Fig. 3: Ovarian cancer cells show enhanced migration toward CCL6/CCL23 by activating Myosin9 via the Cofilin signaling in vitro.

a, b Transwell migration assay of ID8/SKOV3ip.1 cells toward varying concentrations of CCL6 and CCL23 with representative crystal violet staining. Statistical significance (*p < 0.05) was determined for all conditions versus the serum-free media control by ordinary one-way ANOVA analysis. c Quantitation of cell proliferation of ID8/SKOV3ip.1 cells in the presence of CCL6/CCL23 (100 ng/mL–200 ng/mL) by MTS assay. 10% FBS was used as the positive control. d, e Transwell migration assays with ID8 cells in the presence of anti-CCL6 (2.5 μg/mL and 5 μg/mL; n = 3) toward mouse omental conditioned media and SKOV3ip.1 cells toward human omental conditioned media in the presence of anti-CCL23 (2.5 μg/mL–5 μg/mL; n = 3). Statistical significance was determined by two-way ANOVA analysis for each condition corresponding to the serum-free (SF) control (*p < 0.05). f Western blot analysis of ID8 treated with CCL6 (200 ng/mL) and SKOV3ip.1 treated with CCL23 (200 ng/mL) (n = 3). g, h Immunofluorescence staining for phospho-cofilin (red), actin cytoskeleton (green), and nuclei (blue, DAPI) on ID8 and SKOV3ip.1 ovarian cancer cells treated with CCL6 or CCL23, respectively, at different time points (n = 3); (scale bar = 20 µm). i Schematic representation of the CCR1-CCL6/CCL23 signaling pathway leading to increased migratory potential of the ovarian cancer cells. j Transwell migration assay of SKOV3ip.1 toward CCL23 treated with BEZ235 (PI3K Inhibitor) (**p < 0.008). k, l Transwell migration assay of ID8 toward CCL6 and SKOV3ip.1 cells toward CCL23 treated with trametinib (MEK inhibitor). m, n Transwell migration assay of ID8 toward CCL6 and SKOV3ip.1 cells toward CCL23 treated with blebbistatin (myosin inhibitor). Statistical significance was determined by two-way ANOVA analysis with Tukey’s multiple comparison t-test with each condition compared to the 200 ng/mL CCL23 treatment condition.