Supplemental Digital Content
Supplementary Methods
Cell cultures
Jurkat T-cells (European Collection of Cell Cultures) and primary T-cells were cultured in 6well-plates in RPMI-1640 medium (Sigma-Aldrich, Taufkirchen, Germany) supplemented
with 10% heat-inactivated fetal calf serum (FCS; Biochrom, Berlin, Germany), penicillin (100
IU/ml), streptomycin (100 µg/ml), sodium pyruvate and L-glutamine (Gibco) at 37°C in a
humified atmosphere of 5% CO2 in air. HEK-293 cells (European Collection of Cell Cultures)
were grown in Dulbecco’s modified Eagle medium (Lonza, Walkersville, MD) supplemented
with 10% heat-inactivated FCS, 1 % penicillin/streptomycin, 1 % NEAA and L-glutamine at
37°C in 5 % CO2.
Vector cloning
For cloning of the reporter construct, a GR-α 3’-UTR 776 bp-fragment, containing the
predicted target sites of miRNA-124, was amplified by PCR from human genomic DNA with
the primers given in Table 2 (synthesized by Metabion, Martinsried, Germany). The PCR
product was first ligated into the pSC-B amp/kan vector (UltraBlunt PCR Cloning Kit,
Stratagene) according to the manufacturer’s protocol and then cloned into the PmeI-NotI
restriction sites of the multiple cloning region of the psiCHECKTM-2 vector. This reporter
vector system contains both Renilla reniformis luciferase (Rluc) and Photinus pyalis (firefly)
luciferase (Fluc) on a single plasmid with the MCS located downstream of the firefly
encoding region.
For cloning of a miR-124 expression vector, a 599 bp-fragment containing the miRNA-124-2
stem-loop along with upstream 232 bp and downstream 258 bp of flanking DNA was
amplified by PCR from human genomic DNA (primers are listed in Table 2). Cycling
conditions comprised an initial denaturation step (95°C for 3 min), 35 cycles with 95°C for 30
s, 60°C for 30 s and 72°C for 30 s, and a final extension step of 72°C for 3 min. PCR products
were analysed by agarosegelelectrophoresis, ligated into the pSC-B amp/kan vector
(UltraBlunt PCR Cloning Kit, Stratagene) according to the manufacturer’s protocol and
subsequently cloned into the BglII-PstI restriction sites of the multiple cloning region of the
pmR-ZsGreen1 vector. Finally, the construct was verified by sequence analysis (MWG
Biotech, Ebersberg, Germany); functionality was tested by transfection experiments with
subsequent analysis of miR-124 by means of qPCR.
Determination of transfection efficiency
The transfection efficiency was analysed using the Attune® Cytometer (Applied Biosystems).
Briefly, 1x106 transfected cells were transiently transfected either with 1 µg pmR-ZsGreen 1
vector, or with 50 nm Cy™3 dye-labeled Pre-miR™ Negative Control (Ambion) or with 50
nm DY-547-labeled siGLO RISC-Free Control siRNA (Dharmacon). 24 h after
electroporation cells were harvested, washed twice with PBS and transferred to 5 ml
polystyrene tubes in a total volume of 1 ml PBS. The fluorescent signal was detected using
488nm excitation with either a 530nm/30nm bandpass emission filter (ZsGreen 1) or a
575nm/24nm bandpass emission filter (Cy3 and DY-547). At least 10000 events per tube
were measured. The transfection efficiency was calculated as the percentage of fluorescent
cells in the total number of cells.
