Journal: Respiratory Research

Article Title: CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts

doi: 10.1186/s12931-021-01677-0

Figure Lengend Snippet: Effect of lysophosphatidic acid (LPA) on fibrotic growth factor expression in the small airway epithelial basal cells (SAE BC). Primary SAE BC from each of 3 non-smoking individuals were plated in triplicate in the presence or absence of 1.0 μg/ml LPA and evaluated for expression of connective tissue growth factor ( CTGF ), endothelin-1 ( EDN1 /ET-1), transforming growth factor beta ( TGFB1 ), and platelet derived growth factor family member B ( PDGFB ). a mRNA. After a 3 h LPA exposure, RNA was harvested and evaluated by qRT-PCR normalized to 18S RNA. b Protein. After a 3 h LPA exposure, LPA was removed and cells were incubated with basal media without growth factor supplements for 24 h. Conditioned media was harvested and evaluated for growth factor content by ELISA. Each sample was assessed in triplicate; data are expressed as the mean value of the 3 donors ± SE. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human COL1A1 ELISA kit (MyBioSource, La Jolla, CA), ENPP2 human ELISA kit (R&D Systems), human LPA ELISA Kit (Echelon Biosciences) were performed according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts

doi: 10.1186/s12931-021-01677-0

Figure Lengend Snippet: Effect of CREB inhibitor (CREB inh) on growth factor mRNA and protein levels. Primary basal cells from each of three non-smoking donors were plated in triplicate in the presence or absence of 1 μg/ml LPA, 200 nM CREB inhibitor 666-15 and a combination of LPA and CREB inhibitor 666-15. a mRNA expression levels. Shown are connective tissue growth factor ( CTGF ), endothelin-1 ( EDN1 ), transforming growth factor beta ( TGFB1 ) and platelet derived growth factor B ( PDGFB ) mRNA levels determined after 3 h of LPA exposure with and without CREB inhibitor (666-15) using qRT-PCR using 18S RNA to normalize the samples. b Protein levels. Shown are protein levels in conditioned media of LPA with and without CREB inhibitor 666-15, LPA with or without CREB inhibitor. After 3 h, the stimuli/inhibitor were removed and cells were incubated with non-supplemented basal media for 24 h. Conditioned media was harvested and evaluated for growth factor levels by ELISA. Data are expressed as the mean value of the 3 donors ± SEM. ND not detected. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human COL1A1 ELISA kit (MyBioSource, La Jolla, CA), ENPP2 human ELISA kit (R&D Systems), human LPA ELISA Kit (Echelon Biosciences) were performed according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts

doi: 10.1186/s12931-021-01677-0

Figure Lengend Snippet: Effect of ERK1/2 inhibitor (ERK1/2 inh) on growth factor expression. Primary basal cells from each of 3 non-smoking donors were plated in triplicate in the presence or absence of 1 μg/ml LPA, 5 µM ERK1/2 inhibitor (LY32149966) and a combination of LPA and ERK1/2 inhibitor (LY32149966). a mRNA. Shown are expression levels of connective tissue growth factor ( CTGF ), endothelin-1 ( EDN1 ), transforming growth factor beta ( TGFB1 ) and platelet derived growth factor B ( PDGFB ) determined after 3 h of LPA exposure with and without ERK1/2 inhibitor (LY32149966) using qRT-PCR using 18S RNA to normalize the samples. b Protein. Shown are growth factor levels in the conditioned media. After 3 h of LPA with and without CREB inhibitor (666-15) exposure, LPA with or without ERK1/2 inhibitor was removed and cells were incubated with un-supplemented basal media for 24 h. Conditioned media was harvested and evaluated for growth factor content by ELISA. Data are expressed as the mean value of the 3 donors ± SEM. ND—not detected. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human COL1A1 ELISA kit (MyBioSource, La Jolla, CA), ENPP2 human ELISA kit (R&D Systems), human LPA ELISA Kit (Echelon Biosciences) were performed according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts

