cell culture human normal lung fibroblast  (ATCC)

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative Western blot showing TRPV4, phosphorylated AKT [p-AKT (Ser473)], total AKT (t-AKT), and GAPDH (loading control) in lysates of human lung fibroblast (HLF) treated with TRPV4 or scrambled siRNA ±TGF-β as indicated. (B) Quantification of p-AKT (Ser473) relative to total AKT as in (A) by densitometry. *P<0.05 difference between untreated and +TGF-β conditions. Band densities were measured with respect to the untreated scrambled siRNA condition. (C) Representative Western blot showing p-AKT (Ser473) and total AKT (t-AKT) in lysates from HLFs treated with the TRPV4 antagonist RN-1734 ± TGF-β as indicated. (D) Quantification of p-AKT (Ser473) relative to total AKT as in (C). **P<0.01 difference between untreated and +TGF-β conditions. Band densities were measured with respect to the untreated condition. (E) Lipid kinase activity of PI3Kγ and PI3Kα immunoprecipitated from plasma membrane fractions of HLFs treated with the TRPV4 antagonist RN-1734 ±TGF-β as indicated. PI3Kγ and PI3Kα were assayed for their capacity to generate PIP by thin layer chromatography. The line between the untreated and +TGF-β conditions indicates non-contiguous lanes from the same blot. (F) Quantification of PIP densities as in (E). *P<0.05 as indicated. PIP densities were measured with respect to the untreated condition for each PI3K isoform. All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, **P≤0.01, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Control, Activity Assay, Immunoprecipitation, Clinical Proteomics, Membrane, Thin Layer Chromatography

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative fluorescence images showing α-SMA (green) and F-actin (red) in TRPV4 KO and PI3Kγ KO mouse lung fibroblasts (MLFs) treated with empty vector (control) lentivirus (LV), TRPV4 LV, or PI3Kγ LV, then stimulated with TGF-β. Non-transfected WT MLF were used as a positive control. Scale bar, 100 μm. (B) Quantification of cells with α-SMA–positive stress fibers in (A). Data are presented as % myofibroblasts (means ± SEM). N = 3 independent experiments with at least 30 cells per condition. *P<0.05 and ***P<0.005 as indicated. (C) Representative Western blot showing TRPV4, PI3Kγ, and α-SMA in TRPV4 KO MLFs transfected with PI3Kγ LV or TRPV4 LV then stimulated with TGF-β. (D) Quantification α-SMA:GAPDH band density ratios (means ± SEM) in (C). N=3 independent experiments. **P<0.01 difference between untreated and +TGF-β conditions with TRPV4 LV. (E) Representative Western blot showing TRPV4, PI3Kγ, and α-SMA in PI3Kγ KO MLFs transfected with PI3Kγ LV or TRPV4 LV then stimulated with TGF-β. GAPDH is a loading control. (F) Quantification α-SMA:GAPDH band density ratios (means ± SEM) in (E). N=3 independent experiments. *P<0.05 difference between untreated and +TGF-β conditions with PI3Kγ LV. All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, **P≤0.01, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Fluorescence, Plasmid Preparation, Control, Transfection, Positive Control, Western Blot

