analysis and densitometry using anti- EGFR (#05–104, Upstate Biotechnology Inc.) or anti-a-actin (A-2066, Sigma) anti- bodies. The data in graphs (A) and (B) are the mean 6 S.E. of three independent electroporations, with each experiment performed in triplicate. The data in (C) and (D) are representative of the electro- poration experiments. Statistical analysis was performed by Student’s t test as com- pared with control cells: *, p , 0.05, **, p , 0.01.

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: Expressing, Control, Incubation, Cell Migration Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Aging Fibroblasts Present Reduced Epidermal Growth Factor (EGF) Responsiveness Due to Preferential Loss of EGF Receptors

doi: 10.1074/jbc.m000008200

Figure Lengend Snippet: FIG. 9. Effect of increased EGFR signaling capacity on early passage of Hs68 fibroblasts. A and B. EGFR (10 mg/107 cells) or GFP (20 mg/107 cells as control) plasmid were introduced into P5 (PDR42) Hs68 cells. The cells were incu- bated for 48 h in DME containing 0.1% dialyzed FBS before analyses as described under “Experimental Procedures.” A, cell migration assay; B, immunoblot analysis with anti-phosphotyrosine antibody phos- photyrosine (PY-20, Transduction Labo- ratories), anti-EGFR (Upstate Biotech- nology Inc.) or anti-a-actin (Sigma) antibodies. The data are the mean 6 S.E. of two independent electroporations, with each experiment performed in triplicate. The blots shown are representative of the electroporation experiments. C, in vitro wound healing assays were performed with P5 of Hs68 in the absence and pres- ence of EGF (1 or 10 nM). The data are the mean 6 S.E. of more than two independ- ent studies, each performed in triplicate. There were no statistical differences in motility responses between the EGF treatments in A and C.

Article Snippet: Reagents—Hs68 and other normal human diploid fibroblasts were purchased from American Type Culture Collection (ATCC, Rockville, MD): 23-week male fetus CRL-1475 (obtained at passage 8; hereafter referred to as P8), 1-month-old male CRL-1489 (P8), 17-year-old male CRL-7315 (P5), 83-year-old male CRL-7815 (P3); 10-month-old female CRL-1497 (P6); 84-year-old female CRL-7321 (P3).

Techniques: Control, Plasmid Preparation, Cell Migration Assay, Western Blot, Transduction, Electroporation, In Vitro

Journal: The Journal of Cell Biology

Article Title: Ip-10 Inhibits Epidermal Growth Factor–Induced Motility by Decreasing Epidermal Growth Factor Receptor–Mediated Calpain Activity

doi:

Figure Lengend Snippet: Hs68 Cellular Morphometry

Article Snippet: Hs68 normal human diploid fibroblasts were purchased from American Type Culture Collection.

Techniques:

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Detection of the OSMR on lung fibroblasts. Cells were stained with no antibody or with anti-OSMR followed by FITC-labelled goat anti-rabbit IgG. Immunostaining was then analysed by flow cytometry.

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: Staining, Immunostaining, Flow Cytometry

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Proliferation of human lung fibroblasts in response to OSM. Cells were exposed to OSM at concentrations ranging from 0.5 – 100 ng ml−1 for 24, 48 or 72 h. Proliferative effect was examined using an MTS assay (A) and confirmed by [3H]-TdR uptake (B). The proliferative effect of OSM was completely abrogated by incubation of cells with neutralizing antibodies against OSMR and gp 130. Results are means of four separate experiments performed in triplicate. *P<0.05 compared to cells in serum free conditions.

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: MTS Assay, Incubation

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Cell counts of human lung fibroblasts after incubation with OSM

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: Incubation

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Detection of cyclin D1 expression in lung fibroblasts. Immunohistochemical staining for cyclin D1 was performed on unstimulated cells (D) or cells exposed to OSM (2 ng ml−1) for 24 h (E). The specificity of the cyclin D1 antibody was confirmed by substituting the primary antibody with non-immune mouse IgG1 (F). Cells were also stained with DAPI in order to enumerate cell nuclei (A – C). Immunofluorescent images from 1 μm sections were obtained by scanning laser confocal microscopy at 40×magnification.

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: Expressing, Immunohistochemical staining, Staining, Confocal Microscopy

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Effects of the tyrosine kinase inhibitor (genestein) and p42/44 MAPK inhibitor (PD98059) on OSM-induced proliferation. Fibroblasts were seeded in 24 well plates and incubated with genestein (10 μM) or PD98059 (50 μM) in the presence or in the absence of 2 ng ml−1 OSM. At 24 and 48 h cell numbers were assessed using an MTS assay. Results are means of four separate experiments performed in triplicate. *P<0.05 versus cells exposed to OSM 2 ng ml−1. #P<0.05 compared to 24 h time point.

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: Incubation, MTS Assay

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Effects of the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor nimesulide on OSM-induced proliferation, COX expression and PGE2 release. (a) Cells were treated with indomethacin (5 μM) or the selective COX-2 inhibitor nimesulide (5 μM) prior to incubation with OSM (2 ng ml−1). Proliferative responses to OSM were not influenced by either drug at 24 or 48 h. Detection of COX-1 and COX-2 protein. Fibroblasts were incubated for either 6 or 24 h with OSM (2 ng ml−1) or IL-1β (2 ng ml−1) and expression of both COX-1 and COX-2 was determined by Western analysis (b). PGE2 release in response to OSM. PGE2 release was measured by EIA in supernatants taken from fibroblasts treated with OSM (2 ng ml−1) for 24 and 48 h (c). Results are shown as means of four separate experiments. *P<0.05 compared to cells in serum free medium.

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: Expressing, Incubation, Western Blot

Journal:

Article Title: Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts

doi: 10.1038/sj.bjp.0704769

Figure Lengend Snippet: Effect of OSM on human lung fibroblast pro-collagen production. Fibroblasts were grown to confluence and incubated with 10 or 100 ng ml−1 OSM for 48 h and the production of hyp measured by HPLC. Data are means of six independent experiments. *P<0.05 compared to cells in serum free medium.

Article Snippet: The normal diploid human foetal lung fibroblast cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A) and was used between passages 11 and 24.

Techniques: Incubation

Journal: Cell Death & Disease

Article Title: Characterization of novel markers of senescence and their prognostic potential in cancer

doi: 10.1038/cddis.2014.489

Figure Lengend Snippet: Expression and localization of senescence markers. Immunofluorescent images of selected targets in EJp16 and EJp21 uninduced (Control) or 4 days after tet removal (Senescent), as well as early passage IMR90 human fibroblasts compared with those entering replicative senescence after serial passaging. Nucleus are stained with DAPI (blue)

Article Snippet: IMR90 (human fibroblasts derived from lungs of a 16-week female foetus) and normal human diploid fibroblasts (Cellworks, San Jose, CA, USA) were maintained in DMEM supplemented with 10% FBS, and penicillin–streptomycin (50 unit/ml) until they reached replicative senescence.

Techniques: Expressing, Control, Passaging, Staining

Journal: Cell Death & Disease

Article Title: Characterization of novel markers of senescence and their prognostic potential in cancer

doi: 10.1038/cddis.2014.489

Figure Lengend Snippet: Defining a new FACS-based protocol for the detection of senescent cells. ( a ) Representative plot analysis of fluorescence levels in control and senescent EJp16, HT1080p21-9 and human diploid fibroblasts (HDF) stained with fluorescently tagged antibodies against B2MG, DEP1 and NOTCH3, as measured by flow cytometry. Senescent cells were analysed after 5 days of p16 or p21 expression. Numbers indicate mean fluorescent intensity (MFI) values. ( b ) Average fold increases of MFI of the same cells when senescence is induced. Experiments were performed in triplicate. Error bars show S.D.

Article Snippet: IMR90 (human fibroblasts derived from lungs of a 16-week female foetus) and normal human diploid fibroblasts (Cellworks, San Jose, CA, USA) were maintained in DMEM supplemented with 10% FBS, and penicillin–streptomycin (50 unit/ml) until they reached replicative senescence.

Techniques: Fluorescence, Control, Staining, Flow Cytometry, Expressing