doi: 10.1186/s12931-021-01677-0

Figure Lengend Snippet: Effect of EGFR inhibitor (EGFR inh) on growth factor mRNA levels. Primary basal cells (passage 3) from each of three non-smoking donors were plated in triplicate in the presence or absence of 1 μg/ml LPA, 10 µM EGFR inhibitor (AG1478), and a combination of LPA and EGFR inhibitor. a mRNA expression levels shown are mRNA levels of connective tissue growth factor ( CTGF ), endothelin-1( EDN1 ), transforming growth factor beta ( TGFB1 ), and platelet derived growth factor B were determined after 3 h of LPA exposure with and without the inhibitor using qRT-PCR using 18S RNA to normalize the samples. b Protein levels in the conditioned media. After 3 h of LPA with and without EGFR inhibitor (AG1478) exposure, LPA with or without inhibitor was removed and cells were incubated with non-supplemented basal media for 24 h. Conditioned media was harvested and evaluated for growth factor content by ELISA. Data are expressed as the mean value of the 3 donors ± SEM. ND—not detected. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human COL1A1 ELISA kit (MyBioSource, La Jolla, CA), ENPP2 human ELISA kit (R&D Systems), human LPA ELISA Kit (Echelon Biosciences) were performed according to the manufacturer’s instructions.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts

doi: 10.1186/s12931-021-01677-0

Figure Lengend Snippet: Effect of CREB inhibitor (CREB inh) on fibroblast expression in response to basal cell conditioned medium. Primary normal human lung fibroblasts (NHLF) were treated with conditioned media from each of 3 non-smoking donors obtained in the presence or absence of 1 μg/ml LPA. For each condition, conditioned medium collected from cultures that were naive or treated with vehicle control (DMSO) or 200 nM CREB inhibitor (666-15) were collected and transferred to NHLF. Fibroblast expression of collagen type 1 ( COL1A1 ) and smooth muscle actin ( ACTA2 ) was assessed after 24 h in conditioned medium. a mRNA levels. RNA was harvested and evaluated by qRT-PCR using 18S RNA to normalize the samples. b Protein levels. At the same time point, conditioned media was collected and assessed for COL1A1 secretion. Cells were solubilized with a RIPA buffer and evaluated for alpha smooth muscle myosin by ELISA. Data are expressed as the mean value of the 3 donors ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human COL1A1 ELISA kit (MyBioSource, La Jolla, CA), ENPP2 human ELISA kit (R&D Systems), human LPA ELISA Kit (Echelon Biosciences) were performed according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts

doi: 10.1186/s12931-021-01677-0

Figure Lengend Snippet: Effect of CREB inhibitor (CREB inh) on autotaxin ( ENPP2 ) expression, secretion, and activity in fibroblasts in response to medium conditioned by LPA-treated basal cells. Primary NHLF were treated with conditioned media from each of 3 non-smoking donors obtained in the presence or absence of 1.0 μg/ml LPA. For each condition, conditioned medium collected from cultures that were naive or treated with vehicle control (DMSO) or 200 nM CREB inhibitor (666-15) were collected and transferred to NHLF. Autotaxin, an enzyme responsible for the extracellular production of LPA, in encoded by ENPP2 . Expression of the ENPP2 gene was assessed in fibroblasts 24 h after exposure to conditioned medium. Autotaxin protein levels and LPA levels were assessed in cell culture medium 24 h after exposure to conditioned medium. a ENPP2 mRNA levels. mRNA was measured by qRT-PCR using 18S RNA to normalize the samples. b Autotaxin protein levels. Protein levels were measured in cell culture medium by ELISA. c LPA levels. LPA was measured in cell culture medium by ELISA. Data are expressed as the mean value of the 3 donors ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human COL1A1 ELISA kit (MyBioSource, La Jolla, CA), ENPP2 human ELISA kit (R&D Systems), human LPA ELISA Kit (Echelon Biosciences) were performed according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay