Fig. 6

Increased expression of Anxa2^WT or Anxa2^Y23D in Rack1-silenced cells recovered cell invasion ability. a, b Increased expression of Anxa2^WT, Anxa2^Y23A, and Anxa2^Y23D in Rack1 stably silenced MCF-7/ADR cells (a) and MDA-MB-468/EPR cells (b). Lentivirus expressing Anxa2^WT, as well as its two mutants, were used to infect Rack1 stably silenced cells, in which the expression of Rack1 has been stably transfected with an shRNA targeting its noncoding region. Then, the cells were lysed and analyzed by Western blotting using anti-Anxa2, anti-GFP, and anti-Rack1 antibodies. β-actin was used as a loading control. c, d Transwell assays showed that overexpression of Anxa2^WT or Anxa2^Y23D, not Anxa2^Y23A, in Rack1-silenced MCF-7/ADR (c) and MDA-MB-468/EPR cells (d) partially rescued the cell migration and invasion abilities. Data are shown as mean ± SD; n = 6. Statistical analysis was performed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ^nsP > 0.05 indicate no statistical significance. e Src inhibitor KX2-391 efficiently inhibited the expression of pY23-Anxa2 in Anxa2^WT-expressing cells, while had no significant effect on the level of pY23-Anxa2 in Anxa2^Y23D-expressing cells as detected by Western blotting. f Transwell assay showed that the migration ability in Anxa2^WT-expressing cells can be quenched by Src inhibitor, while Src inhibition has little effect on the migration ability in Anxa2^Y23D-expressing cells. Data are shown as mean ± SD; n = 6. Statistical analysis was performed by one-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001, and ^nsP > 0.05 indicate no statistical significance