Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 is required for TGF-β1–mediated differentiation of NHLFs and DHLFs. A) mLFs isolated from WT and TRPV4-KO mice were treated with TGF-β1 (2 ng/ml) for 24, 48, and 96 h, and expression of α-SMA and FN was analyzed by Western blotting. Note that time-dependent TGF-β1 response seen in WT fibroblasts was attenuated in TRPV4-KO fibroblasts. B) Effect of TGF-β1 treatment on TRPV4-dependent calcium flux in NHLFs. NHLFs were loaded with Fluo-4, and calcium flux was measured as described in Materials and Methods in response to a specific TRPV4 activator, GSK1016790A (100 nM), and quantified. C–E) TRPV4 protein was knocked down in NHLFs by transfecting them with siRNA against TRPV4 (20 nM). NHLFs transfected with nonspecific (NS) siRNA were used as control. Twenty-four hours after transfection, NHLFs were treated with TGF-β1 (2 ng/ml; 48 h). C) TRPV4 transcript was analyzed by real-time PCR. D) α-SMA localization was determined by immunofluorescence (red). E) α-SMA expression was analyzed by Western blotting and quantified. F) TRPV4-mediated calcium flux in NHLFs and DHLFs. Calcium flux, indicative of TRPV4 activity, was analyzed in NHLFs and DHLFs from same passage in response to GSK and quantified. G–I) Differential responses of NHLFs and DHLFs to TGF-β1. G) α-SMA transcript levels both at basal and in response to TGF-β1 (2 ng/ml; 48 h) were analyzed by real-time PCR in NHLFs and DHLFs. H, I) Effect of RN1734, a selective TRPV4 antagonist on TGF-β1–induced fibroblast differentiation in NHLFs and DHLFs. H) Both NHLFs and DHLFs were stimulated with or without TGF-β1 (2 ng/ml; 48 h) in the presence or absence of a TRPV4 inhibitor RN1734 (10, 30, and 50 μM), and α-SMA protein expression was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab. I) Blots were quantified using AlphaView software. Results are means ± sem from 3 independent experiments (B, C, E–G, I). *P < 0.05; **P < 0.01.

Article Snippet: Validated primary normal human lung fibroblasts (NHLFs; CC-2512) and diseased [asthma; male, 27 yr old, diagnosed at age 7 and on medication (Proventil, albuterol; Merck, Darmstadt, Germany)] human lung fibroblasts (DHLFs; 00194912) were obtained from Lonza (Walkersville, MD, USA).

Techniques: Isolation, Expressing, Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Activity Assay, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 mediates matrix synthesis in fibroblasts. A, B) NHLFs were treated with TGF-β1 (2 ng/ml; indicated time) with or without TRPV4 inhibitor RN1734 (30 μM preincubation) and collagen 1A1 (A) and FN (B) transcripts were analyzed by real-time PCR. C) FN protein accumulation was analyzed by immunofluorescence in cells without permeabilizing with Triton X-100. D, E) FN protein expression was determined by Western blotting (D) and quantified (E). F–I) DHLFs exhibit higher basal and TGF-β1–induced matrix transcripts and protein levels compared with NHLFs. F, G) NHLFs and DHLFs were treated with TGF-β1 (2 ng/ml; 72 h), and Col1A1 (F) and FN (G) transcripts were analyzed by real-time PCR. H, I) FN protein expression was determined by western blotting (H) and quantified in the presence or absence of RN1734 (30 μM; I). Blots were stripped and reprobed with GAPDH Ab. Blots were quantified using AlphaView software (ProteinSimple). Results are means ± sem from at least 3 independent experiments (A, B, E, G, I). *P < 0.05; **P < 0.01.

Article Snippet: Validated primary normal human lung fibroblasts (NHLFs; CC-2512) and diseased [asthma; male, 27 yr old, diagnosed at age 7 and on medication (Proventil, albuterol; Merck, Darmstadt, Germany)] human lung fibroblasts (DHLFs; 00194912) were obtained from Lonza (Walkersville, MD, USA).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Western Blot, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: Hypothetical model depicting TRPV4 in the regulation of fibroblast differentiation and airway remodeling.

