Figure 2. Evaluation of the Delta-24-ACT oncolytic effects in vitro.

(A) Assessment of viral protein expression (fiber and E1A) in NP53, XFM, SU-DIPG IV, and TP54 cells by Western blotting. Cells were infected with Delta-24-ACT at the indicated MOIs, and whole-cell lysates were collected 48 hours later. GRB2 was used as a protein-loading control. (B) Oncolytic effects of Delta-24-ACT on murine and human DIPG cells. To quantify the oncolytic effects of Delta-24-RGD or Delta-24-ACT on cells, they were infected at the indicated MOIs, and viability was evaluated 5 days later by MTS assays. Values indicate the percentages of viable cells in infected cultures compared with noninfected cultures (mean ± SD, n = 3 each group). (C) Evaluation of the E1A viral protein in vivo. Viral protein expression was evaluated in vivo in mice bearing either XFM or NP53 cells 3 days after mock or Delta-24-ACT treatment. Representative micrographs are shown. Scale bar: 20 μm. (D and E) Concentrations of the damage-associated molecular pattern (DAMP) markers ATP and HMGB1 in supernatants obtained from NP53 and XFM cultures at 72 hours after Delta-ACT (n = 4) or mock (n = 4) infection. Data are shown as the mean ± SEM (Mann-Whitney test), and P values are shown above bars. (F) Representative fluorescence microscopy images of NP53 cells at 4 hours after infection with Delta-24-ACT or mock infection. Calreticulin (CRT) at the cell surface was detected by immunofluorescence (green) and nuclei (blue; DAPI). Arrows denote the calreticulin location in the cell. Original magnification, ×40. Flow cytometric quantification of membrane calreticulin⁺ cells after Delta-24-ACT infection. Data are shown as the mean ± SEM (n = 4 each group; Mann-Whitney test), and P values are shown above bars.