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: BioChem

Article Title: pH-Selective Reactions to Selectively Reduce Cancer Cell Proliferation: Effect of CaS Nanostructures in Human Skin Melanoma and Benign Fibroblasts

doi: 10.3390/biochem3010002

Figure Lengend Snippet: The percentage of live malignant (red) and benign fibroblasts (blue) as a function of the percentage (%) of CaS dispersion used.

Article Snippet: Human skin normal fibroblasts were prepared from the ATCC CCD 1090 sk CRL-2106 cell line.

Techniques: Dispersion

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Effect of MA-based samples on cell viability. MTT assay was performed at 595 nm after 2 ( a ) and 24 ( b ) hours of human gingival fibroblasts’ incubation in the presence of MA-based CAD/CAM and MA-based 3D-printed samples. Results are presented as mean ± SD ( n = 13). *** p < 0.001 compared to control, and # p < 0.05 and ### p < 0.001 compared to MA-based CAD/CAM samples.

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: MTT Assay, Incubation, Control

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Effect on cell morphology after exposure to MA-based samples. Phase-contrast images of human gingival fibroblasts incubated for 2 and 24 h in the presence of MA-based CAD/CAM and MA-based 3D-printed samples and fluorescence staining of live (stained by calcein-AM in green) and dead (stained by ethidium homodimer-1 in red) cells (scale bar is the same for all images: 100 µm). Note the apoptotic features of fibroblasts incubated for 24 h with MA-based 3D samples (membrane blebbing indicated by arrows and cell shrinkage indicated by arrowhead). Scale bar: 100 µm.

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Incubation, Fluorescence, Staining, Membrane

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Effect of cell membrane integrity after exposure to MA-based samples. LDH release was recorded at 490 nm after 2 ( a ) and 24 ( b ) hours of human gingival fibroblasts’ incubation in the presence of MA-based CAD/CAM and MA-based 3D-printed samples. Results are presented as mean ± SD ( n = 13). * p < 0.05 and ** p < 0.01 compared to control.

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Membrane, Incubation, Control

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Cell toxicity after exposure to MA-based samples. NO level was measured at 550 nm after 2 ( a ) and 24 ( b ) hours of human gingival fibroblasts’ incubation in the presence of MA-based CAD/CAM and MA-based 3D-printed samples. Results are presented as mean ± SD ( n = 13).

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Incubation

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Analysis of oxidative stress and apoptosis after exposure to MA-based samples. Levels of GSH ( a ), ROS ( b ), and caspase 3/7 activity ( c ) were measured after 2 and 24 h of human gingival fibroblasts’ incubation in the presence of MA-based CAD/CAM and MA-based 3D-printed samples. Representative images of DCFDA and NucView staining in green of ROS and caspase 3/7 activity, respectively, are shown ( d ). Results are calculated as mean ± SD ( n = 3) and replicates are represented as black dots. ** p < 0.01 compared to control. Scale bar: 100 µm.

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Activity Assay, Incubation, Staining, Control

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Analysis of autophagy after exposure to MA-based samples. ( a ) Representative images of Autophagy Sensors staining in green of LC3B expression are shown after 24 h of fibroblasts’ incubation with MA-based 3D samples. Note the autophagic features of fibroblasts (perinuclear-formed autophagosomes associated with LC3B protein indicated by yellow arrows). Scale bar: 100 µm. ( b ) The fluorescence intensity of LC3B expression was quantified after 24 h of human gingival fibroblasts’ incubation in the presence of MA-based CAD/CAM and MA-based 3D-printed samples. Results are calculated as mean ± SD ( n = 15) and expressed relative to control (unexposed cells). Replicates are represented as black dots. *** p < 0.001 compared to control.

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Staining, Expressing, Incubation, Fluorescence, Control

Journal: Journal of Functional Biomaterials

Article Title: Analysis of Gingival Fibroblasts Behaviour in the Presence of 3D-Printed versus Milled Methacrylate-Based Dental Resins—Do We Have a Winner?

doi: 10.3390/jfb15060147

Figure Lengend Snippet: Effect of MA-based samples on MMP-2 expression. Levels of MMP-2 ( a , b ) were measured after 2 and 24 h of human gingival fibroblasts’ incubation in the presence of MA-based CAD/CAM and MA-based 3D-printed samples. Results are presented as mean ± SD ( n = 3). ** p < 0.01 and *** p < 0.001 compared to control, and ### p < 0.001 compared to MA-based CAD/CAM samples.

Article Snippet: The HGF-1 cell line (catalog no. CRL-2014) of human gingival fibroblasts, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) with fetal bovine serum (at a final concentration of 10%) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Incubation, Control