Article Snippet: Validated primary normal human lung fibroblasts (NHLFs; CC-2512) and diseased [asthma; male, 27 yr old, diagnosed at age 7 and on medication (Proventil, albuterol; Merck, Darmstadt, Germany)] human lung fibroblasts (DHLFs; 00194912) were obtained from Lonza (Walkersville, MD, USA).

Techniques:

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: IPF lung fibroblasts showed increased sensitivity to TGF-β1-stimulated PAI-1 release. Time course of PAI-1 release from non-stimulated ( A ) and TGF-β1-stimulated ( B ) fibroblasts derived from control (white) and IPF (grey) patients. PAI-1 release from control ( C ) and IPF ( D ) fibroblasts at 72 h following exposure to the indicated TGF-β1 concentrations. Data represents the mean +/− SEM of five individuals. Statistical significance was performed using 1-way analysis of variance (ANOVA) with a Dunnett’s test for time courses ( A , B ) where * p < 0.05, ** p < 0.01, *** p < 0.001 and *** p < 0.0001. The logEC 50 for each individual was determined in GraphPad Prism and comparison between control and IPF groups was performed using a unpaired t-test. The EC 50 was calculated from the mean logEC50 values.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Derivative Assay, Control, Comparison

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: Non-stimulated IPF lung fibroblasts show a reduced proliferative response. Proliferation in non-stimulated control (white) and IPF fibroblasts (grey) was measured 72 h using cell count ( A ). Time course of proliferation in PDGF-stimulated (from control ( B ) and IPF ( C ) patients). Proliferation in control ( D ) and IPF ( E ) fibroblasts at 72 h following exposure to the indicated PDGF concentrations. Data represents the mean +/− SEM of five individuals. Statistical significance was performed using 1-way analysis of variance (ANOVA) with a Dunnett’s or Tukey’s test for time courses ( A – C ) where * p < 0.05, ** p < 0.01 and **** p < 0.0001. The logEC 50 for each individual was determined in GraphPad Prism and comparison between control and IPF groups was performed using a unpaired t-test. The EC 50 was calculated from the mean logEC50 values.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Control, Cell Counting, Comparison

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: IPF lung fibroblasts show a reduced IL-1β-stimulated inflammatory response. Time course of IL-6 release from non-stimulated ( A ) and IL-1β-stimulated ( B , D ) and TNFα-stimulated ( C , E ) fibroblasts derived from control (white) and IPF (grey) patients. IL-6 release from control and IPF fibroblasts at 24 h following exposure to the indicated IL-1β ( D ) and TNF-α ( E ) concentrations. Data represents the mean +/− SEM of five individuals. Statistical significance was performed using an unpaired t-test where * p < 0.05 and ** p < 0.01. The logEC 50 for each individual was determined in GraphPad Prism and comparison between control and IPF groups was performed using an unpaired t-test. The EC 50 was calculated from the mean logEC50 values.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Derivative Assay, Control, Comparison

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: Differential expression of the histone epigenetic mark between control and IPF fibroblasts. ChIP sequencing was employed to examine the differential expression of H3K4me1, a marker of primed promoter and enhancer regions. ( A ) examples of the H3K4me1 regions associated with CCL8 and MRAP, ( B ) unsupervised hierarchical clustering was calculated within the DiffBind programme and ( C ) pathways analysis of H3K4me1 associated genes was undertaken using DAVID. Total expression of the histone marks H3K4me1, H3K4me3 and H3K27ac in control and IPF fibroblasts were measured by Western blotting ( D ) and then quantified by densitometry using b-actin as an internal loading control ( E ) where data is the mean +/− SEM of 5 donors and statistical difference was examined using an unpaired T-test. The section D contains cropped gels and the original uncropped gels can be viewed in Supplemental Fig. .

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Quantitative Proteomics, Control, ChIP-sequencing, Marker, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: Transcriptome analysis shows differential expression of long intergenic RNA between control and IPF fibroblasts. ( A ) The differential expression of various classes of genes was examined in 5 control and 5 IPF fibroblasts samples in the presence and absence of TGF-β1 stimulated at 24 h. ( B ) Table showing the genes with highest fold changes in non-stimulated fibroblasts at 24 h. ( C ) The differential expression of the two lincRNAs, LINC00960 and LINC01140 was confirmed by qRT-PCR (n = 5). ( D ) LINC00960 and LINC01140 expression was confirmed by comparison with RNA sequencing data and the epigenetic marks associated with H3K4me3 (active promoters) and H3K27ac (active transcription). ( E ) Expression of LINC00960 and LINC01140 in the lung biopsies of control (n = 19) and IPF patients (n = 20). Data in ( D , E) are the mean +/− SEM and statistical significance was performed using an unpaired t-test where * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Quantitative Proteomics, Control, Quantitative RT-PCR, Expressing, Comparison, RNA Sequencing

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: LincRNAs and the regulation of TGF-β1-stimulated PAI-1 release. Control and IPF fibroblasts were transfected with LNA antisense sequences against LINC00960, LINC01140 or scrambled controls overnight. Cell were then stimulated with TGF-β1 for 72 h prior to ( A ) isolation of mRNA and measurement of LINC00960 or LINC01140 by qRT-PCR or ( B ) measurement of supernatant PAI-1 by ELISA. Data represents the mean +/− SEM of five control or IPF individuals. Statistical significance was performed using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett’s test where * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Control, Transfection, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: LincRNAs and the regulation of the PDGF-stimulated proliferation. Control and IPF fibroblasts were transfected with LNA antisense sequences against LINC00960, LINC01140 or scrambled controls overnight. The cell number was determined at 0 h ( A ) or in non-stimulated ( B ) and PDGF-stimulated ( C ) samples at 72 h. Data represents the mean +/− SEM of five control or IPF individuals. Statistical significance was performed using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett’s test where * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Control, Transfection

Journal: Scientific Reports

Article Title: Long intergenic non-coding RNAs regulate human lung fibroblast function: Implications for idiopathic pulmonary fibrosis

doi: 10.1038/s41598-019-42292-w

Figure Lengend Snippet: LincRNAs and the regulation of the IL-1β-stimulated IL-6 release. Control and IPF fibroblasts were transfected with LNA antisense sequences against LINC00960, LINC01140 or scrambled control overnight. Cell were then stimulated with IL-1β for 24 h prior to isolation of RNA and measurement of LINC00960 or LINC01140 ( A ) and IL-6 ( B ) by qRT-PCR or ( C ) measure of released IL-6 by ELISA. Data represents the mean +/− SEM of five control or IPF individuals. Statistical significance was performed using the repeat measures 1-way analysis of variance (ANOVA) with a Dunnett’s test where * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Control (age = 50 ± 3 y; 3 male and 2 females) and IPF fibroblasts (age = 62 ± 1 y; 3 male and 2 females) were obtained from Professor Carol Ferghali-Bostwick (Medical University of South Carolina, USA) and the Coriell Institute of Medical Research (Camden, New Jersey, USA).

Techniques: Control, Transfection, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: DNA damage is a hallmark of commercial primary IPF fibroblasts in vitro , but not Cdkn2a /p16 ink4a expression or a fibrotic secretome . ( A ) Primary NHLF and IPF cells ( n = 3 different donors, each with 2 technical replicates) were cultured at the same density overnight on standard tissue culture plates in low-serum growth medium and were subsequently fixed. Immunocytochemistry was performed to fluorescently label nuclei (blue) and p21 Waf1/Cip1 (orange). Images were acquired with a 40X water objective using an Operetta High Content Screening instrument and intensity of nuclear p21 Waf1/Cip1 was quantified and calculated as a positive percentage of each population. ( B ) NHLF and IPF cells were treated and imaged as in ( A ), and immunocytochemistry was performed to fluorescently label nuclei (blue) and DNA damage via nuclear γH2A.X (Ser139) foci (orange). The number of nuclear foci per cell was quantified and cells with one or more were reported as positive. ( C ) Following overnight culture in standard tissue culture plates, NHLF and IPF cells were lysed, and qPCR was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2 −ΔΔCt method versus NHLF cells. ( D ) Following overnight culture as in ( C ), cell culture supernatants were collected and assayed for determination of the concentration of common fibrosis-related secreted proteins by MSD kits. Statistical analysis was performed using an unpaired t -test ( A , B ) or a two-way ANOVA with a Bonferroni post-test ( C , D ) in GraphPad Prism: * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: Primary normal human lung fibroblasts (NHLF) were purchased from Lonza and ATCC, and diseased IPF lung fibroblasts were purchased from BioIVT, Lonza, or ATCC.