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative immunoblots showing collagen-1 in WT, TRPV4 KO, and PI3Kγ KO mouse lung fibroblasts (MLFs) treated ±TGF-β. Black lines indicate non-contiguous lanes from the same blot. GAPDH is a loading control. (B) Quantification of collagen-1:GAPDH band density ratios (means ± SEM) from (A). N=3 independent experiments. *P<0.05 compared with untreated WT MLF, TRPV4 KO MLF ±TGF-β, or PI3Kγ KO MLF ±TGF-β. (C) Representative traction force intensity images and corresponding phase images with the region of interest outlined in red for WT, TRPV4 KO, and PI3Kγ KO MLFs treated ±TGF-β. Red and orange, areas of high contraction; blue, areas of low contraction. Scale bar, 50 μm. (D) Quantification of root mean square (RMS) traction force (means ± SEM) results from (C). N=3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with untreated WT MLF, TRPV4 KO MLF ±TGF-β, or PI3Kγ KO MLF ±TGF-β. (E) Quantification of WT and PI3Kγ KO MLFs that differentiated into myofibroblasts when plated on polyacrylamide gels of varying stiffness and treated with TGF-β, as measured by α-smooth muscle actin (α-SMA) in stress fibers. Data shown as % of WT MLF on glass. N=3 independent experiments with at least 30 cells per condition *P<0.05 between WT and PI3Kγ KO MLF at 8 kPa, ***P<0.005 between WT and PI3Kγ KO MLF at 25kPa and glass. (F) Quantification of WT and PI3Kγ KO MLFs plated on polyacrylamide gels of varying stiffness ad treated with TGF-β that showed a TRPV4 plasma membrane:cytoplasm ratio >1 as measured by TRPV4 immunofluorescence. Data shown as % of WT MLF on glass. N=3 independent experiments with at least 30 cells per condition. ***P<0.005 between WT and PI3Kγ KO MLF at 8 kPa, 25 kPa, and glass. All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Control, Clinical Proteomics, Membrane, Immunofluorescence

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Domain structure of WT and mutant PI3Kγ constructs consisting of only the non-catalytic N-terminal domain (N-term) or lacking both the N-terminal domain and the ATP binding site in the catalytic domain (N-del). (B) Immunoblotting of His-tagged N-del or N-term forms of PI3Kγ coupled to Ni-NTA beads incubated with lysates of human lung fibroblasts (HLFs). Blots were probed with an antibody recognizing TRPV4, then stripped and reprobed for PI3Kγ. N=3 independent experiments. (C) Direct interaction between immobilized purified 6-His-TRPV4 and purified 6-His-N-term-PI3Kγ was measured by surface plasmon resonance. N=3 independent experiments. (D) Representative fluorescence images showing TRPV4 in PI3Kγ KO murine lung fibroblasts (MLFs) transfected with N-del or N-term PI3Kγ lentivirus (LV) and treated with TGF-β and the corresponding plot profiles of TRPV4 immunofluorescence. White arrows indicate TRPV4 at the plasma membrane; orange lines indicate regions where plot profiles were obtained; white boxes indicate higher magnification insets. Scale bar, 50 μm. (E) Quantification of results from (D). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with PI3Kγ KO MLF + N-del PI3Kγ LV by Student’s t-test. (F) Western blotting of plasma membrane fractions of PI3Kγ KO MLFs transfected with lentivirus encoding WT PI3Kγ, N-term PI3Kγ, or N-del PI3Kγ, then treated with TGF-β as indicated. Blots were probed for TRPV4, PI3Kγ, and flotillin-1 (loading control). N=3 independent experiments. (G) Representative fluorescence images showing α-SMA in PI3Kγ KO MLFs transfected with control LV (empty vector), WT PI3Kγ LV, N-term PI3Kγ LV or N-del PI3Kγ LV, then stimulated with TGF-β. White boxes indicate areas enlarged in insets. Scale bar, 100 μm. (H) Quantification of results from (G). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 untreated vs +TGF-β conditions by ANOVA followed by Student-Newman-Keuls multiple comparisons test. All graphs show means ± SEM from N = 3 independent experiments.

