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

Journal: BMC Microbiology

Article Title: Heat-killed Lacticaseibacillus paracasei GMNL-653 ameliorates human scalp health by regulating scalp microbiome

doi: 10.1186/s12866-023-02870-5

Figure Lengend Snippet: Treatment of heat-killed GMNL-653 in Hs68 fibroblasts increases the mRNA expressions of growth factors. 1.5 × 10 5 cells/well of human fibroblast Hs68 cells were seeded in wells of 6-well-plate for attachment and then cultured with serum free medium for 24 h. Cells were treated with indicated concentration of heat-killed GMNL-653 for 24 h. The mRNA expression of IGF-1R ( A ), VEGF ( B ), IGF-1 ( C ), and KGF ( D ) were determined by quantified RT-PCR ( n = 5). Data were presented as the relative fold changes (mean ± SEM) in compared to non-GMNL-653 treatment control (Ctrl) after normalization with the house-keeping of β-actin. * p < 0.05

Article Snippet: Human foreskin fibroblast Hs68 cells and human epidermal keratinocyte HaCaT cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin under steady-state conditions at 37 °C and 5% CO 2 in the humidified incubator.

Techniques: Cell Culture, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction