1. Lats2 functions at the G1–S checkpoint in U2OS cells.
Lats2 functions at the G1–S checkpoint in U2OS cells. (A) Confirmation of the successful knockdown of Lats2 in U2OS/siLats2 cells. (i) Western blot analyses were conducted using the indicated samples and antibodies. (ii) Bar graphs represent the intensity ratio of each band relative to the amount of GAPDH, a loading control. (B) Myc–Lats2R was not knocked down by siRNA targeting Lats2. U2OS cells were co-transfected with indicated DNA and siRNA in the indicated combinations. siGL2, siRNA targeting GL2. (C) Myc–Lats2R proteins are stably expressed in U2OS/siLats2 cells. (D) Experimental design for E. Cells were treated with nocodazol (80 ng/ml) for 16 hours. Then, the mitotic cells were collected by shake-off, transferred to new dishes, and incubated for 8 hours. Following this incubation, cells were irradiated with UV (50 J/m2), and collected after the indicated periods of time. (E) FACS analysis of indicated cells monitoring cell cycle progression. 2N and 4N refer to diploid and tetraploid, respectively. Arrow or arrowheads indicates the G2–M peak before or after UV irradiation, respectively. (F) Frequency of sub-G1 cells in U2OS/siLats2/Myc–Lats2R(WT), U2OS/siLats2/Myc–Lats2R(S408A) and U2OS/siLats2/Myc–Lats2R(S408D) cells at 24 hours and 48 hours after UV irradiation. Means and standard deviations were obtained from three independent experiments.
Nobuhiro Okada et al. J Cell Sci 2011;124:57-67
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