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Construct, Binding Assay, Western Blot, Incubation, Purification, SPR Assay, Fluorescence, Transfection, Immunofluorescence, Clinical Proteomics, Membrane, Control, Plasmid Preparation

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative confocal images showing TRPV4 in wild-type (WT) and PI3Kγ knockout (KO) murine lung fibroblasts (MLFs) ±TGF-β as indicated and the corresponding plot profiles of the TRPV4 immunofluorescence. Orange lines indicate regions where plot profiles were obtained. White boxes indicate areas shown in higher magnification in insets. (B) Quantification of plasma membrane:cytoplasm fluorescence for experiments in (A). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with untreated WT MLF or with PI3Kγ KO MLF ± TGF-β. (C) Representative confocal images showing PI3Kγ in WT and TRPV4 KO MLFs ±TGF-β and the corresponding plot profiles of the PI3Kγ immunofluorescence. (D) Quantification of results in (C). N = 3 independent experiments with at least 30 cells per condition. ***P<0.005 compared with untreated WT MLF or with TRPV4 KO MLF ±TGF-β. (E) Representative immunoblots for TRPV4 and β1 integrin (loading control) from a surface biotinylation assay in WT and PI3Kγ KO MLF ±TGF-β. (F) Quantification of results from (E). N=3 independent experiments. **P<0.01 compared with untreated WT MLF or with PI3Kγ KO MLF ± TGF-β. (G) Representative immunoblots for PI3Kγ and flotillin-1 (loading control) from the plasma membrane fractions of WT and TRPV4 KO MLF ± TGF-β. The line between WT and TRPV4 KO MLF conditions indicates non-contiguous lanes from the same blot. (H) Quantification of results from (G). N=3 independent experiments. ***P<0.005 compared with untreated WT MLF or with TRPV4 KO ±TGF-β. All graphs show means ± SEM from N = 3 independent experiments. **P≤0.01, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test. Scale bars, 50 μm.

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Knock-Out, Immunofluorescence, Clinical Proteomics, Membrane, Fluorescence, Western Blot, Control, Surface Biotinylation Assay

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) The binding of purified PI3Kγ to purified 6-His-TRPV4 coupled to Ni-NTA beads was assessed by immunoblotting with an antibody specific for PI3Kγ. The blots were stripped and reprobed with an antibody for TRPV4. Control indicates beads alone (no protein bound to beads), N=3 independent experiments. (B) The direct interaction between immobilized purified 6-His-TRPV4 and purified 6-His-PI3Kγ was measured by surface plasmon resonance. N=3 independent experiments. (C) The binding of purified 6-His-PI3Kγ coupled to Ni-NTA beads to endogenous TRPV4 from human lung fibroblast (HLF) lysates was assessed by Western blotting. Blots were stripped and reprobed with antibodies specific for PI3Kγ, GRK2 (positive control), and TRPV2 (negative control). Control indicates beads alone (no protein bound to beads), N=3 independent experiments. (D) PI3Kγ immunoprecipitates (IP) from plasma membrane and cytosolic fractions of HLFs treated with TGF-β as indicated were immunoblotted for PI3Kγ and TRPV4. The line between the plasma membrane and cytosolic fractions indicates non-contiguous lanes from the same blot. Total whole cell lysate of cells treated ±TGF-β (input) was immunoblotted for TRPV4 and PI3Kγ. GAPDH is a loading control. (E) Quantification of TRPV4 in plasma membrane and cytosolic fractions normalized to TRPV4 in whole cell lysate as in (D). Data represent means ± SEM. N=3 independent experiments. *P≤0.05 between untreated and TGF-β conditions, using ANOVA followed by Student-Newman-Keuls multiple comparisons test.

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Purification, Western Blot, Control, SPR Assay, Positive Control, Negative Control, Clinical Proteomics, Membrane

Journal: Science signaling

Article Title: Translocation of TRPV4-PI3Kγ complexes to the plasma membrane drives myofibroblast transdifferentiation

