(B) Nuli-1 cells treated with media or ZM241385; (C) Pretreatment with ZM241385 ±. CGS21680; (D) CSG21680 ± CSE or CSE alone; and (E) Pretreatment with ...
Smoke Extract Impairs Adenosine Wound Healing: Implications of Smoke-Generated Reactive Oxygen Species Diane S. Allen-Gipson, Matthew C. Zimmerman, Hui Zhang, Glenda Castellanos, Jennifer K. O’Malley, Horacio Alvarez-Ramirez, KusumKharbanda, Joseph H. Sisson, and Todd A. Wyatt Online Data Supplement
Supplemental Data Methods LHC basal and M199 medium were purchased from Biofluids (Rockville, MD). Streptomycin, penicillin, protease (type IV), fetal bovine serum and fungizone were purchased from Life Technologies (Grand Island, NY). The Type I collagen gel matrix, Vitrogen 100, was purchased from Cohesion (Palo Alto, CA). CPCA,(5-(N-cyclopropyl)-carboxamido-adenosine) and CGS21680
(
4‐[2‐[[6‐Amino‐9‐(N‐ethyl‐‐D‐ribofuranuronamidosyl)‐9H‐purin‐2‐
yl]amino]ethyl]benzene propanoic acid hydrochloride), A2A receptor agonists were purchased from TocrisBioScience/R & D System, Inc (Minneapolis, MN). Adenosine, N-acetylcysteine (NAC; non-selective antioxidant); bovine serum albumin (BSA), and all other reagents not listed were purchased from Sigma (St. Louis, MO).
Figure Legends
Figure E1. Smoke Extract Stimulated Adenosine Receptor Transcription and Adenosine A2A Receptor Protein Expression in Human Bronchial Epithelial Cells, BEAS-2B. (A) Transcript levels of A1, A2A, A2B, and A3 adenosine receptors were quantified using real-time Taqman PCR of RNA isolated from BEAS-2B cells treated with 5% smoke extract for 30 min. Values were normalized to (human) In-house Ribosomal RNA. Data is representative of one experiment conducted in triplicate and repeated twice with similar results. (B) Representative bands from Western blot analysis to detect smoke extract-induced A2A adenosine receptor protein
with a 44.7 kDa molecular mass. (C) Relative A2A receptor protein expression, normalized to beta actin. Data represent means SD of 2 separate experiments performed in duplicate.
Figure E2. Adenovirus-Mediated Overexpression of SOD1 or SOD2 Had No Effect on Smoke Extract-mediated Inhibition of Adenosine-stimulated Wound Closure.Smoke extract blocks CPCA-mediated wound closure in (A) Non-transfected; (B) AdEmpty; (C) SOD1 and (D) SOD2 BEAS-2B cells in serum free media as compared to CPCA alone (*P < 0.05). Error bars represent SE of 3 separate experiments performed in duplicate.
FigureE3.Smoke Extract Inhibits A2A-stimulated Wound Closure. (A) Representative of time course during smoke extract inhibition of A2A-stimulated wound closure. (B) Nuli-1 cells treated with media or ZM241385; (C) Pretreatment with ZM241385 ± CGS21680; (D) CSG21680 ± CSE or CSE alone; and (E) Pretreatment with ZM241285 ± CSE + CGS21680 or CSE alone. Each condition was conducted in eight individual wells and experiment repeated at least two different times. Impedance was measured at 4000 Hz, normalized and plotted as a function of time.
12 Fold Increase (Ribosomal units)
5% CSE
Media
#
#
10 8
#
#
6 4 2 0
A1
A2A
A2B
A3
CONTROL
% of Original Wound Closure
150 125
(A)
100 75 50 M199- serum (cltr)
25
CPCA 10-5 M(cltr) 5% CSE(cltr) CPCA 10-5M+ 5% CSE(cltr)
0 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours
EMPTY
150 125
(B)
100 75 50
M199-serum (AdEmpty) CPCA 10-5 M (AdEmpty)
25
5% CSE(AdEmpty) CPCA 10-5 M+ 5% CSE(AdEmpty)
0 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours
AdSOD1
% of Original Wound Area
150 125
(C)
100 75 50 25
M199-serum (AdSOD1) CPCA 10-5 M (AdSOD1) 5% CSE(AdSOD1) CPCA 10-5 M+ 5% CSE(AdSOD1)
0 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours
AdSOD2
150 125
(D)
100 75 50 M199-serum (AdSOD2)
25
CPCA 10-5 M (AdSOD2) 5% CSE(AdSOD2) CPCA 10-5 M+ 5% CSE(AdSOD2)
0 0 2 4 6 8 10 12 14 16 18 20 22 24 Hours
