SUPPLEMENTARY MATERIAL
MATERIALS AND METHODS
Stable shRNA transfection
To stably down-regulate δ-ENaC expression, NCI-H441 and Calu-3 cells were transfected
using commercially available shRNA lentiviral particles (SC-42421-V, Santa Cruz
Biotechnology). As negative control, control shRNA lentiviral particles (SC-108080, Santa
Cruz Biotechnology), carrying constructs encoding for negative scrambled shRNA, were
used. NCI-H441 and Calu-3 cells were seeded at 100,000/cm2 in 24-well plates 24 h prior to
lentiviral infection. The next day, medium was replaced with medium containing 5 µg/ml
Polybrene (Santa Cruz Biotechnology), and lentiviral particles were added to the cells
(7.5×103 infectious units of virus (IFU) per well). Forty eight hours after the transduction,
cells were split at a 1:3 ratio. Following overnight incubation in complete medium, puromycin
containing medium was applied onto the cells to select shRNA expressing puromycinresistant cells. Puromycin concentrations of 2.5 µg/ml and 7.5 µg/ml were previously shown
to be sufficient for selection of NCI-H441 and Calu-3 cells, respectively. Selected puromycinresistant clones were expanded and were used for expression and functional assays.
Immunofluorescence microscopy (IFM)
Transwell-grown human alveolar epithelial cell monolayers were fixed by 10 min incubation
with ice-cold methanol, followed by permeabilisation with 0.1% Triton X-100 for 8 min.
Unspecific binding sites were blocked by incubation with PBS containing 5% BSA for 30 min
at 37°C. After 60 min incubation at 37°C with a 1:50 dilution of the rat anti-δ-ENaC antibody
in PBS containing 1% BSA, the cell layers were washed three times with PBS (1% BSA).
Then, the cell layers were incubated with a 1:300 dilution of a mouse anti-ZO-1 antibody
(610966, BD Biosciences, Oxford, UK) and the washing procedure was repeated.
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Subsequently, cells were incubated for 30 min at 37°C with a 1:1000 dilution of the respective
Alexa Fluor-labelled F(ab')2 fragments in PBS containing 1% BSA. Nuclei were
counterstained with Hoechst 33342 (1 μg/ml in PBS). The specimens were then washed three
times with PBS (1% BSA) and embedded in FluorSave anti-fade medium (Merck,
Nottingham, UK). Images were obtained using a confocal laser scanning microscope (CLSM,
Zeiss LSM 710, Göttingen, Germany).
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RESULTS
Figure S1. Calu-3, NCI-H441 and ATI-like cell lysates were tested for δ-ENaC expression
using the commercially available polyclonal antibody (anti-δ-N). Lysates of non-transfected
(ctrl) and mock-transfected (mock) HEK-293 cells were used as negative controls; δ1-ENaCtransfected (δ1-ENaC) HEK-293 cells as positive controls. The anti-δ-N antibody showed
identical band patterns for non-transfected, mock-transfected and δ1-ENaC over-expressing
HEK-293 cells. High intensity bands were detected at 48 and 100 kDa. Additionally, several
bands of lower intensity (at 36, 52, 80, 140 kDa) were observed. No additional band or
increased signal intensity was observed in δ1-ENaC over-expressing HEK-293 cell lysates. In
the lysates of Calu-3, NCI-H441 and ATI cells, multiple bands were observed. The antibody
detected a protein at the molecular weight of ~85 kDa in the respiratory cell lysates (which
could correspond to the glycosylated full-length δ-ENaC protein as described by others [3, 4,
5, 6].
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Figure S2. RNAi against δ-ENaC in NCI-H441 and Calu-3 cell lines. Representative Western
blots showing expression of δ-ENaC in non-transfected as well as cells stably transfected with
scrambled shRNA and shRNA, designed to knockdown δ-ENaC (mock-transfected and
shRNA, respectively). Cell lysates were probed with anti-δ-C antibody. Beta-actin was used as
loading control. Intensities of protein bands at 100 kDa and of those corresponding to the full
length protein (i.e. 70 and 75 kDa, respectively) were not significantly changed.
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Figure S3. Electrophysiological effects of RNAi against δ-ENaC in NCI-H441 and Calu-3
cell lines. The δ-ENaC inhibitor, Evans blue (EB) and the activators, capsazepine and icilin
were used on cell monolayers of non-transfected cells (black bars) as well as on monolayers
stably transfected with scrambled shRNA (light grey bars) and shRNA designed to silence δENaC (dark grey bars). Short circuit currents (ISC) resulting after application of the relevant
compounds were measured and normalised to the ISC measured prior to the addition of the
substances (basal ISC). Results are expressed as means ± SD (n = 3-15).
