Figure 2

Effects of RA-XII on 4T1 cell motility and migration.

(A) Cells were treated with or without RA-XII at 50 and 100 nM for 24 hours in scratch wound assay. Representative photographs showed the effects of RA-XII at 50 and 100 nM on cancer cell motility and quantified analysis summarized the percentage of the change in open wound area at 24-hour compared with those at 0 h (mean + SD of 3 independent experiments with 6 replicates each). (B) Cells were treated with or without RA-XII at 50 or 100 nM for 4 hours in transwell migration assay. Representative photographs showed the stained migrated cancer cells on the lower side of the membrane after incubation. Quantified analysis summarized the number of migrated cells on the lower chambers (mean + SD of 3 independent experiments with duplicates each) and expressed as the percentage of the control. (C) Representative western blots showed the effect of RA-XII at 100 nM on cofilin and p-cofilin expressions at various time-points. (D) Representative western blots revealed the effect of RA-XII at different doses after 24-hour treatment. The cropped blots have been obtained under the same experimental conditions and the full-length blots were shown in Supplementary information. The histograms in (C,D) showed the expressions of the target proteins which were normalized with corresponding β-actin protein expression and expressed as fold of control (mean + SD of 3-4 independent experiments). (E) Representative flow cytometric histograms showed the inhibitory effect of RA-XII (50 or 100 nM treated for 24 hours) on the expressions of chemokine receptor, CCR7 on 4T1 cell surface. Cells were stained with PE-conjugated CCR7 antibodies. The histograms were expressed as fold of untreated controls (mean + SD of 3-4 independent experiments). Statistical differences were determined by One-way ANOVA, followed by Dunnett Test, with *p < 0.05, **p < 0.01, ***p < 0.001 against untreated controls.