FIGURE 8.

Induction of mature LAT-miRNA by adriamycin. (A) Adriamycin treatment induces mature LAT-miRNA expression. MSB-1 cells derived from a Gallid herpes virus-2 (GaHV-2)-induced lymphoma were treated with adriamycin for 8 h. RNA was extracted with Trizol and analyzed by quantitative primer-extension PCR assay, with the Biorad analysis kit. Expression profiles for cluster 2 miRNAs (mdv1-miR-M7, -M8 and -M6) were recorded. The expressions of non-p53-specific mdv1-miR-M4 and p53-specific p21 were evaluated as negative and positive controls, respectively. Fold induction after adriamycin treatment is shown for each RNA. Each value corresponds to the mean of three independent assays performed simultaneously. Errors bars indicate the SEM of three replicates. (B) Western blot analysis of p53 and the GAPDH level after adriamycin treatment. MSB-1 cells were treated with adriamycin (0.2 μg/mL) for 8, 12, and 24 h. Each blot was probed with mouse anti-p53 (HP64) and mouse anti-GaPDH primary antibodies, followed by rabbit anti-mouse horseradish-conjugated secondary antibody. GaPDH was used to check for equal protein loading. Relative signal intensities for p53 were calculated with respect to the GaPDH loading control, with Adobe Photoshop element 6 software. The values reported under the blots correspond to the respective level of p53 detected after adriamycin treatment, relative to the signal for p53 without adriamycin treatment.