Download 1 Supplementary materials and methods Reagents and Western

Supplementary materials and methods
Reagents and Western blotting
Lipopolysaccharide (LPS, Sigma) and TGF-β1 (R&D Systems) were resolved in water.
Chemicals (Sigma) and solvents for secretion experiments: ionomycin, glyburide, probenicid,
reserpine (all DMSO) and brefeldin A (ethanol).
Western blotting was performed according standard techniques using reducing sample
buffer with EDTA/ß-mercaptoethanol. Probes were subjected to SDS-PAGE and proteins
transferred to nitrocellulose membranes. Membranes were blocked with 2.5% BSA.
Polyclonal anti-YB-1 antibodies raised with recombinant full-length YB-1 protein (kind
gift by Capowski and Malter, University of Wisconsin Medical School, Madison, WI 53792,
USA) or peptide-derived anti-YB-1 antibodies (N-term., antikoerper-online) were used at a
dilution of 1:1,000 for Western blotting. Polyclonal anti-MIF antibody (Kleemann et al.,
2000) was used at a dilution of 1:1,000 for Western blotting. Rab7 and GAPDH antibodies
were obtained from Sigma and used at a dilution of 1:1,000 for Western blotting.
Secondary antibodies were horse radish peroxidase-linked anti-rabbit and -mouse antibodies
(Amersham, 1:2,000 to 1:5,000).
Immunohistochemistry
Immunohistochemistry and double immunofluorescence analyses have been performed
according to standard procedures. Paraffin-embedded sections were stained with hematoxylin
and eosin. YB-1 staining was performed using a polyclonal anti-YB-1 antibody (van Roeyen
at al., 2005) and a suitable secondary antibody using diaminobenzidine as chromogen. For
double immunofluorescence staining polyclonal anti-YB-1 and monoclonal anti-CD68 (ED-1)
antibodies (Santa Cruz biotechnolgies) were used with suitable AlexaFluor™-conjugated
secondary antibodies.
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Peptide design
The peptide used for migration assay corresponds to a motif within the cold shock domain
(CGFINRNDTKEDVFVHQ), a control peptide with the sequence HVAGNPGGDAAPAAC
was added at similar concentrations.
Recombinant protein preparation
Recombinant YB-1 protein (rYB-1) was prepared and purified as reported (En-Nia et al.,
2005).
Confocal laser scanning microscopy and live cell imaging
Confocal laser scanning microscopy was carried out at a Zeiss LSM510 Meta confocal
microscope (Zeiss, Germany) using a water immersion objective (63x). Excitation for GFP
was performed at 488 nm and for TRITC at 543 nm with a detection range for GFP from 500
to 540 nm and for TRITC from 550 to 600 nm). For stress-fiber staining MC were grown on
coverslips for 36h before stimulation and fixed at the indicated time points using 4%
paraformaldehyde in PBS. Cells were further processed as described previously (Raffetseder
et al., 2003) and stained with 1
M TRITC-phalloidin (Sigma) prior to mounting.
For live cell imaging rMC were grown on coverslips and transfected with indicated
plasmids. 36 h after transfection medium was substituted by a FCS-reduced medium. 2 h later
cells were transferred to a thermostat- (37°C) and CO2-controlled perfusion chamber. Cells
were visualized every 2 minutes for up to 2 h.
Cell proliferation and scratch wound assay
Human epithelial kidney (HK-2), rat MC, BT20 and MCF12 cells were seeded in 96 well
plates at a density of 5x10³ cells per well. Cells were serum-starved for 24 h before
stimulation with 10% FCS, epidermal growth factor (EGF), rYB-1 protein and/or monoclonal
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anti-YB-1 antibody that lasted for 48 h. 5-bromo-2’-deoxyuridine (BrdU) was added for the
last 24 h and BrdU-incorporation determined using a cell proliferation assay (Roche).
