Supplementary Information Supplementary Materials and Methods Bioplex assay
Cytokines concentrations in BAL fluid were determined by multiplex cytokine array assays (Bioplex, Bio-Rad, Hercules, CA). The assay was performed according to the manufacturer's instructions. Cytokine concentrations were automatically calculated with Bio-Plex Manager software by using a standard curve derived from a recombinant cytokine standard.
Supplementary Figure Legends
Figure S1. Expression of tight junction protein zonula occludens-1 (ZO-1) and occludin is
decreased in mouse lungs after LPS administration for 3 days compared with naïve mouse lungs.
Figure S2. Electroporation-mediated gene transfer of α1 subunit of ENaC increases α1-ENaC
expression in previously injured mouse lungs by quantitative polymerase chain reaction.
Statistical analysis was by one way ANOVA (mean ± SEM; n = 6). **P < 0.01 compared to
pCDNA3.
Figure S3. Overexpression of β1-Na,K-ATPase, but not α1-ENaC, increases expression of tight
junction proteins ZO-1 in 16HBE14o- cells and HPAEC. Immunofluorescence staining of ZO-1
was followed after 16HBE14o- cells (a) or HPAEC (b) transfected with β1-Na,K-ATPase or α1- ENaC for 48 h, and representative photographs were taken using confocal microscope. Arrows show tight junction formation and magnification is 400 X.
Supplementary Table S1. Cytokines concentrations in BAL fluid after gene transfer of α1- ENaC or β1-Pump in pre-existing injured lungs (pg/ml).
Naive LPS
pcDNA3
+ LPS
α1-ENaC
+ LPS
β1-Pump
+ LPS
Combo
+ LPS
IL-1α 40±3 234±34 220±15 245±23 254±44 206±21
IL-1β 43±0.4 247±12 237±22 257±19 269±31 224±11
IL-2 40±1 65±2 61±3 63±2 65±6 61±4
IL-3 27±0.2 88±11 83±11 87±12 103±13 95±16
IL-4 19±1 79±4 80±4 76±3 89±7 80±8
IL-5 19±1 29±2 29±1 32±2 30±2 30±2
IL-6 124±11 3999±102 3834±309 4468±268 3858±372 3976±495
IL-9 19±1 39±2 37±2 40±2 41±4 37±2
IL-10 34±0.1 78±4 76±6 73±5 87±10 72±5
IL-12 (p40) 153±8 4689±697 4122±483 4578±311 4013±636 4167±678
IL-12 (p70) 69±1 602±93 553±128 564±96 627±176 569±79
IL-13 23±0.2 62±4 58±3 60±4 62±8 58±2
IL-17 28±0.3 530±118 542±74 570±127 528±77 537±79
Eotaxin 28±0.2 71±1 68±5 72±3 78±7 66±3
G-CSF 22±1 5461±709 4899±393 5155±554 5261±975 4828±325
GM-CSF 57±1 91±2 89±6 87±3 108±17 84±3
IFN-γ 23±1 96±16 83±6 85±13 101±25 82±14
KC 74±1 2951±704 2463±460 2699±497 3077±382 2654±428
MCP-1 16±0.2 131±21 101±17 125±24 149±26 115±25
MIP-1α 186±5 3403±339 3411±496 3624±189 3854±383 3094±126
MIP-1β 25±1 1207±267 1101±179 1093±130 1405±149 1138±148
RANTES 53±1 7068±726 6783±517 7760±1008 7367±1403 7226±1104
TNF-α 32±1 370±29 343±30 402±72 376±76 398±98
Supplementary Table S2. Primers used for qPCR
Forward primer / Reverse primerα1-ENaC / 5’- TCCTGCAACCAGGCGAATTACTCT-3’ / 5’-AGGGACAGACCGTTGTTGATTCCA-3’
β1-Na,K-ATPase / 5’- GGCAAACTCCTGCAGCCCAA-3’ / 5’-CCCTGAAAACGGTCTTTCTCACTGTACC-3’
Mouse GAPDH ZO-1
Occludin
Human GAPDH / 5’-ACCCAGAAGACTGTGGATGG-3’
5’- AGGTCTTCGCAGCTCCAAGAGAAA-3’
5’-TAAATCCACGCCGGTTCCTGAAGT-3’
5’-TCGACAGTCAGCCGCATCTTCTTT-3’ / 5’- GGATGCAGGGATGATGTTCT-3’
5’- ACAGGCCTCAGAAATCCAGCTTCT-3’
5’- AGGTGTCTCAAAGTTACCACCGCT-3’
5’- ACCAAATCCGTTGACTCCGACCTT-3’
Supplementary Fig. S1
Supplementary Fig. S2
Supplementary Fig. S3a
Supplementary Fig. S3b