RNA half-life measurements in SkMC.

<p>Normal myoblasts were treated with a combination of actinomycin-D and α-aminitin to inhibit transcription. Myoblasts were harvested at different time-points after treatment (0, 0.5, 4, 8, 16 and 24 h) and RNA was extracted. Synthesized cDNAs were subjected to RT-PCR analysis to measure RNA half-lives as previously described (17,18). <i>MYC</i>, a short-lived RNA and the long-lived 18S RNA were used as controls. Graphical representation of the average percent of RNA plotted against time from two independent experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048825#pone.0048825.s002" target="_blank">Figure S2</a>.</p

Number and severity of splice defects increase when MBNL1 is silenced incrementally from ∼79% to ∼98%.

<p>SkMC were transfected with siRNAs directed against MBNL1 and cell samples on each subsequent day post-siRNA transfection for a period of 5 days, were divided into 4 aliquots where one aliquot was used to measure MBNL1 levels and total RNA was extracted from each of the three other aliquots. Scrambled siRNA transfected samples were harvested on Day 5, the last time point of the experiment. <b>(A)</b> Total protein (10 µg) was analyzed by western blot to measure the silencing achieved for MBNL1 at 24 h intervals for 5 days. Blots were probed for GAPDH as an internal control. <b>(B)</b> Synthesized cDNAs were subjected to PCR analysis to study RNA splicing as indicated with <i>GAPDH</i> RNA as an internal control. In each case the levels of exon inclusion obtained in the experiment shown are indicated. <b>(C)</b> The results of RNA splicing as a function of MBNL1 levels in SkMC are tabulated.</p

Splice defects in <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> skeletal muscle.

<p>Lower limb skeletal muscles from adult wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice were harvested and divided into 2 aliquots. One aliquot was used to measure Mbnl1 levels and the other aliquot was used study RNA splicing. <b>(A)</b> Western blot analysis of steady-state Mbnl1 levels in skeletal muscle of wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice are shown with Gapdh as an internal loading control. <b>(B)</b> cDNAs synthesized from skeletal muscle of wild-type, <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> mice were subjected to PCR analysis as indicated with <i>Gapdh</i> RNA as an internal control. In each case the levels of exon inclusion obtained in the experiment shown are indicated. <b>(C)</b> The results of RNA splicing examined as a function of Mbnl1 levels are tabulated.</p

Incremental depletion of MBNL1 results in an increase of both the number and severity of RNA splice defects.

<p>RNA splice defects that manifest with the depletion of MBNL1 in SkMC and in <i>Mbnl1^+/ΔE3</i> and <i>Mbnl1^ΔE3/ΔE3</i> skeletal muscle are shown. Line thickness represents the severity of the splice defect.</p

Fragment A exhibits enhancer activity in the reverse orientation only.

<p>(<b>A</b>) Relative luciferase activity for positive control, fragment A in the forward, and fragment A in the reverse orientation. Light columns indicate activity in HCT-116, dark columns indicate activity in SW480. (<b>B</b>) Fragments B through G, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111914#pone-0111914-g001" target="_blank">figure 1B</a> as red stripes do not show enhancer activity in either orientation in HCT-116. Positive controls are shown for each group of constructs analyzed on the same plate. Fragment E contains the tagSNP rs4939827. (<b>C</b>) Activity of fragment A in the reverse orientation with all SNPs in the fragment with their C allele, with each allele changed independently to the alternate allele, shown as fold change relative to the construct containing the four C alleles. All four alleles result in a statistically significant reduction in activity: rs6507874 T <i>p</i> = 8.25×10⁻³ (HTC-116), <i>p</i> = 3.30×10⁻² (SW480); rs6507875 G <i>p</i> = 3.40×10⁻² (HCT-116), <i>p = </i>6.79×10⁻³ (SW480); rs8085824 T <i>p = </i>1.20×10⁻² (HCT-116), <i>p = </i>3.12×10⁻³ (SW480); rs58920878 G <i>p = </i>2.09×10⁻³ (HCT-116), <i>p = </i>3.05×10⁻³ (SW480).</p

Fragment A Enhancer is responsive to BMP4 stimulation but not TGFβ1.

<p>(<b>A</b>) Enhancer activity was measured by luciferase assay for the fragment A containing the major (CGTC) and minor (TCCG) haplotypes in serum starved HCT-116 and SW480 cells incubated with TGFβ1 for 6 hours. Samples are plotted as a fold change in activity relative to non-treated cells on the same plate. (<b>B</b>) Enhancer activity was stimulated by 6 hours of BMP4 treatment for the CGTC haplotype in HCT-116 (<i>p</i> = 4.14×10⁻³) and SW480 cells (<i>p</i> = 1.22×10⁻¹⁰). The TCCG haplotype in fragment A shows lower levels of stimulation, and only in SW480 (<i>p</i> = 1.61×10⁻⁶). (HCT-116 <i>p</i> = 0.38). Effect of BMP is plotted as fold change over untreated samples for each haplotype on the same plate. (<b>C</b>) Enhancer activity comparison between the CGTC and TCCG haplotypes following BMP4 treatment. Relative luciferase activity was plotted for each haplotype in HCT-116 and SW480 using the same experimental dataset as (B).</p

Differential protein binding by SNP alleles using EMSA.

<p>(<b>A</b>) Nuclear extracts from SW480, HCT-116, and RKO cell lines were incubated with IR-dye labeled 33mers centered on rs6507874 C (red label) and T (green labels) prior to native EMSA. Lanes 1 and 2 show probe without nuclear extract. Lanes 3, 4, 9, 10, 15, and 16 show each labeled probe binding to nuclear proteins individually with cell line as noted above. Lanes 5–8, 11–14, and 17–20 are a 1∶1 competition with labeled C and T allele probes. Lanes 6, 12 and 18 contain 200-fold excess unlabeled C allele competitor. Lanes 7, 13, and 19 contain 200-fold excess unlabeled T allele competitor. Lanes 8, 14, and 20 contain 200-fold excess competitor of an unmatching sequence with similar nucleotide content. Bands specific for one allele and lost upon competition are marked with arrows. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111914#pone.0111914.s002" target="_blank">Figure S2</a> for red (700) channel image of the C probe and the green (800) channel of the T probe in black and white. (<b>B</b>) As in panel A, with rs6507875 C allele (red probe) and G allele (green). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111914#pone.0111914.s003" target="_blank">Figure S3</a> for red (700) channel image of the C probe and the green (800) channel of the G probe in black and white. (<b>C</b>) As in panel A, with rs8085824 C allele (red) and T allele (green). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111914#pone.0111914.s004" target="_blank">Figure S4</a> for red (700) channel image of the C probe and the green (800) channel of the T probe in black and white. (<b>D</b>) As in panel A, with rs58920878 C allele (red) and G allele (green). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111914#pone.0111914.s005" target="_blank">Figure S5</a> for red (700) channel image of the C probe and the green (800) channel of the G probe in black and white.</p

Candidate functional SNPs are association with <i>SMAD7</i> gene expression levels.

<p>Fold change (FC) of SMAD7 expression was measured in normal colon tissue samples from surveillance colonoscopies. Rank-based non-parametric analysis was used to estimate the effect of each extra minor allele for rs6507874, rs6507875, rs8085824, and rs4939827 (additive model) on gene expression adjusting for gender, age and race. Two-sided <i>p</i>-values were obtained from a likelihood ratio test. Log₂(ΔΔCt) were plotted as a function of genotypes using a box plot – dot plot overlay. Number of samples is noted under each genotype. A statistically significant association was found for rs8085824.</p