A scratch wound assay was performed with normal human epidermal keratinocytes from
foreskin specimen. Cells were fed with keratinocyte growth medium (KGM bullet kit
CC3111, Cambrex), keratinocytes in the first two passages were used. NHEK were cultured
in chamber slides until they reached 90% of confluence and then a scratch wound was made.
In silico analyses
In silico analyses for potential protein modifications were performed with the SignalP 3.0
program for N-terminal signal peptide or signal peptidase II cleavage sites and SecretomeP
software (all CBS, Technical University of Denmark) for non-classical secretion.
Cell viability and LDH activity
A trypan blue exclusion assay was performed and LDH activity in supernatants was measured
to control for non-specific cell death.
MIF ELISA
An ELISA for MIF was performed as described previously (Flieger et al., 2003) using
conditioned supernatants.
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Supplementary results
Suppl. Fig. 1. Mesangial cells and monocytes secrete YB-1 upon LPS stimulation
Rat MC release YB-1 upon stimulation with PDGF-BB and LPS within 1 h (left panel).
Quantification demonstrates that the secreted amount increases three-fold over constitutive
release (right panel) (A.). Similarly MM6 cells as monocytic cell line secrete cold shock
protein YB-1 after LPS-challenge in a dose- and time-dependent manner within a narrow
LPS-range (1 to 7.5 ng/ml) (B., C.), which parallels MIF secretion (lower panels in B. and
C.). Time response curves indicate the augmented release of YB-1 following LPS stimulation
within 4 h in this cell system (D.).
Suppl. Fig. 2. Inflammatory stimuli induce the secretion of HA-YB-1 protein by MC
A. Schematic processing overview performed with conditioned medium after cell stimulation.
After pelleting of cells and debris (‘P1’) supernatants (‘S1’) were passed through a 0.2 µm
filter and vesicular structures were enriched by ultracentrifugation in pellet ‘P2’ with
corresponding supernatant (‘S2’).
B. - D. Immunoblotting for HA-tagged proteins in respective ‘S1’, ‘S2’ and ‘P2’ fractions of
processed cell medium after growth under control conditions and elevated glucose
concentrations (B.), challenge with TGF-β (C.) or H2O2 (D.). Full-length HA-YB-1 (<YB-1)
as well as N-terminal fragments thereof are indicated by #1, #2 and #3.
Suppl. Fig. 3: Colocalization of YB-1 and Rab7 in LPS-stimulated rMC
rMC were co-transfected with GFP-Rab7 and dsRed-YB-1 (or dsRed as control) and
stimulated with LPS for 2-3h. Colocalization was assessed by confocal laser scanning
microscopy. In unstimulated cells dsRed-YB-1 was equally distributed within the cytoplasm
(upper panel), whereas LPS-stimulation led to accumulation of YB-1 in vesicular structures
that partially colocalize with GFP-Rab7 (middle panel and inset). Localization of dsRed under
LPS-stimulation was unaffected.
Suppl. Fig. 4: Extracellular YB-1 exhibits pro-proliferative effects on different cells
A. and B. HK-2 cells and rMC were assayed for BrdU incorporation after addition of FCS
(10% v/v, positive control), rYB-1, monoclonal anti-YB-1 antibody (2 µg/ml) and the
combination of rYB-1 with antibody.
C. Primary human epidermal keratinocytes were grown to 90% confluence. A scratch
approximating 200 µm was introduced and closure of the scratch was monitored after 48 h.
Monoclonal anti-YB-1 antibody or isotype-matched non-specific control antibody were added
at the indicated increasing concentrations. The scratch introduced at time point 0 is depicted
graphically in the lower left part of the figure.
Supplementary videos
Videos were obtained by live cell imaging of rMC expressing YB-1-GFP (video 1) or GFP
alone (video 2). Cells were stimulated under temperature- and CO2-controlled conditions with
LPS (10 ng/ml). During the observation period of 2 h pictures were taken every 2 minutes.
LPS-stimulation resulted in the formation of YB-1-GFP enriched intracellular vesicles,
whereas the GFP distribution was unaltered.