Techniques: In Vitro, Expressing, Cell Culture, Immunocytochemistry, High Content Screening, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: Summary of in vitro and ex vivo systems to mimic alveolar epithelial and fibroblast damage and dysfunction in the IPF lung. While IPF fibroblasts and bleomycin treated NHLFs exhibit some hallmarks of senescence, these cells do not appear to demonstrate a phenotype of Cdkn2a /p16 ink4a expression, ECM deposition, or secretion of fibrotic mediators such as TIMP1. Rather, the SASP of senescent, aberrant epithelial cells drives a fibrotic phenotype in NHLFs that is consistent with progressive fibrosis. Development of senolytic agents presents an opportunity for therapeutic impact early in disease pathogenesis and with an orthogonal mechanism than the fibroblast targeting standard of care, Nintedanib. Image created with https://www.biorender.com/ .

Article Snippet: Primary normal human lung fibroblasts (NHLF) were purchased from Lonza and ATCC, and diseased IPF lung fibroblasts were purchased from BioIVT, Lonza, or ATCC.

Techniques: In Vitro, Ex Vivo, Expressing

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

doi: 10.1164/rccm.201612-2480OC

Figure Lengend Snippet: Transforming growth factor-β1 (TGF-β1)–stimulated normal human lung fibroblasts (NHLFs) show altered metabolism and high concentrations of extracellular mitochondrial DNA. (A and B) NHLFs stimulated with 5 ng/ml of TGF-β1 for 7 days (right) demonstrated the previously reported increase in glycolysis relative to unstimulated cells (left) as measured by significant elevations in (A) extracellular acidification rate (ECAR) and (B) ratio of ECAR to oxygen consumption rate (OCR). Data are presented as mean ECAR (mpH/min) and mean (±SEM) ratio of ECAR to OCR, respectively. (C) A standard curve was developed from serial dilutions of a commercially available plasmid containing the sequence of the human MT-ATP6 gene. (D) Relative to supernatants obtained from NHLFs cultured with normal medium (left), the mean MT-ATP6 copy number is significantly increased in the supernatant of NHLFs stimulated with 5 ng/ml of TGF-β1 for 7 days (right). Data are presented graphically as log base 10 of the raw values (MT-ATP6 copies per microliter of supernatant) with mean ± SEM. A graph including the raw values is presented in Figure E2D. CT = cycle threshold.

Article Snippet: NHLFs and IPF fibroblasts obtained from Lonza (Allendale, NJ) and Asterand Bioscience (Detroit, MI) were used between passages 3 and 7 and stimulated with and without 5 ng/ml of recombinant human TGF-β1 as previously described ( 14 ).

Techniques: Plasmid Preparation, Sequencing, Cell Culture

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

doi: 10.1164/rccm.201612-2480OC

Figure Lengend Snippet: Transforming growth factor-β1 (TGF-β1) stimulation of normal human lung fibroblasts (NHLFs) reduces mitochondrial mass without affecting cell viability. (A) NHLFs were stimulated with 5 ng/ml of TGF-β1 for 7 days, at which point mitochondrial mass was determined using polymerase chain reaction–based comparison of DNA derived from mitochondria (assessed by the MT-ATP6 gene) and the genome (measured by the β-actin gene). Relative to unstimulated cells (left), there was a significant decline in the mean ratio of mitochondrial DNA (mtDNA) to genomic DNA (gDNA) in TGF-β1–treated cells (right). Data are presented as the mean (±SEM) fold change in ratio of mtDNA to gDNA. (B) Cell counts in NHLF cultures stimulated with (right) and without (left) 5 ng/ml of TGF-β1 for 7 days were unchanged across both conditions. Data are presented as mean (±SEM) fold change in cell count. (C) Assessment of viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed similar fold changes in absorbance at a wavelength of 540 nm between NHLFs stimulated with (right) and without (left) 5 ng/ml of TGF-β1 for 7 days. Data are presented as the mean (±SEM) fold change in absorbance. (D) Stimulation of NHLFs with 0.5 ng/ml of mtDNA for 48 hours significantly increased α-smooth muscle actin (α-SMA) expression, relative to β-actin, by NHLFs. Data are presented as mean (±SEM) α-smooth muscle actin expression relative to β-actin.

