Figure 5.

TNFAIP8 v2 depletion stalls DNA replication and induces p53-dependent cell cycle arrest. (a) TNFAIP8 v2 mRNA levels were quantified in p53+ and p53-silenced (p53i) A549 cells untransduced (‘−') or transduced with scramble (‘scri') or TNFAIP8-directed (‘TP8i') shRNA. At right, an immunoblot of v2 under the same conditions is shown. (b) 5-Bromo-2'-deoxyuridine (BrdU) incorporation and 7AAD staining were quantified using flow cytometry in p53+ and p53i A549 cells transduced with scri or TP8i shRNA. BrdU+ cells (S phase), yellow; G1, green; G2, purple. (c) Quantification of BrdU intensity of cells in (b). (d) Cell cycle profile based on 7AAD staining of cells in (a). G2-phase cells (purple) are defined as cells with double the nuclear 7AAD stain intensity compared with G1-phase cells (green). S-phase cells are BrdU+ (yellow). (e and f) mRNA levels of PCNA (RT-PCR) (e) and cyclins D1, E1, and E2 (Nanostring) (f) of cells under indicated treatments. (g) p21 quantified by RT-PCR and immunoblot in p53+ A549 cells transduced with either scri or TP8i shRNA, followed by transfection of scramble (‘scri') or p21 (‘p21i') siRNA. (h) Cell cycle analysis of cells described in (g). Results are representative of three or more independent experiments. *P<0.05