normal adult human lung fibroblasts ccl 210  (ATCC)

Journal: JCI Insight

Article Title: Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions

doi: 10.1172/jci.insight.144799

Figure Lengend Snippet: ( A ) Experimental scheme depicting myofibroblast differentiation of CCL210 fibroblasts with TGF-β (2 ng/mL) for 48 hours, followed by dedifferentiation with PGE 2 (1 μM) or FGF2 (50 ng/mL). ( B ) αSMA protein expression measured by Western blot analysis 5 days following treatment with PGE 2 or FGF2 compared with untreated fibroblast and myofibroblast controls. The histogram depicts mean densitometry values. ( C ) αSMA stress fibers identified by immunofluorescence microscopy using anti-αSMA antibody and FITC-conjugated secondary antibody. Nuclei are stained with DAPI. ( D ) Relative ACTA2 , COL1A1 , and FN1 expression by qPCR in myofibroblasts treated for 24 hours with PGE 2 (1 μM), the EP2 agonist butaprost (500 nM), the adenylyl cyclase activator forskolin (500 nM), the PKA specific cAMP analog 6-BNZ cAMP (2 mM), or the Epac specific cAMP analog 8-pCPT cAMP (2 mM). ( E ) Relative ACTA2 , COL1A1 , and FN1 expression by qPCR in myofibroblasts treated for 48 hours with FGF2 (50 ng/mL) with and without the MEK/ERK inhibitor UO126 (20 μM). ( F ) Schematic detailing PGE 2 signaling cascade via the EP2 receptor and FGF2 signaling through FGF2R via MEK/ERK. PKA mediates the reduction in ACTA2 , COL1A1 , and FN1 elicited by PGE 2 , while MEK/ERK mediates the reduction in ACTA2 and COL1A1 elicited by FGF2. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH . Data are presented as mean ± SEM. Data points in B represent individual replicate samples from 4 separate experiments. Data points in D and E represent paired replicate samples from 3 experiments. Lines indicate conditions being compared. * P < 0.05, compared with untreated myofibroblast, 1-way ANOVA. Diff, differentiation; De-diff, dedifferentiation; AC, adenylyl cyclase.

Article Snippet: CCL210 normal adult human lung fibroblasts were obtained from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Immunofluorescence, Microscopy, Staining

Journal: JCI Insight

Article Title: Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions

doi: 10.1172/jci.insight.144799

Figure Lengend Snippet: CCL210 fibroblasts were differentiated to myofibroblasts with TGF-β (2 ng/mL) and treated with PGE 2 (1 μM), FGF2 (50 ng/mL), or both. ( A ) Cells were stained with the membrane dye PKH26 (2 μM) and examined by fluorescence microscopy 5 days after treatment. ( B ) Kinetics of ACTA2 in untreated, PGE 2 -, FGF2-, and PGE 2 + FGF2–treated myofibroblasts. Fibroblasts were treated with TGF-β for 48 hours, followed by treatment and harvesting for mRNA at the indicated time points. ( C ) Immunofluorescence microscopy and representative Western blot for αSMA in untreated and PGE 2 + FGF2–treated myofibroblasts evaluated at 5 days. The histogram depicts mean densitometry values. ( D ) qPCR analysis of the fibrosis-associated genes ACTA2 , COL1A1 , FN1 , CTGF , VASP , and NOX4 after 24 hours of PGE 2 ± FGF2 compared with untreated myofibroblast control. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH . Data are presented as mean ± SEM; data points represent replicate samples from 3 experiments. Lines indicate conditions being compared. *Statistical significance compared with untreated myofibroblast; + Statistical significance compared with untreated, PGE 2 -, and FGF2-treated myofibroblasts. * P < 0.05 and + P < 0.05. Performed 2-way ANOVA for B , paired 2-tailed t test for C , and 1-way ANOVA for D . Diff, differentiation; De-diff, dedifferentiation.

Article Snippet: CCL210 normal adult human lung fibroblasts were obtained from the American Type Culture Collection.