Quantitative real-time RT-PCR
Quantification of mRNA expression
cDNA was synthesized from equal amounts of total RNA (1000 ng) using Superscript III
reverse transcriptase (Invitrogen) and oligo(dT) and random hexamer primers following the
supplier’s instructions. Quantitative RT-PCR was performed in duplicate on a LightCycler
480 instrument (Roche Diagnostics, Mannheim, Germany) using LightCycler 480 Probes
Master and RealTime ready single assays (Roche Diagnostics) for IL-2 (Assay 100958) and
for the reference genes SDHA (Assay ID 102136) and TBP (Assay ID 101145). The
RealTime ready single assays contain target specific primers and a Universal ProbeLiberay
LNA probe. Assays specifically amplifying the GR-α and GR-β isoform were designed using
the ProbeFinder software (Roche Diagnostics). Primers and Universal ProbeLiberay LNA
probes are listed in Table 2 (primer binding sites are illustrated in Figure 1). The cycling
conditions comprised an initial denaturation phase at 95°C for 5 min, followed by 45 cycles at
95°C for 10 s, 60°C for 30 s and 72°C for 15 s; mRNA expression was normalized against
SDHA and TBP expression, which have been proven to be suitable reference genes under the
experimental conditions (28). PCR amplification efficiencies were determined by serial
dilution standard curves, and relative GR-α and GR-β mRNA expression was calculated by
the LightCycler Relative quantification software using an efficiency-corrected algorithm.
Quantification of miRNA expression
Equal amounts of RNA (10 ng) were reverse transcribed using miRNA-specific stem-loop
primers and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems).
Quantitative real-time PCR was performed in duplicate on the LightCycler 480 instrument,
using LightCycler 480 Probes Master and applying the following cycling conditions:
denaturing at 95°C for 10 min, 45 cycles of 95°C for 15 s and 60°C for 60 s. U6 RNA was
used for normalization of miRNA expression data.
Western Blotting
For preparation of whole cell extracts, 2 million Jurkat cells were transiently transfected with
50 nM pre-miR-124 or pre-miR negative control using Neon Transfection System
(Invitrogene). 24 hours after transfection, cells were harvested, washed with ice cold PBS,
and resuspended in 500 µl lysisbuffer (50 mM Tris, 5mM EDTA, 150 mM NaCl, 1% triton X
100 (v/v), pH 7.3) supplemented with protease inhibitor cocktail set III (Calbiochem).
Subsequently, cell lysates were sonified two times for 30 seconds and incubated on ice for
further 10 minutes. Total protein extracts were finally collected by a centrifugation step for 10
minutes and 15 000 g at 4°C. Protein concentration was determined using Bradford assay
(Bio-Rad). 30 µg of total protein extract were subjected to electrophoresis in a 10 % SDSPAGE and subsequently transferred onto PVDF-membrane. Non-specific binding sites on the
membrane were blocked with 5% non-fat dry milk in PBST (PBS + 0.1% Tween-20).
Antibodies were applied in PBST supplemented with 1% non-fat dry milk. GR-α protein was
detected using anti-glucocorticoid receptor alpha rabbit polyclonal antibody (Thermo
Scientific; 1:1000). Incubation with mouse monoclonal antibody AC-15 to β-actin (Sigma;
1:30000) served as a loading control. Immunoreactive bands were detected using horseradish
peroxidise-labelled goat anti-mouse antibody (Cell Signaling Technologies; 1:10000) or
stabilized goat anti-rabbit HRP conjugate (Pierce; 1:2000). Visualization was achieved using
20 x LumiGlo Reagent and 20 x Peroxide (CST).
Supplementary Table 1: Characteristics of healthy volunteers
Characteristics of healthy volunteers
N
15
Age (years)
40.5 (± 5.5)
Gender (male)
10 (66. 6 %)
Nonsmokers, without suspect of any
acute or chronical disease, blood count
and electrolytes within normal ranges
Supplementary Figure legends
Figure S1: FACS Plots of purified T-cells. Transcripts 106 human T-cells were
simultaneously counterstained with 10 nM Qdot 655 Anti-Human CD4 Antibody Conjugates
and 10 nM Qdot 605 Anti-Human CD8 Antibody Conjugates (both Invitrogen) according to
the manufacturer‘s recommendations. Fluorescence signals were analysed on an Attune®
Acoustic Focusing Cytometer (Invitrogen) using 488 nm excitation with a 640 nm longpass
filter (Qdot 655) and a 405 nm excitation with a 603nm/48nm bandpass filter (Qdot 605).