Figure 7.

NPAT activates histone H4 transcription independent of its cell cycle effects. (A) U2OS or SAOS2 cells were transfected as described in Fig. 6A. The transfected U2OS and SAOS2 cells were lysed at 22 h and 36 h, respectively, after transfection. The samples were analyzed as described in Fig. 6A. The results from three independent experiments are shown. (B) U2OS cells, grown on 10-cm plates, were transfected with 6 μg pCMV (vector) or 6 μg pCMV–NPAT together with 300 ng pGLH4(65) and 300 ng pCMV–lacZ. Five hours after transfection, Nocadazole (final concentration 70 ng/mL) was added into the culture medium to arrest the cells in M phase. Eighteen hours later, cells in M phase were harvested by mitotic shake-off. The cells were washed twice with culture medium and replated. Two and one-half hours after replating, the distribution of the cells in the cell cycle was analyzed by FACS and the effect of NPAT on H4 transcription was analyzed as described in Fig. 6A. The mean results from two independent experiments are shown. (C) U2OS cells were transfected with 50 ng pGLH4 (65) together with pCMV vector (1 μg), p27 (100 ng), NPAT (0.5 μg), or NPAT plus p27 as indicated. Thirty-six hours after transfection, the samples were analyzed as described in Fig. 6A. The data represent the mean from two independent experiments.