Figure 6
From: Ubiquitin-specific protease 22 is a deubiquitinase of CCNB1
Knockdown of USP22 by shRNA inhibits colorectal tumorigenesis. (a) 2×105 cells were seeded in six-well plates. The growth of cells was examined by counting cell numbers every 24 h after plating. Error bars represent data from three independent experiments; P<0.05. (b) The growth of stably expressing HCT116 cells indicated plasmids was examined by WST-1 assay as described in materials and methods. (c) Wild-type or usp22-null MEF cells were subjected to WST-1 assay as in b. (d) Stably expressing HCT116 cells indicated plasmids were seeded in soft agar and cultivated for 3–4 weeks. Colony formation was assayed by light microscopy and representative images are shown. (e) The number of colonies in each plate was counted. Error bars represent data from three independent experiments with a total of three plates per group. P<0.05. (f–h) 1×106 HCT116 or stable USP22 knockdown (KD) cells were injected subcutaneously into nude mice (n=8 per group). Twenty-two days after injection, two pairs of representative mice were photographed (f, top panel). Tumors were isolated and photographed. 7–8 represents no tumors (f, bottom panel). The tumor sizes were measured every other day, and their growth curves are shown (g). Mice were euthanized at day 22, and the tumors were excised and weighed at the end of the experiment (h). P<0.05. (i) Our working model of CCNB1 regulation by USP22. CCNB1, cyclin B1; shRNA, short hairpin RNA; siRNA, small interfering RNA; USP2, ubiquitin-specific protease 22.
