BALB/C nude mouse subcutaneous transplantation tumor experiment and immunohistochemistry of the transplanted tumors.

<p>(<b>a</b>) All the mice from the three groups developed tumors. <i>OVOS2</i> downregulation significantly decreased tumor growth and one tumor spontaneously regressed in the A375/<i>OVOS2</i>-shRNA group. (<b>b</b>) The tumor growth was significantly inhibited in the A375/<i>OVOS2</i>-shRNA group during the observation period (*<i>P</i> < 0.05, <i>P</i> = 0.011 and <i>P</i> = 0.03, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; repeated measure of ANOVA); the weight of tumors in the A375/<i>OVOS2</i>-shRNA group was also significantly reduced (*<i>P</i> < 0.05, <i>P</i> = 0.019 and <i>P</i> = 0.012, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). The results are shown as means ± SD. (<b>c</b>) H&E staining of the three groups (100×). The histopathologic characteristics of the xenograft tumors among the three groups were similar. (<b>d</b>) Number of abnormal mitoses of transplanted tumors in every high power field. The number of abnormal mitoses was much less in the A375/<i>OVOS2</i>–shRNA tumors compared with those in the A375 cell and A375/NC–shRNA tumors (*<i>P</i> < 0.05, <i>P</i> = 0.002 and <i>P</i> = 0.023, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). (<b>e</b>) The downregulation of <i>OVOS2</i> in A375 cells dramatically decreased the percentage of Ki-67 positive cells as shown by immunohistochemistry study (**<i>P</i> < 0.01, ANOVA, LSD multiple comparison). (<b>f</b>) Immunohistochemistry staining of OVOS2 expression in transplanted tumors. OVOS2 was expressed in tumors induced by A375 and A375/NC–shRNA cells, but it was absent in one of the A375/<i>OVOS2</i>–shRNA cell-induced tumors, and the expression level in the group of A375/<i>OVOS2</i>–shRNA was significantly decreased (*<i>P</i> < 0.05, <i>P</i> = 0.009 and <i>P</i> = 0.033, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; Kruskal–Wallis test). (<b>g</b>) The expression of E-cadherin and β-catenin in the xenograft tumors showed that both E-cadherin and β-catenin were significantly upregulated in the A375/<i>OVOS2</i>-shRNA tumor (*<i>P</i> < 0.05, <i>P</i> = 0.015 and <i>P</i> = 0.02 for E-cadherin, <i>P</i> = 0.033 and <i>P</i> = 0.04 for β-catenin, compared with A375/NC-shRNA and untreated A375 cell groups, respectively; ANOVA, LSD multiple comparison). (<b>h</b>) Immunohistochemistry staining for apoptosis indexes of Bax and Bcl-2in transplanted tumors. The ratio of Bax to Bcl-2 was higher in the A375/<i>OVOS2</i>–shRNA group than that in the A375 and A375/NC–shRNA groups; but not reached statistical significant difference (<i>P</i>>0.05, Kruskal–Wallis test).