doi: 10.1126/scisignal.aau1533

Figure Lengend Snippet: (A) Representative plots showing the effects of scrambled, PI3Kα, and PI3Kγ siRNA on Ca2+ influx induced by the TRPV4 agonist GSK1016790A (GSK) in human lung fibroblasts. Ca2+ influx was measured in relative fluorescence units (RFU) using Calcium 5 dye on intact human lung fibroblast (19Lu) monolayers treated with scrambled, PI3Kα, or PI3Kγ siRNA. (B) Quantification of experiments in (A). ***P<0.005 compared with PI3Kα siRNA or scrambled siRNA. (C) Representative immunoblots showing PI3Kα and PI3Kγ in 19Lu cells treated with scrambled, PI3Kα, or PI3Kγ siRNA. GAPDH is a loading control. (D) Quantification of PI3Kα and PI3Kγ band density relative to GAPDH in (C). ***P<0.005 as indicated. (E) Representative plot showing GSK1016790A-induced Ca2+ influx in WT and PI3Kγ KO mouse lung fibroblasts (MLFs) treated ±TGF-β as indicated. (F) Quantification of RFU in 300 nM GSK1016790A conditions in (E). *P<0.05 as indicated. (G) Representative immunoblot showing TRPV4 in WT and PI3Kγ KO MLFs treated ±TGF-β. (H) Quantification of TRPV4 band density relative to GAPDH in (G). All graphs show means ± SEM from N = 3 independent experiments (*P≤0.05, ***P≤0.005, using ANOVA followed by Student-Newman-Keuls multiple comparisons test).

Article Snippet: Cell Culture Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Fluorescence, Western Blot, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation

doi: 10.1165/rcmb.2017-0298OC

Figure Lengend Snippet: Increased CDKN2B methylation was observed in idiopathic pulmonary fibrosis (IPF) patients with radiographic honeycombing and was associated with decreased gene expression. Fibroblasts from normal (nonfibrotic) lungs and the lungs of patients with IPF were assayed for CDKN2B DNA methylation by bisulfite pyrosequencing. (A) Mean CDKN2B DNA methylation (averaged over six CpG sites within region 1 and 19 CpG sites within region 2) were compared with levels of CDKN2B mRNA expression using linear regression. (B) Mean levels of CDKN2B methylation were compared between fibroblasts derived from normal lung and IPF lung with and without radiographic evidence of honeycombing. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. (C and D) Lung sections from histologically normal, nonfibrotic lungs and fibrotic areas of IPF were immunostained for CDKN2B, α-smooth muscle actin (α-SMA), or isotype control. (C) Representative images at ×100 and ×200 magnification from three different patients are shown. Arrows indicate the presence of fibroblastic foci. (D) Representative immunofluorescent images of CDKN2B (red), α-SMA (green), DAPI (blue), and merged images at ×200 magnification from four normal and six IPF tissue sections are shown. (E) Mean fluorescence intensity of CDKN2B (red) was measured from lung sections of four different nonfibrotic and six different patients with IPF. The fluorescence intensity for each tissue section was calculated from the mean intensity of 4–10 images taken from each section. *P < 0.05, Student’s t test. IHC = immunohistochemistry.

Article Snippet: Cell Culture Normal primary human lung fibroblasts (CCL210) and human bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection.

Techniques: Methylation, Gene Expression, DNA Methylation Assay, Expressing, Derivative Assay, Control, Fluorescence, Immunohistochemistry