Article Snippet: NHLFs and IPF fibroblasts obtained from Lonza (Allendale, NJ) and Asterand Bioscience (Detroit, MI) were used between passages 3 and 7 and stimulated with and without 5 ng/ml of recombinant human TGF-β1 as previously described ( 14 ).

Techniques: Polymerase Chain Reaction, Comparison, Derivative Assay, Cell Counting, Expressing

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

doi: 10.1164/rccm.201612-2480OC

Figure Lengend Snippet: Direct contact with stiff surfaces phenocopies exposure to transforming growth factor-β1 in normal human lung fibroblasts (NHLFs). Measurements of cellular metabolism revealed enhanced aerobic glycolysis in NHLFs grown on the 20-kPa hydrogels for 7 days (right) compared with cells grown on the 1-kPa hydrogels (left), as evidenced by significantly elevated (A) extracellular acidification rate (ECAR) and (B) ECAR/oxygen consumption rate (OCR) ratio. Data are shown as mean (±SEM) ECAR (mpH/min) and mean (±SEM) ratio of ECAR/OCR, respectively. (C) After 7 days, relative to supernatant obtained from NHLFs seeded on the 1-kPa hydrogel (left), there was a significant increase in MT-ATP6 concentration in the supernatant of NHLFs seeded on the 20-kPa hydrogel (right). Data are presented graphically as log base 10 of the raw values (MT-ATP6 copies per microliter of supernatant) with mean (±SEM). A graph including the raw values is presented in Figure E3B. NHLFs grown on the 20-kPa hydrogel (right) demonstrated no significant change in (D) cell counts or (E) viability based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay compared with cells grown on the 1-kPa hydrogel (left) for 7 days. Data are presented as mean (±SEM) fold change in cell count and mean (±SEM) fold change in absorbance, respectively.

Article Snippet: NHLFs and IPF fibroblasts obtained from Lonza (Allendale, NJ) and Asterand Bioscience (Detroit, MI) were used between passages 3 and 7 and stimulated with and without 5 ng/ml of recombinant human TGF-β1 as previously described ( 14 ).

Techniques: Concentration Assay, Cell Counting

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis

doi: 10.1164/rccm.201612-2480OC

Figure Lengend Snippet: Idiopathic pulmonary fibrosis (IPF) fibroblasts exhibit enhanced glycolysis and increased extracellular mitochondrial DNA. Compared with normal human lung fibroblasts (NHLFs) (left), IPF fibroblasts (right) displayed enhanced aerobic glycolysis, as measured by a significantly elevated (A) extracellular acidification rate (ECAR) and (B) ECAR/oxygen consumption rate (OCR) ratio. Data are presented as mean (±SEM) ECAR (mpH/min) and mean (±SEM) ratio of ECAR/OCR, respectively. (C) Relative to samples obtained from NHLFs (left), a significant increase in MT-ATP6 concentration was detected in supernatants from IPF fibroblasts (right). Data are presented graphically as log base 10 of the raw values (MT-ATP6 copies per microliter of supernatant) with mean (±SEM). A graph including the raw values is presented in Figure E4B. (D) Cell counts were unchanged between NHLFs (left) and IPF fibroblasts (right). Data are presented as mean (±SEM) fold change in cell count. (E) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated no significant differences in the viability of NHLFs (left) and IPF fibroblasts (right). Data are presented as mean (±SEM) fold change in absorbance.

Article Snippet: NHLFs and IPF fibroblasts obtained from Lonza (Allendale, NJ) and Asterand Bioscience (Detroit, MI) were used between passages 3 and 7 and stimulated with and without 5 ng/ml of recombinant human TGF-β1 as previously described ( 14 ).

Techniques: Concentration Assay, Cell Counting