Figure S4. Immunofluorescence microscopy for δ-ENaC (green) and ZO-1 (red) in
Transwell-grown human alveolar epithelial cells. Cell nuclei were counterstained with
Hoechst 33342 (blue). (a’) Shows a cross section along the grey Y-Z trace and in (b) a 3-D
reconstruction of the monolayer is depicted. Signals for δ-ENaC are localised mainly inside
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the
cells
with
occasional
single
punctate
staining
in
the
apical
membranes.
DISCUSSION
Specificity of anti-δ-ENaC antisera
To study δ-ENaC protein expression, two polyclonal anti-δ-ENaC antibodies were utilised:
the custom-made anti-δ-C [7] as well as the commercially available anti-δ-N antibody [1, 4, 5,
6]. As positive controls, the full-length δ1-ENaC protein, heterologously expressed in HEK293 cells was used. It is noteworthy that the antibodies tested were raised against amino acid
regions present in both δ-ENaC isoforms [2], thus, the δ1-protein was regarded as a suitable
positive control. Cell lysates of non-transfected and mock-transfected HEK-293 cells were
used as negative controls. The protein band at 100 kDa was detected in all positive and
negative control samples with both anti-δ-ENaC antibodies. Hence, this band can be
considered to be unspecific, despite the manufacturer’s specification in the case of anti-δ-N
antibody. Full-length δ1-ENaC protein only appeared as an additional band in the
immunoblots probed with the anti-δ-C antibody. Neither an additional band nor a band of
increased intensity was observed in positive controls probed with anti-δ-N antibody. Thus, the
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specificity of anti-δ-C antibody was confirmed, whereas the anti-δ-N antibody proved to be
unreliable in our hands.
Conformation of cellular localisation of δ-ENaC
To study the localisation of the δ-ENaC protein, human alveolar epithelial cells were grown
on Transwell inserts and co-immunostained using the anti-δ-C antibody and a monoclonal
anti-ZO-1 antibody as a marker of the apical cellular aspect. Immunocytochemical analysis
provided signals for δ-ENaC which were localised mainly inside the cells with occasional
single punctate staining observed in the apical membranes.
REFERENCES
1. Bangel-Ruland N, Sobczak K, Christmann T, Kentrup D, Langhorst H, Kusche-Vihrog K,
Weber WM (2010) Characterization of the epithelial sodium channel delta-subunit in
human nasal epithelium. Am J Respir Cell Mol Biol 42(4):498-505. doi:
10.1165/rcmb.2009-0053OC
2. Giraldez T, Afonso-Oramas D, Cruz-Muros I, Garcia-Marin V, Pagel P, GonzálezHernández T, Alvarez de la Rosa D (2007) Cloning and functional expression of a new
epithelial sodium channel delta subunit isoform differentially expressed in neurons of the
human and monkey telencephalon. J Neurochem 102(4):1304-15. doi: 10.1111/j.14714159.2007.04622.x
3. Haerteis S, Krueger B, Korbmacher C, Rauh R (2009) The delta-subunit of the epithelial
sodium channel (ENaC) enhances channel activity and alters proteolytic ENaC activation.
J Biol Chem 284(42):29024-40. doi: 10.1074/jbc.M109.018945
7
4. Kapoor N, Bartoszewski R, Qadri YJ, Bebok Z, Bubien JK, Fuller CM, Benos DJ (2009)
Knockdown of ASIC1 and epithelial sodium channel subunits inhibits glioblastoma whole
cell current and cell migration. J Biol Chem 284(36):24526-41. doi:
10.1074/jbc.M109.037390
5. Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL
(2009a) Regulation of epithelial sodium channels by cGMP/PKGII. J Physiol
587(11):2663-76. doi: 10.1113/jphysiol.2009.170324
6. Nie HG, Tucker T, Su XF, Na T, Peng JB, Smith PR, Idell S, Ji HL (2009b) Expression
and regulation of epithelial Na+ channels by nucleotides in pleural mesothelial cells. Am J
Respir Cell Mol Biol 40(5):543-54. doi: 10.1165/rcmb.2008-0166OC
7. Wesch D, Althaus M, Miranda P, Cruz-Muros I, Fronius M, González-Hernández T,
Clauss WG, Alvarez de la Rosa D, Giraldez T (2012) Differential N termini in epithelial
Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane. Am J
Physiol Cell Physiol 302(6):C868-79. doi: 10.1152/ajpcell.00255.2011
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