Techniques: Staining, Membrane, Fluorescence, Microscopy, Immunofluorescence, Western Blot, Control

Journal: JCI Insight

Article Title: Myofibroblast dedifferentiation proceeds via distinct transcriptomic and phenotypic transitions

doi: 10.1172/jci.insight.144799

Figure Lengend Snippet: ( A ) CCL210 myofibroblasts were treated with PGE 2 , FGF2, or PGE 2 + FGF2 for 24–72 hours. qPCR analysis of the proliferation gene FOXM1 was performed at 48 hours, while CCNB2 , CCND1 , and CDKN1C were assessed at 72 hours (top panel). qPCR analysis of the antiapoptotic gene SERPINE1 was performed at 24 hours, while BIRC5 and MYC were assessed at 48 hours; the proapoptotic gene CASP9 was assessed at 72 hours. ( B ) Proliferation was assessed 96 hours following treatment with PGE 2 and/or FGF2 by CyQUANT Cell Proliferation Assay. ( C ) Apoptosis sensitivity was assessed by measuring total and cleaved CASP3 and PARP by Western blot analysis in myofibroblasts 5 days following addition of PGE 2 and/or FGF2, followed by treatment with the death receptor ligand anti-Fas. CASP3 was measured 30 minutes and PARP 1 hour following anti-Fas treatment. Densitometry represents ratio of cleaved products to total protein. Relative fold changes of indicated genes measured by qPCR are normalized to GAPDH . Data are presented as mean ± SEM. Data points represent replicate samples from 3 ( A and C ) or 4 ( B ) experiments. Lines indicate conditions being compared. * P < 0.05, compared with untreated myofibroblast; 1-way ANOVA. De-diff, dedifferentiation.

Article Snippet: CCL210 normal adult human lung fibroblasts were obtained from the American Type Culture Collection.

Techniques: CyQUANT Assay, Proliferation Assay, 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 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: 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: 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: 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: 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: 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: 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: Adult normal human lung fibroblasts (19Lu, catalog # CCl-210) were purchased from American Type Culture Collection (ATCC).

Techniques: Fluorescence, Western Blot, Control

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 1. ALI edema fluid has a greater proliferative effect on human lung fibroblasts than does HYDRO edema fluid. Human lung fibroblasts were plated at low density and serum starved for 48 h followed by the addition of the indicated dilutions of pulmonary edema fluid (2-, 20-, and 200-fold in SFM) pooled from either ALI or HYDRO patients. Cell num- ber was determined by direct counting after 96 h. E, ALI edema fluid at indicated dilutions; F, HYDRO edema fluid at indicated dilutions. Data are plotted as percent cell proliferation (mean SD; n 4/condition) of that seen with complete medium (10% SCM) from at least six separate deter- minations. , p 0.05 between ALI and HYDRO fluid-exposed fibroblast proliferation rates.

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 2. IL-1-neutralizing molecules partially abrogate ALI edema fluid-induced fibroblast proliferation. Subconfluent fibroblasts were serum starved for 2 days followed by a 96-h incubation with ALI edema fluid (1/20 dilution) in SFM. Cells were preincubated with IL-1ra (200 ng/ml, 1 h) or the fluid was preincubated with TBP-1, IL-1, or PDGF A/B chain neutralizing Abs (30 min) before their addition to the cells. Cell number from at least three separate determinations was directly counted and plotted as a percentage (mean SD; n 4/condition) of that seen with ALI edema fluid alone. , p 0.05 compared with edema fluid alone. A, Effect of the indicated neutralizing molecules (filled bars) on ALI edema fluid-induced fibroblast proliferation. Open bars represent proliferation seen with 0.4% and 10% SCM internal controls, as indicated. B, Concentration dependence of abrogation of ALI edema fluid-induced proliferation by IL-1ra (f) or TBP-1 (F).

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 6. Abrogation of effect of ALI edema fluid-induced gene ex- pression by IL-1ra. Subconfluent human lung fibroblasts were serum starved and exposed to either ALI or HYDRO fluid (1/20 diluted in SFM) for 8 h with or without preincubation with IL-1ra (1 h, 200 ng/ml). North- ern blotting was performed and band density was quantified on a Phos- phorImager. Representative autoradiograms are presented with the RNA species listed to the left and the fold induction (relative to HYDRO without IL-1ra) listed below each autoradiogram. Lane 1, HYDRO edema fluid alone; lane 2, HYDRO edema fluid IL-1ra; lane 3, ALI edema fluid alone; lane 4, ALI edema fluid IL-1ra.

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 4. Fibroblasts produce soluble mitogenic factors upon incuba- tion with ALI edema fluid. Human lung fibroblasts were incubated with either 1/20 diluted ALI edema fluid or SFM (1% BSA) for 24 h. This fibroblast-conditioned medium was applied to fresh serum-starved fibro- blasts as indicated (1/3, ALI 33%; 1/12, ALI 8% in 1.3% SCM (n 3 wells/condition). The corresponding control medium was the identical con- centration of ALI edema fluid that was not conditioned by exposure to cells. Proliferation was measured by direct counting of trypsinized cells after 72 h. f, Conditioned medium; , nonconditioned medium controls; F, 1.3% SCM alone control (SERUM ALONE). Data are plotted as mean SD from three separate determinations. , p 0.05 vs noncon- ditioned medium controls.

