Additional file 2: Table S2. of Epigenetic effects of casein-derived opioid peptides in SH-SY5Y human neuroblastoma cells

Diseases and disorders generated by IPA analysis of DMTs and DETs corresponding to treatment. The p-value range listed represents the range of significant (P < 0.05) pathways associated with the listed disease or disorder. The number of probes indicates the number of differentially methylated or transcribed transcripts associated with the disease or disorder. (PDF 60.5 kb

Additional file 1: Table S1. of Epigenetic effects of casein-derived opioid peptides in SH-SY5Y human neuroblastoma cells

List of 50 differentially transcribed probes from heat maps used in figure 2. (PDF 66.6 kb

Copy number variation-based read-count normalization of MBD-Seq of glioblastoma multiforme (GBM) tumour samples data using Illumina Infinium 450k platform.

<p>The use of methylated DNA capturing followed by massive parallel sequencing has become a robust method for the study of genome-wide methylation patterns. However, it has recently been demonstrated that the accurate estimation of methylation levels is dependent on incorporating CNV status of the sampled genomic regions (Robinson et al, 2012).</p> <p>We used Illumina multiplex paired-end (PE) sequencing of MBD-captured DNA isolated from GBM samples. The mean number of PE reads per library was 5.3 million [19.3-2 million]. Alignment of quality-controlled (min. Q20) and de-duplicated reads to the human reference genome hg19 was performed using bowtie2, resulting in a mean number of mapped PE reads of 3.8 million [14-1 million]. Our preliminary results indicate differences in the methylation of CpG islands located in promoter regions when comparing normal versus tumour samples. We also performed DNA methylation estimation using the Infinium 450k platform for the same set of samples. Illumina's Infinium HumanMethylation450 technology is based on the OmniBeadChip genotyping technology which has been used to accurately estimate CNVs. We therefore hypothesized that the 450k methylation data (signal intensities) could be used for the estimation of the CNV status. One pre-requisite for this approach is the availability of a canonical set of controls generated from normal tissue. Due to the limited sample size of our data set, we plan to incorporate the 450k methylation and CNV data available from TCGA to:</p> <p>1. create the canonical set of controls of 450k data from normal tissues of TCGA samples</p> <p>2. perform CNV estimation of TCGA samples based on 450k methylation data</p> <p>3. verify the accuracy of the 450k-based approach against TCGA CNV data</p> <p>The use of TCGA data in this methodology will allow our laboratory to evaluate the robustness of this approach before applying it to our own datasets.</p

List of putatively glycosylated membrane proteins with <i>N-</i> and/or <i>O</i>-glycans located on the cell surface, identified after RSA affinity chromatography and LC-MS/MS analysis.

<p>List of putatively glycosylated membrane proteins with <i>N-</i> and/or <i>O</i>-glycans located on the cell surface, identified after RSA affinity chromatography and LC-MS/MS analysis.</p

Effect of RSA to induce apoptosis in <i>Drosophila melanogaster</i> S2 cells.

<p>(A) DNA fragmentation in S2 cells. Cells were treated with 0.7 µM RSA compared to control (untreated) cells. Ten micrograms of extracted DNA was loaded on the 2% agarose gel. (B,C) Nuclear condensation assay: Upon treatment with 0.7 µM RSA, the nuclei of the S2 cells were stained with Hoechst. Typically, treated cells (C) showed a normal, non-fragmented nucleus similar to the untreated control cells (B).</p

Effect by lectins on cell proliferation in <i>Drosophila melanogaster</i> S2 cells.

<p>(A) Effect by RSA, GNA, PNA and WGA when cells were treated with lectin at 0.7 µM for 24 h. (B) Inhibitory effect of sugars on the activity of RSA on S2 cells. 0.7 µM RSA was pre-incubated with 100 µM of the specific sugar GalNAc or the non-specific sugar mannose (negative control) and PBS in the control treatments for 1 h. Then the mixtures of RSA and sugars were added to S2 cells and incubated for 24 h at 27°C. Cell proliferation was measured using an MTT assay. Data are presented as mean percentages of cell proliferation inhibition ± SE compared to the control, and based on four repeats. Values are followed by a different letter (a-c) are significantly different (<i>post hoc</i> Tukey-Kramer test with p = 0.05).</p

Inhibition of RSA activity after pre-incubation of the S2 cells with different kinase inhibitors.

<p>Cells were pre-incubated with three inhibitors (individually): 10 µM of SB203580 (p38 MAP kinase inhibitor), 50 µM of PD98059 [MAP kinase, (MEK) inhibitor] and 50 µM of AG490 (JAK inhibitor) for 1 h before exposure to 0.3 µM RSA for 24 h. Values are given as means ± SEM based on two independent repetitions. Values are followed by a different letter (a-c) are significantly different (<i>post hoc</i> Tukey-Kramer test with p = 0.05).</p

Binding and uptake of lectins in <i>Drosophila melanogaster</i> S2 cells.

<p>Confocal microscopy S2 cells incubated with different lectins: S2 cells were incubated with 0.7 µM FITC-lectin for 1 h. (A) RSA- FITC (B) GNA-FITC (C) WGA-FITC and (D) PNA-FITC. Scale bars are 2.5 µM.</p

Effect of RSA on S2 cells.

<p>(A) Control, (B) Treated cells with 0.7 µM RSA, (C) S2 cell number at time zero (at the beginning of the assay) and after 4 days incubation in the presence and absence of 0.7 µM RSA. Cell numbers increased 5.2 and 2.8 fold in control and treated cells, respectively, which showed clearly that RSA inhibited cell proliferation. (D) Concentration-response curves of S2 cells challenged with RSA for 4 days after sigmoid curve fitting in Prism v4. Cell number was measured using an MTT assay.</p

Yield independent genome-wide kit evaluation using Infinium HumanMethylation27 BeadChip data for external validation.

<p>Fractions of mapped MBD-seq fragments corresponding with specific Infinium methylation degrees (binned per 5%) for two cell lines (A, DU145; B, PC3) and all kits (violet, MethylMagnet; yellow, MethylCollector; blue, MethylCollector Ultra (MC Ultra); red, MethylCap; green, MethylMiner) with indication of the background profile (black, fractions of all Infinium methylation values measured for specific cell line). Additionally, the same fractions after division by the corresponding background profile fractions are plotted (C, DU145; D, PC3).</p