A versatile system to study replication-independent nucleosome dynamics in mammals. (A) Schematic of TET-inducible expression system to study H3.3 turnover. CMV, cytomegalovirus; rtTA, reverse tetracycline-controlled transactivator; TRE, tetracycline responsive elements. (B) Western blot showing protein levels of transgenic HA/FLAG-H3.3 compared to endogenous H3.3. HA/FLAG-H3.3 expression 24 hours after DOX addition. The band marked with an asterisk is non-specific. The arrow marks transgenic HA/FLAG-H3.3. (C) Time course western blots of HA/FLAG-H3.3 expression. (D) Bromodeoxyuridine (BrdU) immunostaining of NIH/3 T3 cells treated with DNA polymerase inhibitor aphidicolin and DOX across time points of H3.3 induction. DMSO, dimethylsulfoxide. (E) Cell cycle analysis of cells treated with aphidicolin/DOX. Cells were stained with propidium iodide and analyzed by flow cytometry.