Supplemental Methods
Quantification of HBD2-specific mRNA.
Total RNA from HT-29 cells was prepared using RNApureTM according to manufacturer’s
instructions (Peqlab) and mRNA from 1.25 µg total RNA was reversely transcribed using
random hexamer and oligo(dT)15 (Promega).
In the semiquantitative approach, standard PCR (Roche) was performed with 30 cycles
from 62.5 ng cDNA using the specific primer pairs for HBD2. For HBD2, a second round
of PCR using the same primer pair produced detectable amounts of the expected 195 bp
fragment. PCR products were separated by agarose gel electrophoresis and visualized
after ethidium bromide staining. Using the Image J software (National Institutes of Health),
mean band densities representing the 195 bp (HBD2) and 422 bp PCR product (GAPDH)
were evaluated. HBD2 expression was calculated as the quotient of the HBD2 and
GAPDH band mean densities of each sample. RNA preparations were treated with
RNase-free DNase (Sigma-Aldrich) before reverse transcription reaction in order to
exclude relevant DNA contamination. PCR revealed comparable relations of HBD2 and
GAPDH from untreated and DNAse treated templates (not shown).
The TaqMan® gene expression assay for HBD2/DEFB4 (HS00823638m1) or the GAPDH
(4326317E; both from Appleid Biosystems) comprising primer pairs matched for different
genomic regions of two different exons, excluding bias of the genomic DNA amplification.
Cloned HBD2 and the GAPDH fragment derived from the sequence of the respective
gene expression assay cloned in pCR2.1-TOPO (Invitrogen) were used as standards
(pHBD2 and pGAPDH). Quantification was optimized in the range of 102-109 template
molecules per reaction using linearized plasmid DNA with 360 nM of the respective
TaqMan® Gene expression assay for HBD2 and GAPDH in the StepOne Plus
Thermocycler (Applied Biosystems; Supplemental Figure 1). HBD2 and GAPDH specific
amplifications for individual samples from HT-29 cells were performed in PCR reactions
containing both assay, and in parallel to mixed plasmid standards. Copy numbers of
HBD2 or GAPDH mRNA were calculated from the CT values of the samples in relation to
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the respective standard curve. HBD2 expression was calculated as the quotient of the
copy numbers of HBD2 and GAPDH mRNA.
Western-blot analysis.
Detached HT-29 cells, preadipocytes or homogenized mucosal scrapings of human colon/
ileum sections were lysed with 3% SDS in 50 mM Tris-HC, pH 8.0 and immediately
transferred to reducing sample buffer for SDS-PAGE. Heat denatured proteins were
separated by gradient Tris-glycine SDS-PAGE (5-22.5% polyacrylamide). The following
prestained markers were used to estimate protein sizes of the HBD2 peptide (about
4 kDa) and the larger fusion proteins (about 35 kDa): Color Marker Ultra-Low Range
1-26 kDa (Sigma-Aldrich) and Page RulerTM Prestained Protein Ladder 11-170 kDa (MBI
Fermentas). Proteins were transferred to a polyvinylidene fluoride membrane with a pore
size of 0.22 µm (Millipore) by tank blotting (Biometra). Air-dried membranes were
incubated overnight with 1% protein-free reagent (Roche) in TBST buffer containing
50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20 (Sigma-Aldrich). After extensive
washing with TBST, membranes were incubated for 2 h with the polyclonal goat-derived
HBD2-specific antibody (200 ng/mL; sc10854; Santa Cruz Biotechnology) diluted in TBST.
Membranes, again washed twice with TBST, were incubated for 1.5 h with a horseradish
peroxidase-coupled secondary goat-specific antibody (16 ng/mL; 705035147, Jackson
ImmunoResearch). The primary antibody for protein loading control was the murine
monoclonal antibody specific to human β-actin (1:10.000; clone AC-15, Sigma-Aldrich)
followed by a horseradish peroxidase-coupled secondary mouse-specific antibody
(1:1000, P0161, Dako). Antibody binding was detected using the ECLTM system (Roche).
Chemiluminescence was visualized by LAS 1000 (Fujifilm Life Sciences). Exposition times
were 10-30 min for HBD2 and 10 sec-1 min for β-actin.
Cloning of HBD2 for eukaryotic and prokaryotic fusion protein expression.
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HT-29 cells were treated with 10 ng/mL IL-1β for 6 h and cDNA was prepared.
Appropriate terminal restriction sites, inserted by PCR primers, allowed directed cloning of
HBD2 sequences. A complete HBD2 mRNA for constitutive eukaryotic expression of a
secreted
HBD2-EGFP
fusion
protein
was
amplified
using
the
primer
pair
5’-GCTCCCAGCCTCGAGCCATGAGGGTC-3’/
5’-GCTTCTTGGCCGAATTCTGGCTTTTTGC-3’. Using standard recombinant techniques
including sequence verification (Seqlab), the HBD2 construct was cloned as an
XhoI/EcoRI fragment into the vector pEGFP-N2 (pEGFP; Clontech) for expression under
the control of the early promoter/enhancer of the human cytomegalovirus (pCMV). The
resulting pHBD2 vector contained the native HBD2 leader-sequence for secretion of the
HBD2-EGFP fusion protein by transfected mammalian cells.
For prokaryotic expression, a cDNA for mature HBD2 was recovered with the primer pair
5’-CTCTTCCAGGTGGATCCGGTGGTATAGG-3’/
5’-GCTTCTTGGCCGAATTCTGGCTTTTTGC-3’ and cloned as a BamHI/EcoRI fragment
downstream of the GST sequence into the vector pGEX-2T (GE Healthcare). For the
controlled fusion protein production and subsequent HBD2 release, the isopropylbeta-D-thiogalactopyranoside (IPTG) inducible lactose operon promoter (plac) and the
internal thrombin cleavage site (TCS) were used.
Production and purification of GST-HBD2.
E. coli BL21 were transformed with the expression vector for GST or for the GST-HBD2
fusion protein and cultured in LB medium. After initial culture to OD600nm of 0.4-0.5
(GST) or 1.0-1.2 (HBD2-GST) recombinant protein production was induced by 200 mg/mL
IPTG for 4-5 h (GST) or 2 h (HBD2-GST). All following steps were performed on ice and
with pre-cooled solutions and devices. Bacteria were collected by centrifugation (4,000xg,
20 min, 4°C, without brake) and resuspended in phosphate-buffered saline, pH 7.2 (PBS).
Cells were lysed by low energy ultrasonic treatment (Bandelin Electronic) in the presence
of 2 µg/mL DNase I and 50 µg/mL lysozyme (both from Fluka). Clear bacterial lysates
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after high-speed centrifugation (12,000xg, 30 min, 4°C) were subjected to GST TrapTM
affinity chromatography (GE Healthcare) and eluted with a buffer containing 50 mM
Tris-HCl, pH 8.0, 100 mM NaCl and 20 mM gluthathione. If applicable, glutathione was
removed by standard Sephadex G-25 gel filtration (GE Healthcare) and buffer was
replaced for PBS or 50 mM Tris-HCl, pH 8.0. Protein concentrations of glutathione-free
GST or GST-HBD2 preparations were determined using the bicinchoninic-acid assay
(Pierce).
Product
purity
was
controlled
by
SDS-PAGE
and
staining
with
Coomassie brilliant blue-R250 (Merck) and HBD2 specific western-blot analysis. Final
preparations to be used for the experiments were adjusted to 2.5 mg/mL, shock-frozen
over liquid nitrogen and stored at -80°C.
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