Additional file 1 of CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts
Creators: name: Nathan, Shyam
name: Zhang, Haijun
name: Andreoli, Mirko
name: Leopold, Philip L.
name: Crystal, Ronald G.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology
subject: Virology

description: Additional file 1: Table SI. Study subject demographics. Figure S1. Evaluation of growth factor expression in basal cells in the context of a range of LPA levels. Primary small airway basal cells (passage 3) were obtained from each of three healthy, non-smoking donors and plated in triplicate in the presence or absence of 0.1, 1.0, or 10 μg/ml LPA. Cells were evaluated for expression of CTGF, EDN1, TGFβ1, and PDGFA, PDGFB, and PDGFC. After a 3 h of LPA exposure, RNA was harvested and evaluated by qRT-PCR using 18S RNA to normalize the results. Data are expressed as the mean value of the 3 donors ± SE. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S2. Expression of LPA receptors in healthy non-smoker BC. Primary small airway basal cells (passage 3) were plated in triplicate and evaluated for expression of lysophosphatidic receptor family members: LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, LPAR6 and sphingosine-1-phosphophate receptor family members: S1PR1, S1PR2, S1PR3, S1PR4, S1PR5. RNA was harvested and evaluated by qRT-PCR using 18S RNA to normalize the results. Data are expressed as the mean value of the 3 donors ± SE. Figure S3. Human phospho-kinase array coordinates. Reference spots for the R&amp;D Systems Proteome Profilier Human Phospho-kinase Array including kinases that did not respond significantly to LPA treatment. Figure S4. Assessment of SAE BC toxicity following exposure to kinase inhibitors. Basal cell LDH release was assessed using an LDH enzyme activity assay in cell culture medium 24 h after a 4 h exposure to kinase inhibitors. Data are normalized to cell culture volume tested. There was no toxicity observed at the levels of inhibitors used (“NS” – not significant; p &gt; 0.2 compared to naive). Figure S5. Effect of ERK1/2, and EGFR inhibitors on fibroblast response to basal cell conditioned medium. Primary normal human lung fibroblasts (NHLF, passage 5) were treated with BC-conditioned media from each of three non-smoking donors obtained in the presence or absence of 1 μg/ml LPA. Conditioned medium was collected from cultures that were naive or treated with 5 µM ERK1/2 inhibitor (LY32149966), or EGFR inhibitor (AG1478). Conditioned medium was then transferred to naive NHLF. After 24 h, expression of collagen type 1 (COL1A1) and smooth muscle actin (ACTA2) in fibroblasts was assessed after 24 h in conditioned medium. RNA was harvested and evaluated by qRT-PCR using 18S RNA to normalize the samples. A. Medium collected following treatment with ERK1/2 inhibitor. B. Medium collected following treatment with P70 S6 kinase inhibitor. C. Medium collected following treatment with EGFR inhibitor. Data are expressed as the mean value of the 3 donors ± SE. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S6. Evaluation of growth factor expression in basal cells in the context of the presence or absence of 1 μg/ml LPA and a range of CREB inhibitor (666-15) concentrations. Primary small airway basal cells (passage 3) were obtained from each of three healthy, non-smoking donors and plated in triplicate presence or absence of 1 μg/ml LPA, and a combination of LPA and 200 nM, 1 μM or 2 μM CREB inhibitor (666-15). Cells were evaluated for CTGF, EDN1, TGFβ1, and PDGFB. After a 3 h of LPA exposure, RNA was harvested and evaluated by qRT-PCR using 18S RNA to normalize the results. Data are expressed as the mean value of the 3 donors ± SE. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S7. Evaluation of growth factor expression in basal cells in the context of the presence or absence of 1 μg/ml LPA and a range of ERK inhibitor (LY32149966) concentrations. Primary small airway basal cells (passage 3) were obtained from each of three healthy, non-smoking donors and plated in triplicate presence or absence of 1 μg/ml LPA, and a combination of LPA and 5 nM, 50 nM, 500 nM, 1 μM, 5 μM or 10 μM ERK1/2 inhibitor (LY32149966). Cells were evaluated for CTGF, EDN1, TGFβ1, and PDGFB. After a 3 h of LPA exposure, RNA was harvested and evaluated by qRT-PCR using 18S RNA to normalize the results. Data are expressed as the mean value of the 3 donors ± SE. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001.