human foreskin fibroblasts a1f  (ATCC)

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: ( A-C ) Embryonic Foreskin Fibroblasts (A1F), Human Adult Dermal Fibroblasts (HDF), and Human Adult Kidney Fibroblasts (HKF) were treated with increasing concentrations of fibronectin DAMPs, FnEDA and FnIII-1c. After 4 h, conditioned medium was collected and IL-8 concentration was determined by ELISA. ( D-F ) Cells were incubated with 5 μM FnEDA and 5 μM FnIII-1c (A1F, HDF) or 5 μM FnEDA, 10 μM FnIII-1c (HKF) individually or in combination for the indicated times. Student’s t-test was used to compare the expected additive (Add) IL-8 concentration (x—x) to the actual IL-8 concentration when both DAMPs were combined (black boxes). The data represent the mean ± s.e.m. of 3 independent experiments performed in triplicate. (*P≤0.05, **P≤0.01, ***P≤0.001).

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: A1F ( A,D ), HDF ( B,E ), and HKF ( C,F ) cells were treated with 1 μM of the TAK1 inhibitor 5Z-7-Oxozeaenol (5O), or the inactive analog 5Z-Zeaenol (5Z) for 1 h prior to incubation with either FnEDA (20 μM) or III-1c (20 μM), individually or in combination for an additional hour. Control cells (C) were treated with PBS/DSMO. Cells were lysed and phosphorylation of TAK1 (T184/187) was determined by western blot ( A-C ) and normalized to GAPDH ( D-F ). Blots are representative of 3 independent experiments. Data represent the mean ± s.e.m of 3 independent experiments; 1-Way ANOVA w/Tukey Post-hoc test was used for multiple comparisons. (*P≤0.05, **P≤0.01, ***P≤0.001).

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Incubation, Western Blot

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: A1F ( A-C ), HDF ( D-F ), and HKF ( G-I ) cells were treated with increasing concentrations of either the TAK1 inhibitor 5Z-7-Oxozeaenol (5O) or the inactive analog 5Z-Zeaenol (5Z) for 1 h prior to incubation with either 5 μM FnEDA or FnIII-1c ( A-F ) or 5 μM FnEDA, 10 μM FnIII-1c ( G-I ), individually or in combination, for an additional 4 h. Conditioned medium was collected and IL-8 concentration was determined by ELISA. IL-8 concentration is expressed as a percent of control (no inhibitor). The data represent the mean ± s.e.m of 3 independent experiments performed in triplicate. Student’s t-test was used to compare IL-8 levels at each concentration of 5O vs 5Z. (*P≤0.05, **P≤0.01, ***P≤0.001).

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: A1F ( A-C ), HDF ( D-F ), and HKF ( G-I ) cells were treated with siRNA targeting TAK1 (TAK1) or non-targeting control (C) followed by treatment with either 5 μM FnEDA or FnIII-1c ( A-F ) or 5 μM FnEDA, 10 μM FnIII-1c ( G-I ), individually or in combination, for 4 h. Cells were lysed and TAK1 expression was analyzed ( A,D,G ) and quantified ( B,E,H ) by WES. Conditioned medium was collected and IL-8 concentration was determined by ELISA ( C,F,I ). The data represent the mean ± s.e.m of 3 independent experiments performed in triplicate. One Way ANOVA w/Tukey Post-hoc test was used for multiple comparisons. (*P≤0.05, **P≤0.01, ***P≤0.001).

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: A1F cells were treated with either PBS/DMSO ( C ), 1 μM of the TAK1 inhibitor (5O), or the inactive analog (5Z) for 1 h prior to incubation with FnEDA (20 μM) or FnIII-1c (20 μM), individually or in combination for an additional hour. Cells were lysed and levels of phospho-IKKα/β and phospho-NF-κB were visualized ( A-C ) and quantified using WES ( D-E ). Phosphorylation of MAPKs, ERK, JNK and p38, were visualized ( F-H ) and quantified using WES ( I-K ). Data represent the mean ± s.e.m. of 3 independent experiments. One Way ANOVA w/Tukey Post-hoc test was used for multiple comparisons. (*P≤0.05, **P≤0.01, ***P≤0.001).

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Incubation

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: Fibroblasts were treated for 3 h with 5 μM FnEDA, 5 μM Fn-III-1c (A1F, HDF) or 5 μM FnEDA, 10 μM FnIII-1c (HKF). PBS served as control. RNA was isolated and cDNA was generated and applied to a Human Inflammatory Response and Autoimmunity PCR Array. A list of genes with ≥5-fold increase in expression compared to PBS-treated control cells was generated for FnEDA ( A ) and FnIII-1c ( B ) treated cells. The Venn-diagrams illustrate the overlap among the three cell types in the FnEDA ( C ) and FnIII-1c ( D ) induced pro-inflammatory genes. The number and names of commonly induced (in overlapping areas) and unique (inside each circle) genes are shown.

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Isolation, Generated, Expressing

Journal: PLOS ONE

Article Title: Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases

doi: 10.1371/journal.pone.0286390

Figure Lengend Snippet: Fibroblast cell lines were pretreated with inhibitors to TAK1 (1 μM 5O), ERK1/2 (10 μM Temuterkib), JNK1/2/3 (10 μM JNK IX), or p38 (10 μM SB202190) for 1 h followed by addition of FnEDA ( A ) or FnIII-1c ( B ) for an additional 3 h. A1F and HDF cells received 5 μM FnEDA or 5μM FnIII-1c. The HKF cells received 5 μM FnEDA or 10 μM FnIII-1c. PBS/ DMSO served as control. RNA was isolated and cDNA was generated and applied to a Human Inflammatory Response and Autoimmunity PCR Array. Fold regulation of gene expression for each cell line was determined using RT-PCR and a list of genes with ≥5-fold decrease in expression compared to control treated cells were generated. Heatmaps were generated to illustrate FnEDA ( A ) and FnIII-1c ( B ) induced genes that are regulated by TAK1 or the MAP kinases p38, ERK, and JNK. Genes not induced by fibronectin DAMPS are shown in shaded boxes.

Article Snippet: Human foreskin fibroblasts (A1F) [ ] and adult human dermal fibroblasts (HDF-ATCC, Manassas, VA-#PCS201-012) were grown and maintained in complete medium (Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA) supplemented with Pen-Strep (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco) in a 8% CO 2 humidified atmosphere at 37°C.

Techniques: Isolation, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction