Figure 1.
RANTES-mediated induction of S100A4 release from 4MEF.
(A) Differential screening of VMR-CM and CSML0-CM by a cytokine antibody array. Upregulated cytokines are marked with white rectangles. (B) Western blot analysis of S100A4 released into CM in response to increasing concentrations of recombinant RANTES added to CSML0-CM (lane 2–8) and the inhibitory effect of rabbit anti-RANTES antibodies on RANTES-mediated S100A4 release (lane 6–7). Rabbit IgG was used as a negative control (lane 8). (C) Western blot analysis of S100A4 in CM of 4MEF in response to VMR-CM and anti-RANTES antibodies. (D) A representative experiment (qPCR) demonstrating lack of S100A4 transcriptional activation in 4MEF in response to various treatments.
Figure 2.
Mechanism of S100A4 externalization.
(A) Western blot analysis of S100A4 in 4MEF CM. Brefeldin A did not affect S100A4 secretion. (B) Double immunofluorescence of 4MEF with anti-S100A4 and anti-LAMP1 (lysosomal marker) antibodies. (C) Western blot of S100A4 in CM from stimulated 4MEF before and after microparticle depletion. (D) Sandwich ELISA of S100A4 in microparticles released from 4MEF in response to VMR-CM, CSML0-CM, 10 and 20 ng/ml RANTES in CSML0-CM. (Inset) Appearance of S100A4-positive microparticle-like structures in fibroblasts stimulated with VMR-CM. (E) Immunofluorescence analysis of macroparticle-containing fraction (100K pellet) from CM of cells treated with CSML0-CM and CSML0-CM+RANTES labeled with lipophilic dye FM®1–43FX (live, green) and anti-S100A4 antibodies (fixed, red).
Figure 3.
Cytoskeleton-associated transport of S100A4 in microparticles.
(A) Sandwich ELISA of S100A4 in microparticles released from CSML100 cells treated with CSML0-CM, 20 ng/ml RANTES in CSML0-CM, 20 ng/ml RANTES in CSML0-CM+Y27632, 20 ng/ml RANTES in CSML0-CM+Cytochalasin D, VMR-CM, and VMR-CM+Cytochalasin D. (B) Immunofluorescence live imaging of plasma membrane structure of adherent CSML100 cells labeled with the lipophilic dye FM®-1–43FX by different treatments as indicated. (C) Cells after the same treatments were visualized with double-immunofluorescence with anti-S100A4 antibodies (green) and rhodamine phalloidin (red). Scale bar = 50 µM.
Figure 4.
Functional significance of extracellular S100A4.
(A) Distribution of microparticles isolated from S100A4-positive 4MEF cells added to 5MEF and VMR cells. Immunofluorescence staining was performed with anti-S100A4 antibodies (green), rhodamine phalloidin (red), and nuclear staining with TO-Pro (TP3) (pink). (B) Immunofluorescence detection of FN (green) in 5MEF cells in response to S100A4+/+ and S100A4−/− microparticles from 4MEF and 5MEF cells, and 1µg/ml of the recombinant oligomeric S100A4 protein, respectively. (C) Detection of FN by Western blot analysis of cell lysates from 5MEF treated with S100A4−/− and S100A4+/+ microparticles and recombinant S100A4. As a control cell lysate from non-treated cells were used. FN band corresponding to molecular weight of approximately 250 kDa is indicated. (D) Effects of S100A4 microparticles on wound healing in 5MEF cells. Conditioned media from 4MEF and 5MEF cells before (a) and after (b) 100,000×g centrifugation and isolated microparticles (c) were added to scratched monolayers of 5MEF cells. Time-course kinetics of residual wounds are depicted in the graphs. (d) Wound healing assay with 4MEF cells. The residual size of scratches 12 h after “healing” is presented. Three different batches (#1, 2 and 3) of affinity purified polyclonal anti-S100A4 antibodies were used.
Figure 5.
S100A4-mediated activation of cytokines in VMR cells.
(A) Cytokine antibody array analysis of CM from VMR cells, unstimulated and stimulated with 0.5 µg/ml recombinant oligomeric S100A4. Upregulated cytokines are marked with white rectangles. (B) Kinetics of RANTES transactivation in response to 0.5 µg/ml recombinant oligomeric S100A4 (qPCR). (C) Western blot analysis of S100A4-mediated upregulation of RANTES in VMR-CM.
Table 1.
Survival rate and metastases among animal groups of A/Sn wild type mice injected with VMR-vector and -RANTES cell lines.
Figure 6.
S100A4-associated RANTES-driven metastatatic capacity of tumor cells.
(A) Metastatic burden indicated as a number of lesions per area unit in lungs of wild type (S100A4+/+) and S100A4−/− A/Sn mice inoculated i/v with VMR-mock and -RANTES cells. n = 6 per group. (B) Metastatic burden in liver in wild type (S100A4+/+) and S100A4−/− A/Sn mice inoculated i/v with VMR-mock and -RANTES cells. n = 6 per group. (C) Lung and liver metastatic burden in wild type A/Sn mice inoculated i/v with CSML0-mock and -RANTES cells. n = 5 per group, p = 0.1060 (lung metastases) and p = 0.7241(liver metastases). (D) S100A4 concentration in the serum of A/Sn mice inoculated with VMR-mock and -RANTES cells determined by sandwich ELISA.