Effect of exon mutations on exon 16 splicing during MELC induction as well as in HeLa and L8 cell lines. (A) Diagram of exon 16 minigene construct, GE15, GE42, and exonic mutations (EX-1, EX-2, EX-2.1, EX-3, EX-3.1, and EX-3.2) within exon 16. The wild-type and mutated sequences are indicated. (B) Standardization of semiquantitative RT-PCR. Agarose gel electrophoretic analysis of RT-PCR performed around exon 16 from WT-transfected HeLa at an increasing number of amplification cycles. + indicates exon 16 inclusion; and -, exon 16 exclusion. The minimal number of cycles, 22 cycles, that is sufficient for detecting clearly visible bands was subsequently used to analyze the spliced products. (C) Analysis of exonic mutations on exon 16 splicing by RT-PCR. The minigene and its mutation construct stably transfected MELCs were induced to differentiate with DMSO as described in “Materials and methods.” Total RNA was collected from cells as indicated, subjected to RT-PCR, and analyzed by 2% agarose gel. + indicates exon 16 inclusion; and -, exon 16 exclusion. (D) Effect of EX-2.1, EX-3.1, and EX-3.2 on exon 16 splicing by RT-PCR. + indicates exon 16 inclusion; and -, exon 16 exclusion. Endo. indicates endogenous exon 16 splicing patterns.