Figure 4.

Overexpression of Roma Results in S Phase Arrest

(A) EpH4 cells were transfected with a Roma-FLAG expression construct and cultured under puromycin selection. Doxycycline was used to induce Roma-FLAG expression. Cell counts were monitored over the course of a week and represented in a graph.

(B) Representative images of cells under the indicated conditions.

(C) EpH4 cells transfected with a Roma-FLAG expression construct were treated with doxycycline at a series of time points and then released from doxycycline and pulsed with BrdU followed by flow cytometry analysis.

(D) Protein extracts were prepared from EpH4 cells at conditions stated and immunoblot analysis of cell-cycle checkpoint, and DNA damage response markers was conducted.

(E) RNA was extracted from EpH4 cells cultured in conditions stated followed by PCR analysis of p21Cip1.

(F) EpH4 cells cultured in conditions stated were treated with p21Cip1 siRNA and pulsed with BrdU followed by cell-cycle analysis using flow cytometry.

See also Figure S4.