Effect of PQ treatment on splicing of <i>SMN</i>. <i>A,</i>

<p>Diagrammatic representation of the <i>SMN</i> gene. Exonic and intronic sequences are depicted as in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone-0049595-g002" target="_blank">Figure 2A</a>). Dotted lines indicate exon skipping that generated novel splice variants. Annealing positions of primers used for MESDA are shown. <b><i>B,</i></b> Detection of multiple exon skipping events in endogenous <i>SMN</i> in the presence (+) and absence (−) of PQ. Names of cell lines used are given on the top of the gel. Total RNA was prepared from cells treated with 1 mM of PQ for 24 hours. Splice products were analyzed by RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone-0049595-g003" target="_blank">Figure 3B</a>. Diagrammatic representation of splice variants is given on the left of the gel; their sizes are indicated on the right of the gel with names of skipped exons given in brackets. The identity of splice variants was confirmed by sequencing. Note that 513-nucleotide-long Δ5,6,7 variant moves slower than 519-nucleotide-long Δ3,7 variant. Novel splice variants are marked by stars. FL, full length; NS, non-specific. For intra-lane comparison of bands, pictograms were generated for each lane (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone.0049595.s002" target="_blank">Figure S2</a>). <b><i>C,</i></b> Splicing patterns of endogenous <i>SMN</i> exon 7 in the presence (+) or absence (−) of PQ. Names of cell lines used are given on the top of the gel. Total RNA was prepared from cells treated with 1 mM of PQ for 24 hours. Spliced products were analyzed by RT-PCR as described in panel B except primers P25 and P31 were used for PCR amplification step. To distinguish transcripts originated from <i>SMN2</i>, PCR products were digested with DdeI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone.0049595-Singh3" target="_blank">[55]</a>. <i>SMN1</i> and <i>SMN2</i> transcripts are indicted on the right of the gel. Exon 7-included (+) and exon 7-skipped (−) products are indicated on the left of the gel. The percentage of <i>SMN2</i> exon 7 skipping was calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049595#pone-0049595-g002" target="_blank">Figure 2B</a>. <b><i>D,</i></b> Western blot showing translated products generated from <i>SMN</i> in GM20384 and GM03813 cells treated with PQ for 24 h. We loaded 50 and 80 µg of protein from GM20384 and GM03813 cell lysates, respectively. Primary antibodies used for probing are indicated on the left of the gel. Although the predicted molecular mass of SMN is 32 kDa, it migrates as 40 kDa band due to posttranslational modifications.