Journal: Nucleic Acids Research
Article Title: Cellular and viral chromatin proteins are positive factors in the regulation of adenovirus gene expression
Figure Lengend Snippet: Ad-core/TP-DNA transfection assays. ( A ) ChIP assays of viral chromatin in transfected cells. HeLa cells were transfected with TP-DNA (left three panels) or Ad-core (right three panels). At 8, 16 and 24 hpt, cells were collected, and cell lysates were subjected to ChIP assays using indicated antibodies. Semi-quantitative PCR was performed using primer sets for E1A pro, E4 pro or MLP and immunoprecipitated DNA as templates. PCR products were separated on a 7% polyacrylamide gel and visualized by staining with EtBr. Input DNAs (lanes 13–15 and 28–30) were purified from 2% of cell lysates used for ChIP. ( B ) Semi-quantitative RT–PCR assays. HeLa cells were transfected with Ad-core (Ad; lanes 2, 5, 8 and 11), TP-DNA (TP; lanes 1, 4, 7 and 10), or mock-DNA (Mo; lanes 3, 6, 9 and 12), and total RNAs were purified at 12 (lanes 1–3 and 7–9) and 24 hpt (lanes 4–6 and 10–12). cDNAs were synthesized without (lanes 1–6) or with (lanes 7–12) reverse transcription. Semi-quantitative PCR was performed using primer sets for E1A, E4, MLP and β-actin mRNAs. PCR products were analyzed as described in the legend for (A). ( C ) The amount of nuclear and cytoplasmic viral DNA in transfected cells. Ad-core and TP-DNA were used for transfection (100 ng DNA) into HeLa cells. Transfected cells were cultured in the presence of HU to prevent viral DNA replication. At 12 (lower panel, lanes 1–12) and 24 hpt (lanes 13–30), nuclear and cytoplasmic fractions were prepared from Ad-core- (lower panel, lanes 1–6 and 13–18), TP-DNA- (lanes 7–12 and 19–24), and mock-transfected cells (lanes 25–30) as described in ‘Materials and Methods’ section. To confirm the separation of each fraction, nuclear (upper panels, lanes 1, 3, 5, 7 and 9) and cytoplasmic fractions (lanes 2, 4, 6, 8 and 10) were subjected to western blot analyses using antihistone H3 and anti-Hsp90 antibodies for nuclear and cytoplasmic marker proteins, respectively. The viral DNA of nuclear (lower panel, lanes 1–3, 7–9, 13–15, 19–21 and 25–27) and cytoplasmic fractions (lanes 4–6, 10–12, 16–18, 22–24 and 28–30) was amplified by semi-quantitative PCR with a primer set for the MLP region. PCR products were analyzed as described in the legend for (A). For each fraction, 4-fold serial dilution (1, 1/4 and 1/16 volume) was used as templates for PCR.
Article Snippet: HeLa cells were transfected with Ad-core, TP-DNA, or the reconstituted complexes (100 ng DNA) using GeneJuice (Novagen) according to the manufacturer’s protocol.
Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction, Staining, Purification, Quantitative RT-PCR, Synthesized, Cell Culture, Western Blot, Marker, Amplification, Serial Dilution