CD44 expression is essential for Rb knockdown-activated CCM in vitro.

<p>(A) MCF7ras cells were co-infected with a lentiviral construct expressing puromycin resistance gene and Rb or control shRNA. After selection, cells were infected with shRNA against CD44 or a scrambled sequence in an EGFP-expressing vector. Lysates from all double knockdown cells were examined by western blot with anti-CD44 and anti-Rb antibodies. β-tubulin was used as a loading control. (B) Growth of MCF7ras cells with double knockdown of Rb and CD44 was analyzed by MTT assay, <i>n</i> = 3 experiments, data are presented as mean ± SD. Controls included single Rb knockdowns, and cells with shRNA against scrambled sequence with or without further infection with CD44 shRNA. (C) CCM of double knockdown cells. The analysis was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080590#pone-0080590-g001" target="_blank">Figure 1B</a>. Data are presented as mean ± SD. The experiment was performed three times in triplicate. Scale bar, 100 μm; unequal variance Student's t-test, ** p<0.01, *** p<0.001. (D) Immunofluorescent analysis of F-actin, CD44 and E-cadherin expression in cells with single and double Rb/CD44 knockdowns. Scale bar, 40 μm.