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation

doi: 10.1165/rcmb.2017-0298OC

Figure Lengend Snippet: Loss of CDKN2B in fibroblasts resulted in a decrease in cell proliferation. (A) Normal lung fibroblasts (CCL210) were treated with either control siRNA or siRNA against CDKN2B for 48 hours, and expression of various CDK inhibitors was assayed by RT-PCR (n = 4). (B–F) CCL210 fibroblasts were treated with either control or CDKN2B siRNA for 48 hours before treatment with either medium alone, platelet-derived growth factor (PDGF) (20 ng/ml), fibroblast growth factor (FGF) (25 ng/ml), transforming growth factor (TGF)-β1 (2 ng/ml), or Dulbecco’s modified Eagle medium (DMEM) with 10% serum for 24 hours. (B) Expression of CDKN2B was evaluated by immunoblot. Representative blot of four independent experiments is shown. (C) Cell proliferation was assayed by the CyQuant assay, as described in Methods, with values expressed as arbitrary fluorescent units. Data from a single experiment, which is representative of four independent experiments, are shown. Each experiment was performed in six replicate wells, with mean and SE measurements shown. (D) Levels of CCNB1 (cyclin B1) and CCND1 (cyclin D1) mRNA were assayed by RT-PCR (n = 8). (E) Cyclin B1 (n = 2) and (F) cyclin D1 (n = 3) protein were assayed by immunoblot with representative immunoblots shown. (G) Fibroblasts from wild-type and Cdkn2b−/− mice were assayed for cyclin D1 protein expression at baseline and in the presence of FGF (25 ng/ml). The mean of the relative densitometric values from three independent experiments are indicated below. (H) CDKN2B mRNA levels were assayed from fibroblasts of wild-type, Cdkn2b+/−, and Cdkn2b−/− mice in the presence or absence of TGF-β1 (2 ng/ml) or FGF (25 ng/ml). (I) Fibroblasts from wild-type, Cdkn2b+/−, and Cdkn2b−/− mice were assayed for proliferation by CyQuant assay in the presence of medium alone, FGF (25 ng/ml), PDGF (20 ng/ml), and 10% serum (representative experiment from four independent experiments). (J and K) BEAS-2B, a bronchial epithelial cell line, was treated with control or CDKN2B siRNA. Proliferation of cells in (J) bronchial epithelial growth medium (BEGM) was assayed by CyQuant assay (n = 3) and by expression of (K) cyclin D1 (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test (A, D, J, and K), one-way ANOVA with Tukey’s multiple comparisons test (C and F). arb = arbitrary; cont si = control siRNA; Rel exp = relative expression.

Article Snippet: Cell Culture Normal primary human lung fibroblasts (CCL210) and human bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection.

Techniques: Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Modification, Western Blot, CyQUANT Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation

doi: 10.1165/rcmb.2017-0298OC

Figure Lengend Snippet: A decrease in CDKN2B expression in fibroblasts was associated with an increase in myofibroblast differentiation. (A) Levels of COL1A1 mRNA were assayed in normal fibroblasts (CCL210) treated with control siRNA or CDKN2B siRNA (n = 8). (B and D) CCL210 fibroblasts were treated with control or CDKN2B siRNA for 48 hours before treatment with TGF-β1 (2 ng/ml) for 24 hours. Levels of (B) collagen I protein (n = 4) and (D) α-SMA (n = 3) were assayed by immunoblot. Representative blots from each experiment are shown. (C) Fibroblasts from normal (nonfibrotic) and IPF lungs were treated with control or CDKN2B siRNA before treatment with TGF-β1 (2 ng/ml) and levels of ACTA2 (α-SMA) mRNA were assayed by RT-PCR (n = 4). (E and F) CCL210 fibroblasts were transfected with a plasmid that overexpressed CDKN2B or control plasmid (pAdTrack-CMV). Levels of (E) CDKN2B mRNA (n = 1) and (F) ACTA2 (n = 3) were assayed by RT-PCR. (G) Fibroblasts from IPF and nonfibrotic control lungs were transfected with pAdTrack-CMV or CDKN2B overexpression plasmid and levels of ACTA2 mRNA was assayed by RT-PCR (n = 4 for nonfibrotic and n = 3 for IPF). (H) CCL210 fibroblasts were transfected with pAdTrack-CMV or CDKN2B overexpression plasmid and α-SMA protein expression was assayed by immunoblot (representative blot from two independent experiments). (I) Fibroblasts were cultured from the lungs of wild-type (wt), Cdkn2b−/−, and Cdkn2b+/− mice and treated with or without TGF-β1 (2 ng/ml) for 24 hours. Levels of α-SMA protein were assayed by immunoblot. Mean densitometry from three independent experiments and a representative blot are shown. *P < 0.05, **P < 0.01, Student’s t test (A, D, and F), one-way ANOVA with Tukey’s multiple comparisons test (B, C, G, and I). NS = not significant.

