Fig 3. Decreased ALDH1B1 expression inhibits spheroid formation and tumor growth.

SW480 cells were transfected with ALDH1B1 shRNA or scramble shRNA to develop stable cell colonies. (A) ALDH1B1 expression was examined by Western blotting and quantified by densitometric analysis. Numbers at the bottom of ALDH1B1 Western blot bands are the average densitometry of three replicates expressed relative to scramble shRNA-transfected (control) cells. Rec, recombinant ALDH1B1; CT, Control SW480 cells without transfection. (B) Spheroid formation was measured 10 d after plating of cells. Representative phase contrast micrographs (left) and histograms (right) showing the number and volume of spheroids developing from SW480 cells transfected with ALDH1B1 shRNA (open bars) or with scramble shRNA (control) (closed bars) vectors. Data are presented as mean ± SEM (n = 3). # P < 0.01, Student’s unpaired t-test, compared with scramble shRNA. (C) Growth of tumors induced by injection of SW480 cells transfected with ALDH1B1 shRNA (circles) or scramble shRNA (squares)) into the left and right flanks of female athymic (nu+/nu+) mice. Tumor volume was calculated as (length × width² × 0.52). Data are presented as mean ± SEM (n = 10 tumors per group). * P < 0.05, Student’s unpaired t-test, compared with tumors in scramble shRNA group at same time point.