Expression of cellular S-phase specific genes in infected IMR-90 cells.

<p>(A) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>pm</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. At the indicated time-points, total RNA was extracted using the TRIzol reagent and mRNA levels for the indicated genes were determined by qRT-PCR. GAPDH was used as a loading reference and mock-infected cells were used as a control and were set to 1. Error bars represent SD of 4 biological replicates. (B) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>pm</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. One hour prior to the indicated time point cells were pulsed with EdU for 1 hour, fixed at the indicated time point, and stained for EdU and E1A as described in the Materials and Methods. Data represents percentage of infected cells that were positive for EdU staining. Error bars represent SD of 5 biological replicates.</p

Virus growth and effects on cell morphology in arrested lung fibroblasts IMR-90.

<p>(A) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>pm</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. Virus titres were determined on 293 cells at the indicated time points. Inset shows E1A levels at 24 hours after infection. Error bars represent standard deviation (SD) of 4 replicate experiments. (B) Representative images of infected cells from A and mock-infected cells that were treated the same as infected cells, minus addition of virus. Images were taken prior to harvest of cells for titre determination and were taken at 100X magnification using phase-contrast optics.</p

Viral protein levels after infection of arrested IMR-90 cells.

<p>(A) IMR-90 cells were arrested by contact inhibition for three days. After which cells were infected with HAdV5 <i>dl</i>309, <i>dl</i>520, or <i>dl</i>975 for 1 hour in serum-free media. Media that was removed from the cells was saved and replaced after 1 hour. At the indicated time-points, cells were lysed and 20μg total cellular lysate was resolved by SDS-PAGE on Novex BOLT 4–12% gradient minigel. E1A was detected with a combination of M58 and M73 monoclonal antibodies and visualized using a secondary HRP-conjugated anti-mouse antibody (Jackson Immunoresearch). (B) Same as A except probed for the 72kDa E2 DNA-binding protein. Secondary HRP-conjugated anti-mouse antibody (Jackson Immunoresearch) was used for detection. (C) Same as A except probed with antibody recognizing viral structural proteins (Abcam). Secondary anti-rabbit HRP-conjugated antibody was used for detection (Jackson Immunoresearch). (D) Same as A except probed for cellular actin (Abcam) as a loading control. Secondary anti-rabbit HRP-conjugated antibody was used for detection (Jackson Immunoresearch).</p