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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. 1 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 2 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. 3 4 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.