Stable clones of FE25 cells transfected with shRNA of CNN1 (shCNN1) or negative control (shNC) were analyzed for protein expression (A), cell proliferation (B), Transwell migration (C), cell anoikis (D), anchorage-independent growth of colony with diameter > 20 μm (E) and xenograft tumorigenesis in NSG mice (F) as stated in Materials and Methods. In the anoikis assay, two independent stable clones of shCNN1 (shCNN1#3 and shCNN1#10) were analyzed. In AIG assay, colonies with diameter larger than 20 μm were counted. Xenograft tumors were observed 5.5 month after s.c. or i.p. injection of CNN1 knock-downed FE25 cells (FE25 shCNN1) (n = 5, 2 with s.c, 3 with i.p.) or CNN1-normal FE25 cells (FE25 CTL) (n = 10, 5 transfected with shNC, 5 without). Three of the shNC-transfected FE25 cells were i.p. injected. The others were s.c. injected. *p < 0.05, **p < 0.01, ***p < 0.001.