Effect of PNA2512 on the splicing of introns 2, 3 and 4. JAR cells were transfected with PNA2512 or mismatch PNA (PNA2733) for 24 h and subjected to RT-PCR analysis using extracted total RNA. (A) Location of PNA2512 and detected splice sites ((a), (b), (c), (d) and (e)) are indicated. (B) Cells were transfected with PNA2512 (2 μM) and subjected to RT-PCR analysis. N: no PNA treatment. P: treated with PNA2512 (2 μM). The structures or names of RT-PCR products are shown on the right of each picture. Primer sets (see table 2) for the detection of each splice site by RT-PCR (product length) are as follows: (a), A (161 bp) and H (β-actin, 223 bp); (b), C (120 bp) and H (β-actin, 223 bp); (c), D (169 bp) and H (β-actin, 223 bp); (d), F (151 bp) and H (β-actin, 223 bp); (e), G (215 bp) and I (β-actin, 166 bp). The numbers under the figure indicate the relative amount (normalized to β-actin) of the target mdm2 splicing variants. (C) Effect of PNA2512 on the skipping of exon4. Cells were treated with PNA2512 or its mismatch PNA (PNA2733) at the indicated concentrations and subjected to RT-PCR analysis. For the RT-PCR, Exo3S and Exo5AS (primers in Table 2) were used. Locations of the normally spliced form (Normal (207 bp)) and exon4-skipped form (skipped (131 bp)) are indicated on the right. Numbers under each lane indicate the amount of the skipped form relative to the sum of skipped and normal form.