Additional file 1: of A screening protocol for child abuse at out-of-hours primary care locations: a descriptive study

Screening protocol child abuse, Primair Huisartsenposten, represented by a flow chart for the general practitioners (whole screening protocol consisting of 12 pages is available on request). (TIF 119 kb

Additional file 5: Table S6. of Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease

Upstream regulators. (XLSX 135 kb

Additional file 7: Table S3. of Systematic analysis of chromatin interactions at disease associated loci links novel candidate genes to inflammatory bowel disease

Publicly available datasets. (XLSX 11 kb

VPA and NAM treatment modulates H3K27 acetylation at the promoters of myeloid specific genes.

<p>UCB-derived CD34+ cells were differentiated towards megakaryocytes. At day 4 of differentiation, cells were treated overnight with 200μM, or 5mM NAM. Next, lysates were prepared, followed by ChIP-sequencing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196400#sec002" target="_blank">Methods</a>). Data represent a Venn diagram comparing the number of genes identified based on a >2-fold increase or decrease in H3K27 acetylation levels compared to the control (A). Gene ontology analysis of up- and down-regulated genes by VPA (B) and NAM (C) treatment. From the same cells, RNA was isolated, followed by cDNA synthesis and qPCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196400#sec002" target="_blank">Methods</a>). Data represent the relative mRNA level of GATA1, RUNX1, LMO2 and RUNX2 compared to the control (D). Data are representative for 3 experiments. Error bars represent SEM, * p<0.05, ** p<0.01.</p

qPCR primers.

<p>qPCR primers.</p

VPA and NAM treatment inhibits megakaryocyte progenitor proliferation.

<p>UCB-derived CD34+ cells were differentiated towards megakaryocytes for 11 days in the absence or presence of 100–200μM VPA, or 1-5mM NAM. (A) At all culture time points, trypan blue negative cells were counted. Data represent the fold expansion of megakaryocyte precursors during development. (B) At day 7 and 11, the percentage of apoptotic cells was determined by FACS. Data represent the percentage of Annexin V-positive cells, compared to the control. (C) Absolute numbers of CD61/CD42b positive megakaryocytes were calculated after 11 days of differentiation. Data are representative for 4 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.</p

VPA treatment increases the absolute number of MEP.

<p>UCB-derived CD34+ cells were differentiated towards megakaryocytes for 4 days in the absence or presence of 200μM VPA, or 5mM NAM. Myeloid progenitor staining was performed according to Manz <i>et al</i>. (46), and distinct progenitor populations were analysed by FACS. MEP were gated from the Lin- CD34+, CD38+ CD123- and CD45RA- cell population. Data represent (A) the absolute numbers of CD34+ cells and MEP, compared to the control, and (B) the percentage of CD34+ cells and MEP, compared to the control. Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.</p

KDACi treatment differentially modulates megakaryocyte development.

<p>(A) From the peripheral blood the absolute number of platelets was analysed in patients treated with VPA (n = 217) together with plasma VPA concentration, measured at the same day. Data represent linear regression analysis of the absolute number of platelets (dependent variable), and plasma VPA concentration (independent variable). (B) UCB-derived CD34+ cells were differentiated towards megakaryocytes in the absence or presence of 10nM TSA, 250μM SB, 200μM VPA, or 1mM NAM for 11 days. Differentiation was determined based on the surface expression of CD61 and CD42b (percentage of double positive cells compared to the control), and (C) cytospin analysis. Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.</p