p8 negatively regulates the cell cycle: precocious S-phase entry in
p8-knockdown myoblasts. (A) Endogenous p8 mRNA levels analyzed by
northern blotting in asynchronous cultures of pools generated by the
expression of four independent shRNAs. p8sh2 and p8sh4 pools showed
significantly reduced p8 RNA levels compared with control cells
transfected with empty vector `C', whereas the p8sh1 pool showed no reduction
and subsequently served as a control shRNA pool. (B) Quantification of
p8 mRNA levels during a timecourse of reactivation in two independent
knockdown pools relative to control cells expressing GFP shRNA:
quantitative real-time RT-PCR reveals suppression of p8 mRNA
throughout the timecourse (control cell mRNA levels at each time point=1). (C)
p8 protein levels are suppressed in p8-knockdown cells (p8-sh) vs GFP-sh
control cells (con-sh) in mid-G1 (6 hours after reactivation). (D) Flow
cytometric cell-cycle analysis of p8-knockdown cells (p8sh2 pool, p8sh4 pool,
p8sh4 clone 12) and control cells containing empty vector. DNA content
associated with each phase of the cell cycle of asynchronously growing
myoblasts (Mb) and G0-synchronized populations reactivated for 6, 12 or 24
hours (R6, R16, R24) is shown. Note that the majority of control cells are
still in G1 at R16, whereas the knockdown cells have already progressed into S
phase (arrowheads), with a reduction in the G1 peak. (E) Quantification of the
proportion of cells in S phase from data shown in D. Distribution of cells
during asynchronous growth and arrest is not affected in the p8-knockdown
lines (Mb and R6 points). However, all shRNA pools enter S phase precociously
at 16 hours of reactivation, ahead of control cells at 24 hours. (F)
Precocious histone expression in p8-knockdown cells confirms FACS analysis.
Northern blot shows asynchronously growing myoblasts (`G'), arrested myoblasts
(`A') and synchronized cells reactivated into the cell cycle for 6 hours
(`R6'). Induction of p8 mRNA at R6 was specifically attenuated in
p8sh4 knockdown cells. Histone mRNA reports for S phase and shows accelerated
expression in the p8sh4 pool. Induction of expression of MyoD mRNA
during G1 re-entry is also blunted in the p8-knockdown cells. (G)
Quantification of normalized levels of histone and p8 mRNA in
knockdown pool p8sh4 compared with p8sh1 control cells. Note the reciprocal
relationship between control and knockdown cells with respect to p8
and histone mRNAs specifically at R6.