Fig 1.
Characterization of Snail-B16F1 stable clones.
Quantitative RT-PCR analysis of human snail (SNAIL) mRNA levels normalized to EF1a. Mean±SD, n = 4, ***p< 0.001. (B) Western blot analysis of Snail in B16F1-WT, in mock-B16F1 and in Snail overexpressing B16F1 cells (clones Snail19-B16F1—high expression of Snail and Snail21-B16F1—low expression of Snail). (C) Morphology (representative pictures) of B16F1-WT, mock-B16F1, and Snail-overexpressing B16F1 cells as observed by phase contrast microscopy (scale bar, 10 μm).
Fig 2.
Characterization of Snail-HT29 stable clones.
Quantitative RT-PCR analysis of human SNAIL mRNA levels normalized to the expression of SNAIL in HT29-WT. Mean±SEM, n = 8, *p< 0.02; **p< 0.01 (B) Western blot analysis of Snail in mock-HT29 and in Snail overexpressing HT29 cells (clones Snail8-HT29-high expression of Snail and Snail3-HT29 –low expression of Snail). (C) Morphology of HT29-WT, mock-HT29 and Snail-overexpressing HT29 cells as observed by phase contrast microscopy (scale bar, 10 μm).
Fig 3.
Effect of Snail overexpression and of lumican on MMP-2,-9 and MMP-14 expression and activity in B16F1 and HT29 cells.
(A) MMP-14 activity in mock and Snail overexpressing B16F1 and HT29 cells (clones Snail19-B16F1 and Snail8-HT29). Mean±SEM, n = 3 experiments performed in duplicate. (B) MMP-2 and -9 expression in mock and Snail overexpressing B16F1 and HT29 cells (clones Snail19-B16F1 and Snail8-HT29). Cell-conditioned concentrated media were analyzed on SDS-polyacrylamide gels containing 1 mg/ml gelatin.
Fig 4.
Effect of lumican on MMP-2,-9 and MMP-14 expression and activity in Snail-B16F1 cells.
(A) Effect of lumican on MMP-2 and -9 activities in Snail overexpressing B16F1 cells (clone Snail19-B16F1). (B) Activity of MMP-14 in Snail-B16F1 cells (clone Snail19-B16F1) incubated 24h in the absence or in the presence of 100 nM lumican as described in Materials and Methods using fluorometric SensoLyte™ 520 MMP-14 Assay Kit. Data given as mean±SD, n = 6. Asterisk (*) indicates statistically significant differences between control and 100 nM lumican incubated Snail overexpressing B16F1 cells, (*** p < 0.001). Significance of differences estimated with block two-way ANOVA, using the cell line and the presence of lumican as grouping variables, and the post hoc Scheffe’s multiple comparisons test and contrast analysis. (C) Western blot analysis of MMP-14 expression in Snail overexpressing B16F1 cells (clone Snail19-B16F1) in the absence or in the presence of 100 nM lumican.
Fig 5.
Lumican inhibits Snail overexpressing B16F1 cell functional properties.
(A) Cell migration of Snail overexpressing B16F1 cells (clone Snail19-B16F1) in presence or absence of lumican (100 nM) after 24h and 48h; n = 2, ***p < 0.001 (B) Colony formation assay of mock and Snail overexpressing B16F1 cells (clone Snail19-B16F1) in absence or presence of lumican (100 nM); Representative images of cell colonies are displayed at the inserts 14 days after seeding. The quantification of the colony diameter was performed using Image Tool software. Graphs represent the mean size of 100 colonies ± SD from two independent experiments. (**p < 0.01, ***p< 0.001). Scale bar inserts: 200 μm.
Fig 6.
Lumican inhibits melanoma primary tumor growth of Snail overexpressing B16F1 cells in vivo.
Lum+/+ and Lum-/- mice on the C57Bl/6 background were injected subcutaneously with 2.5 x 105 of B16F1 cells, mock or Snail overexpressing B16F1 cells (clone Snail19-B16F1). The kinetic of melanoma primary tumor growth is displayed in volume (mm3) till day 15 when mice were sacrificed. * p < 0.05: significantly different from mock-transfected B16F1 cell tumors, Open circle: wild type C57Bl/6 mice injected with mock-B16F1 cells (n = 10); Diamond: lumican deleted C57Bl/6 mice injected with mock B16F1 cells (n = 10); Open square: wild type C57Bl/6 mice injected with Snail overexpressing B16F1 cells (n = 10); Triangle: Lumican deleted C57Bl/6 mice injected with Snail overexpressing B16F1 cells (n = 10).