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 5. Microarray analysis of edema fluid exposed fibroblasts. To- tal RNA was isolated from subconfluent human lung fibroblasts exposed to either ALI or HYDRO fluid (1/20 diluted) for 8 h. The RNA was subjected to 32P-RT-PCR using primer pairs complementary to the 588 different cDNAs present on the cDNA membrane arrays. The resultant 32P-labeled cDNA population was hybridized to membrane arrays and quantified using a PhosphorImager. A, Highly induced genes from representative ALI and HYDRO edema fluid-exposed membranes are indicated at left, with fold induction indicated at right. B, Housekeeping genes from ALI and HYDRO edema fluid-exposed membranes. Lane 5, Ubiquitin; lane 6, phos- pholipase A2; lane 7, hypoxanthine phosphoribosyltransferase; lane 12, G3PDH; lane 13, -1-tubulin; lane 14, HLA class I C4-; lane 19, -actin; lane 20, ribosomal protein L13A; lane 21, ribosomal protein S9; all other lanes, negative controls.

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques: Microarray, Isolation, Reverse Transcription Polymerase Chain Reaction, Membrane, Labeling, Ubiquitin Proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 7. IL-6 protein secretion is induced by ALI edema fluid: specific abrogation by IL-1ra. Subconfluent human lung fibroblasts were serum starved for 48 h, followed by a 24-h incubation with the indicated samples. The conditioned medium was assayed for IL-6 by ELISA. ALI EF, ALI edema fluid (1/20 dilution in SFM); HYDRO EF, HYDRO edema fluid (1/20 dilution in SFM); 0.4% S Alone, 0.4% SCM; 10% S Alone, 10% SCM. Data are plotted as mean SD from four separate determinations (n 3 wells/condition). A, Effect of ALI/HYDRO edema fluid on IL-6 Ag levels in conditioned medium. Open bars, Basal level of IL-6 in nonconditioned medium (edema fluids). Filled bars, 24-h conditioned medium. , p 0.05 for greater than basal HYDRO EF level; denotes p 0.05 for response induction compared with that seen with HYDRO EF. B, Abrogation of ALI EF-induced IL-6 production by TBP-1 (F) or IL-1ra (f). Data are plotted as a percent of the values obtained after incubation with ALI EF in the absence of IL-1ra/TBP-1. , p 0.05 by ANOVA compared with ALI EF alone.

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression.

doi: 10.4049/jimmunol.172.4.2668

Figure Lengend Snippet: FIGURE 8. Autocrine mitogenic effect of IL-1-exposed fibroblasts is dependent on IL-6. DNA synthetic rates at 24 h were measured by 5-bromo- 2-deoxyuridine incorporation, as described in Materials and Methods. Data are plotted as mean SD (n 4/condition) from three separate determinations. A, IL-1 sensitizes fibroblasts to the mitogenic effect of IL-6. Quiescent fibroblasts were incubated with the indicated concentrations of IL-6 with (E) or without (F) submaximal concentrations of IL-1 (10 pg/ml). Data are plotted relative to the mitogenic effect of 10% SCM (set at 100%; f). B, Blockade of IL-6/gp130 abrogates autocrine IL-1-induced fibroblast proliferation. Quiescent fibroblasts were incubated with a 1/3 dilution of 24-h conditioned medium (CM) from fibroblasts incubated with (IL-1 - CM) or without (CM No IL-1) 10 pg/ml of IL-1, and DNA synthetic rates were measured in the presence or absence of indicated antagonists to IL-6 or of control IgG. Data are plotted relative to the autocrine mitogenic effect of IL-1 conditioned medium without added Abs (set at 100%). , p 0.05 by ANOVA compared with IL-1 conditioned medium alone.

Article Snippet: Normal diploid adult human lung fibroblasts (CCL-210) were obtained from the American Type Culture Collection (Gaithersburg, MD) and propagated in Eagle’s MEM with L-glutamine and penicillin/streptomycin (Mediatech/Cellgro; Birmingham, AL) with 10% FBS (Sigma-Aldrich, St. Louis, MO).

Techniques: Incubation, Control