Figure 3. Chemerin stimulates migration of squamous carcinoma cell lines.

Graphs demonstrating the bell-shape curves of chemotactic response to the increased concentrations of Chemerin in (A) SCL-1, (B) SCC-12B2, (C) SCC-13 and (D) A431 cells using the Transwell^® chamber migration assay. Date are shown as mean ± S.D for one of three independent experiments with n = 4 replicate wells; n.s. = non-significant, *p < 0.05, **p < 0.01 and ***p < 0.001 in comparison to random migration control with no Chemerin treatment calculated by Bonferroni post hoc test after ANOVA. The role of Chemerin in mediating cSCC cell migration was confirmed by silencing the Chemerin gene (RARRES2) in senescent fibroblasts and assessing their ability to induce SCL-1 cell migration (E) Transcript level of RARRES2 was quantified by qRT-PCR in senescent fibroblasts 42 h post treatment with siRNAs, confirming the successful gene silencing compared to scrambled control. Data are shown as mean ± S.E.M from three independent experiments. ***p < 0.001 calculated by Bonferroni post hoc test after ANOVA. (F) Chemerin protein level (normalized to 5 × 10⁶ cells/ml) was measured 48 hours after treatment with siRNAs or scrambled control. Data are shown as mean ± S.E.M from four independent experiments; *p < 0.05 calculated by Bonferroni post hoc test after ANOVA. (G) Conditioned media derived from Chemerin-silenced fibroblasts (siRNA 1, 2, 5 and 6) were used to induce the migration of SCL-1 cells using the Transwell^® chamber migration assay. Conditioned media derived from mock-treated fibroblasts (Scrambled) and senescent fibroblasts with no treatment (control) were used as controls. Shown is one representative of four independent experiments, each with triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001 calculated by Bonferroni post hoc test after ANOVA.