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: Scientific Reports

Article Title: Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes

doi: 10.1038/s41598-024-69364-w

Figure Lengend Snippet: Concentration-dependent effects of PA on mitochondrial respiration and glycolysis. ( A ) Oxygen consumption rate (OCR) was measured using Seahorse XFe96 extracellular flux analyzer in control fibroblasts, NoC(V7) (grey circles), and ciBAs converted in the absence of PA, RoFB(V7) (black diamonds). Mitochondrial respiration inhibitors, oligomycin, FCCP, and antimycin A/rotenone, were added during the measurement, as indicated. P values were determined using two-way ANOVA. ( B ) OCR was compared between RoFB(V7) (grey circles) and either RoFB(V7 + PA, 0.5 μg/mL), RoFB(V7 + PA, 4 μg/mL), or RoFB(V7 + PA, 16 μg/mL) (black diamonds). P values were determined using two-way ANOVA. ( C,D ) OCR corresponding to basal respiration, maximal respiration, ATP production, and proton leak was compared. ( E ) Extracellular acidification rate (ECAR) was measured in control fibroblasts, NoC(V7) (grey circles), and ciBAs in the absence of PA, RoFB(V7) (black diamonds). Glucose, oligomycin, and 2-deoxyglucose (2-DG) were sequentially added during the measurement, as indicated. P values were determined using two-way ANOVA. ( F ) ECAR was also compared between the control ciBAs, RoFB(V7) (grey circles), and either RoFB(V7 + PA, 0.5 μg/mL), RoFB(V7 + PA, 4 μg/mL), or RoFB(V7 + PA, 16 μg/mL) (black diamonds). P values were determined using two-way ANOVA. ( G ) ECAR corresponding to glycolysis and glycolytic capacity was calculated. Data represent mean ± SD ( n = 6–8). ( H ) Lactate secretion into culture supernatants was quantified in control fibroblasts and ciBAs treated with PA at various concentrations throughout the experiments. Data represent mean ± SD ( n = 3). ( I ) Mitochondrial membrane potential (MMP) was evaluated by staining with the fluorescent probe, MT-1 dye. The area of the staining for the dye was quantified by ImageJ software. Data represent mean ± SD ( n = 5). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Article Snippet: Other lines of human dermal fibroblasts (HDF35 and HDF54) were also purchased from PromoCell .

Techniques: Concentration Assay, Control, Membrane, Staining, Software, Comparison

Journal: Scientific Reports

Article Title: Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes

doi: 10.1038/s41598-024-69364-w

Figure Lengend Snippet: Comparison of the transcriptome in ciBAs treated with PA at low and high concentrations throughout the conversion. ( A ) Multidimensional scaling analysis graphically indicates the similarity and variability of the transcriptome in the control fibroblast, NoC(V7), ciBAs, RoFB(V7), and ciBAs treated with PA at low and high concentrations, RoFB(V7 + PA, 0.5 μg/mL) and RoFB(V7 + PA, 16 μg/mL). ( B ) Gene ontology (GO) enrichment analysis was performed in upregulated differentially expressed genes (DEGs) in RoFB(V7 + PA, 0.5 μg/mL) compared with RoFB(V7). The top 10 GO terms are represented in the category of biological process. ( C ) GO analysis was performed in downregulated DEGs in RoFB(V7 + PA, 16 μg/mL) compared with RoFB(V7 + PA, 0.5 μg/mL). ( D ) The FPKM values in the RNA-Seq results indicate the transcriptional levels of UCP1 , CKMT1A , CKMT1B , CKMT2 , CKB , ALPL ( TNAP ), SLC6A8 , and CKM genes. ( E ) UCP1 , CKMT1 , CKMT2 , CKB , ALPL ( TNAP ), and SLC6A8 mRNA were measured by qRT-PCR analysis in ciBAs treated with PA as indicated. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * P < 0.05, ** P < 0.01, *** P < 0.001, N.S.; not significant.

Article Snippet: Other lines of human dermal fibroblasts (HDF35 and HDF54) were also purchased from PromoCell .

Techniques: Comparison, Control, RNA Sequencing Assay, Quantitative RT-PCR