Expression of <i>BRD1</i> transcripts and methylation proportions in region 2 and 3 in SH-SY5Y cells following drug treatment.

<p>(a) Expression of <i>BRD1</i> transcripts in SH-SY5Y cells following Lithium, Valproate, and Carbamazepine treatment. The total <i>BRD1</i> expression and expression of transcript variants containing exon 1C, 1B, and 1A were measured in RNA extracted from control cells or following exposure of the cells to the lowest and highest therapeutic dosage of the drugs for 72 hours in SH-SY5Y cells. Data are presented as mean percentages of the mean value of the control group (no drug treatment) +SEM (n = 3/group). ***<i>p</i><0.001, one-way ANOVA with Dunnett's post-hoc tests. (b) Methylation proportions as determined by pyrosequencing of region 2 and 3 following Carbamazepine treatment in SH-SY5Y cells. Methylation proportions were measured by pyrosequencing of region 2 and 3 in DNA extracted from SH-SY5Y cells following exposure to 0 or 0.1 mM Carbamazepine for 72 hours. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 3/group). Group means were compared by unpaired t-test.</p

Methylation proportion changes in the <i>BRD1</i> locus during fetal neocortex development.

<p>Methylation proportions in the <i>BRD1</i> locus during fetal neocortex development. A public available dataset was utilized to correlate DNA methylation proportions with fetal age (n = 179, range = 23–184 days post-conception). DNA methylation data was available for probes from the Illumina Infinium Human Methylation 450K Bead Array. Probes located near the promoter region of <i>BRD1</i> were assessed for correlation between DNA methylation proportions and fetal age. All probes that revealed age related changes in methylation proportions and some that did not are illustrated. Statistical analysis was performed with Pearson’s correlation test.</p

Expression of <i>BRD1</i> transcripts and methylation proportions in region 2 and 3 following Zebularine treatment in HeLa cells.

<p>(a) Expression of <i>BRD1</i> transcripts in HeLa cells following Zebularine treatment. The total <i>BRD1</i> expression and expression of transcript variants containing exon 1C, 1B, and 1A were measured in RNA extracted from HeLa cells following exposure to 0, 0.05, 0.1, or 0.25 mM Zebularine for 72 hours. <i>PGK1</i> and <i>TBP</i> were found to be the most stably expressed reference genes and were used for normalization. Data are presented as mean percentages of the mean value of the control group (no Zebularine treatment) +SEM (n = 3/group). *<i>p</i><0.05, ***<i>p</i><0.001, one-way ANOVA with Dunnett's post-hoc tests. (b) Methylation proportions as determined by pyrosequencing of region 2 and 3 following Zebularine treatment in HeLa cells. Methylation proportions were measured by pyrosequencing of region 2 and 3 in DNA extracted from HeLa cells following exposure to 0 or 0.25 mM Zebularine for 72 hours. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 3/group). *<i>p</i><0.05, ***<i>p</i><0.001, unpaired t-test.</p

Structure of the <i>BRD1</i> promoter regions and methylation proportions in region 1–4 in cell lines.

<p>(a) Structure of the <i>BRD1</i> promoter regions. <i>BRD1</i> comprises 12 coding exons with the first coding exon 1 illustrated in the figure. <i>BRD1</i> was initially described to feature two splice variants, a long and a short resulting from alternative splicing of exon 7 and with transcription initiated from respectively, exon 1 with a 487 bp 5’ UTR (exon 1C) or from a non-coding exon located more than 3 kb upstream of the common exon 1 (exon 1A). We confirm the presence of a third non-coding exon located 1.4 kb upstream exon 1 (exon 1B). Additionaly, the alternative versions of exon 1 (exon 1C, 1B, and 1A) are supported by sequence evidence from different cell types including respectively, GenBank mRNA variant AF005067, CR456408, and AK292428 as well as EST sequences. On the figure the genomic localizations of SNP rs138880 and 6 CpG probes from the Illumina Infinium Human Methylation 450K Bead Array are illustrated: cg15144773, cg21032013, cg02550151, cg15145965, cg06057569, and cg16001335. For DNA methylation analysis, four regions (region 1–4) were analyzed by bisulfite sequencing. The number of CpG sites in each region is: region 1: 33, region 2: 4, region 3: 12, and region 4: 66. Region 2 and 3 was also examined by pyrosequencing. Pyrosequencing of region 2 included CpG sites 3 and 4 from the region 2 bisulfite sequencing assay, including cg15145965 (region 2, CpG site 3) and SNP rs138880. Pyrosequencing of region 3 covered the same region as examined by bisulfite sequencing but because of sequencing limitations it only included CpG sites 1–6 and 9–12. These CpG sites include cg06057569 (region 3, CpG site 6) and cg16001335 (region 3, CpG site 12). The genomic localizations of three CpG islands in the region are shown as well. (b) Methylation proportions as determined by bisulfite sequencing of region 1–4 in HeLa cells. Cells were cultured under standard conditions and DNA was isolated for bisulfite sequencing with primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170121#pone.0170121.s007" target="_blank">S3 Table</a>. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 9–10 clones for each region). For Region 1 and 4 CpG positions were merged when the methylation proportions were equal for adjacent sites. (c) Methylation proportions as determined by bisulfite sequencing of region 1–4 in SH-SY5Y cells using the same conditions as described for HeLa cells. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 9–10 clones for each region).</p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-6

, or treated with pro-inflammatory cytokines 10 ng/ml oncostatin M (OSM) in combination with 20 ng/ml tumour necrosis factor alpha (TNFα) (-◆-). The conditioned medium from four independent wells was measured for the presence of G1/G2 molecules at each collected time-point , or accumulated throughout the study-period. As negative control, explants were frozen and thawed four times in liquid nitrogen (--). The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-3

Thritis (RA) (N = 38) were analyzed in the G1/G2 assay. The concentrations are log-transformed data and the values are mean + SEM. The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t-tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-5

, or treated with pro-inflammatory cytokines 10 ng/ml oncostatin M (OSM) in combination with 20 ng/ml tumour necrosis factor alpha (TNFα) (-◆-). The conditioned medium from four independent wells was measured for the presence of aggrecanase-generated ARGSVI-G2 fragments at each collected time-point , or accumulated throughout the study-period. As negative control, explants were frozen and thawed four times in liquid nitrogen (--). The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-2

Thritis (RA) (N = 38) were analyzed for their anti-CCP level. The activities are log-transformed data and the values are mean + SEM. The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t-tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-4

From patients with Rheumatoid arthritis (RA) (lane 3) and the membrane was stained with the monoclonal antibody F-78 raised against intact bovine aggrecan, binding to G1 and G2, or with BC-3 raised against the aggrecanase-generated ARGSVI sequence. As control, plasma samples from 22 healthy individuals were used (lane 2). A standard molecular weight marker was also run to determine the size of the detected fragments (lane 1).<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p