Article Snippet: Cell Culture Normal primary human lung fibroblasts (CCL210) and human bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection.

Techniques: Expressing, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression, Cell Culture

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation

doi: 10.1165/rcmb.2017-0298OC

Figure Lengend Snippet: Loss of CDKN2B resulted in increased expression of serum response factor (SRF) and myocardin-related transcription factor (MRTF)-A. (A–D) Normal lung fibroblasts treated with either control or CDKN2B siRNA for 48 hours were then treated with TGF-β1 (2 ng/ml) for 24 hours before being assayed for levels of SRF (A, n = 6) and MKL1 (MRTF-A) (C, n = 3) mRNA by RT-PCR and SRF (B) and MRTF-A (D) protein by immunoblot. Representative blots of three independent experiments are shown, with relative densitometry (mean ± SE) indicated. (E and F) Lung fibroblasts from wild-type (wt) and Cdkn2b−/− mice were treated in the presence or absence of TGF-β1 (2 ng/ml) for 24 hours, and expression of SRF (E) and MRTF-A (F) were assayed by immunoblot. Representative blots from three independent experiments are shown. (G) CCL210 fibroblasts were treated with either control or CDKN2B siRNA in the presence or absence of an SRF/MRTF-A inhibitor CCG-203971 (30 μM) for 48 hours. Cells were then treated with or without TGF-β1 (2 ng/ml) for 24 hours and assayed for α-SMA by immunoblot. A representative blot from four independent experiments is shown. *P < 0.05, ***P < 0.001; Student’s t test (B, C, D, and F), one-way ANOVA with Tukey’s multiple comparisons test (A, E, and G). Inh = inhibitor.

Article Snippet: Cell Culture Normal primary human lung fibroblasts (CCL210) and human bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection.

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Loss of CDKN2B Promotes Fibrosis via Increased Fibroblast Differentiation Rather Than Proliferation

doi: 10.1165/rcmb.2017-0298OC

Figure Lengend Snippet: Fibroblasts from fibrotic lung exhibit decreased expression of CDKN2B. (A) Wild-type and Cdkn2b−/− mice were given saline (n = 14 wild type, n = 8 Cdkn2b−/−) or bleomycin (n = 11 wild type, n = 14 Cdkn2b−/−) at Day 0, and whole lungs were assessed at Day 21 for levels of hydroxyproline. (B and C) Wild-type, Cdkn2b+/−, and Cdkn2b−/− mice (three to six mice each) were treated with either saline or bleomycin. On Day 2, BAL was assayed for CXCL1 (B) and whole lung was assayed for levels of Tnf and myeloperoxidase (Mpo) (C) mRNA. Shown are levels of Tnf and Mpo in bleomycin-treated mice (Tnf and Mpo levels in saline-treated mice were undetectable). (D and E) Fibroblasts were cultured from the lungs of wild-type mice 21 days after mice were treated with either saline or bleomycin, and examined for levels of Cdkn2b mRNA (D, n = 3) and CDKN2B protein expression (E). (F) Lung fibroblasts were cultured from mice at Day 21 after saline (n = 5) or bleomycin (n = 12), and different regions of the Cdkn2b gene were assessed for DNA methylation by bisulfite pyrosequencing. (G) Lung sections were obtained from wild-type mice 21 days after treatment with saline or bleomycin, and immunostained for CDKN2B expression. Representative images, at ×100 and ×200 magnification, from three mice in each group are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test (B and C), one-way ANOVA with Tukey’s multiple comparisons test (A).

Article Snippet: Cell Culture Normal primary human lung fibroblasts (CCL210) and human bronchial epithelial cells (BEAS-2B) were obtained from American Type Culture Collection.

Techniques: Expressing, Saline, Cell Culture, DNA Methylation Assay