Fig. 1: Enhanced adenoviral replication by silencing Daxx in mice.

a Cellular Daxx levels in human cancer cell lines (left), mouse cancer cell lines and two human cancer cell lines used as controls (right) were examined by immunoblotting. b Adenoviral infectivity of various mouse and human cancer cell lines was examined using replication-incompetent GFP-expressing adenovirus after 48 h of infection (multiplicity of infection [MOI] of 20; ×100 magnification). c Adenoviral replication was enhanced by Daxx downregulation in mouse or p53-mutant human cancer cell lines using Daxx-specific shRNA-expressing oncolytic adenovirus at an MOI of 100 for 48 h. Panc02 and B16F10-CAR cells were infected at an MOI of 200 for maximal infectivity. The same number of all cancer cell lines (1 × 10⁵ cells) were plated onto 6-well plates and infected with Ad-3484-NC or Ad-3484-shDaxx at an MOI of 100 or 200 for maximal infectivity. After 48 h of infection, each viral soup (3 ml) was titrated after repetitive freeze-thaw cycles. Ad-3484-shhDaxx (shhDaxx, shRNA targeting human Daxx), Ad-3484-shmDaxx (shmDaxx, shRNA targeting mouse Daxx), and Ad-3484-NC (NC, negative control, the scrambled DNA sequence of the shRNA). Ad-3484-NC adenovirus is a replication-competent adenovirus used as a negative control, and 3484 indicates the presence of E1A in the viral genome controlled by the cytomegalovirus (CMV) promoter lacking the E1B region. Error bars represent standard errors from three independent experiments. d BNL mouse cell lines were infected with LNCX-CAR, and a clone expressing CAR was selected for efficient adenoviral replication in mouse cell lines (left). Enhanced adenoviral infection was observed in clone #1 of BNL-CAR after infection with replication-incompetent GFP-expressing adenovirus at an MOI of 20 for 48 h (right, 100 × magnification). e Cellular Daxx levels were examined in B16BL6 cells, CAR-expressing B16BL6 cells (B16BL6-CAR) and B16BL6 cells expressing both CAR and E1B55K (B16BL6-CAR-E1B55K) by immunoblotting. f Cellular Daxx levels and viral production were examined after infection with oncolytic adenovirus expressing shRNA targeting mouse Daxx without E1B55K and/or RCA expressing E1B55K at an MOI of 50 or 100 for 48 h. g To compare the oncolytic activity induced by Ad-3484-NC with that induced by Ad-3484-shhDaxx or Ad-3484-shmDaxx, human and mouse cancer cell lines were infected with each virus at an MOI between 0.1 and 50. Immediately after 293 A cells infected with one of the viruses at an MOI of 0.1 exhibited complete cell lysis, all remaining cells on the plate were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet (upper). Cell viability after infection with each virus was compared by cell death curves in a quantitative graph (lower). Error bars in a, c, e, and g represent standard errors from three independent experiments