MOESM1 of Systemic epigenetic response to recombinant lentiviral vectors independent of proviral integration

Additional file 1. Characteristics of different batches of vector and particles tested on CD34+ cells

Illumina Infinium analysis also detects distinct methylation patterns in the CD34⁺ cells depending on the stimulation and/or lentiviral transduction.

<p><b>A</b>. Dendogram obtained by unsupervised hierarchical clustering of 12 samples (triplicates for each condition) analysed. The LV transduced cells (Group 4) clusters separately of all others (upper part). The heat-map columns under the squares with the sample number represent the methylation level (beta values) of the genes that changed by at least by 20% between the experimental conditions. <b>B</b>. Comparison of the numbers of genes that increased (red) or decreased (green) methylation between the groups. The number of methylated/demethylated genes increases dramatically in LV transduced as compared to the two the non-transduced CD34⁺ cells.</p

High-content image analysis of the heterogeneous population of HP1alpha immunostained cells of the four groups suggests changes in the cell nucleus.

<p><b>A.</b> Examples of immunostaining images used for the analysis of HP1alpha expression. <b>B.</b> Representative example of the PCA analysis of the data extracted from analysis of the images of HP1alpha immunostained cells. Note that the three clusters overlap.</p

The region of chromosome 6p bearing genes methylated differentially between Group 2versus Group 4 cells.

<p>Chr 6 is schematically represented on the top. Target genes with more than 20% gain of methylation are indicated by a small vertical bar as a function of their chromosomal location and their methylation: the darker is the bar higher is the gain of methylation. Note the accumulation of the targets genes in the cluster of histone- and HLA-coding genes.</p

Chromosomal clusters.

<p><b>Legend:</b> Chromosomal regions bearing non-random methylation changes in the genome of Group 2 (cytokine stimulated) cells (left panel) and in Group 4 (LV infected) cells (right panel) compared to the genome of control Group 1 (unstimulated) cells are indicated. Chromosomal number, the nucleotide position of the start and the end of the region of interest, the number of changes/number of analysed genes in the region and the adjusted p-value are indicated. The non-random accumulation of the changes on the chromosome 6 is highly significant. Although in the other chromosomal regions the deviation from the random pattern is at the limit of statistical significance, it is important to note that in LV infected cells the changes occur in the same regions at always higher proportion.</p

High-content image analysis of the heterogeneous population of SIRT1 immunostained cells of the four groups suggests changes in the cell nucleus.

<p><b>A.</b> Examples of images used for the analysis of SIRT1 expression representative of 9 experiments. The groups and colour codes are the same as indicated on the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048943#pone-0048943-g001" target="_blank">Figure 1</a>. Scale = 50 micrometers. Confocal analysis was performed on a LEICA TCS SP2 microscope (Leica Microsystems) with the Leica Confocal Software. Images were acquired with a 40X HCX PL APO Plan Fluor oil objective (NA 1.25) at room temperature. For display, the images were processed using ImageJ a median filter 3 pixels followed by an image adjustment, increasing the brightness was performed equally on all the Red images. The nuclei were stained with DAPI and the SIRT1 antibody was labelled with Alexa 594 <b>B.</b> Upper plot: A representative example of the principal component analysis of the data extracted from the SIRT1 immunostaining images. Individual cells are visualized as points on the scatter plot of the first two principal components describing the variation. They were identified by the colour code according to their group of origin only after their position on the scatter plot is calculated. Note that cells from the same experimental group tend to cluster. The groups 2 and 3 overlap. Lower plot: Representative example of the LDA analysis of the same data. This alternative multiparametric analysis takes into account the existence of four groups and helps to visualize the differences between them. Note the overall similarity of the LDA and PCA scatter plots.</p

Average methylation profiles (beta values) obtained for the CpG-s analysed in each experimental condition.

<p>Each density plot represents the median of three samples.</p

Lentiviral induced DNA methylation changes detected by MeDIP analysis. A

<p>. Venn diagram representing the sets of tags with log₂ ratios >2. Each group is represented by a circle: non-stimulated CD34⁺ cells cells (Group 1; black circle), cytokine pre-stimulated (Group 2; red circle) and LV transduced (Group 4; bleu circle). Intersections represent the tags shared by two or three groups. A color code is used to designe the unique sections and the intersections and the number of tags in each section are indicated on the right side. As a result, the total number of tags identified in a group is equal to the summ of the four sections that fill up the circle. The little overlap between the three groups suggests that the difference in the methylation is extensive. <b>B</b>. Heat-map comparison of the tags with the highest log₂ ratios in the different groups. Left panel shows the first 500 tags with the highest log₂ ratios in Group 1 and the corresponding log₂ ratio in the two other groups. The middle panel shows the 500 tags with the highest log₂ ratios in Group 4 together with the corresponding values in the other groups. On the right panel the log₂ ratios of the first 500 tags in the Group 2 are compared to the other groups. The color code of the log₂ ratios is indicated under the panels. Note that most of the tags with the high log₂ ratio in one group have a different log₂ ratio in the two other groups. The greatest difference is observed between the Group 1 and 4.</p

Schematic outline of the experimental strategy.

<p>Freshly isolated CD34⁺ umbilical cord blood cells were separated into four experimental groups: Group 1: non treated CD34⁺ cells analyzed immediately; Group 2: cells stimulated with cytokines only; Group 3: cells cultured with cytokines and undergoing a mock infection with polybrene only but no LV; Group 4: cells cultured with cytokines, then transduced with polybrene and LV. The cells were either fixed for immunochemical analysis or were frozen for DNA extraction and methylation analysis. In a typical experiment, cells isolated from the same cord blood were split into the different groups. For immunochemical analysis groups of cells deriving from a single cord were compared. For methylation analyses, DNA from several cord blood donors was pooled.</p

High-content image analysis of the heterogeneous population of DNMT1 immunostained cells of the four groups suggests changes in the cell nucleus.

<p><b>A.</b> Examples of immunostaining images used for the analysis of DNMT1 expression representative of 9 experiments. The experimental and image analyse procedures were the same as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048943#pone-0048943-g003" target="_blank">Figure 3</a>. <b>B.</b> Representative example of the PCA analysis of the data extracted from analysis of the images of DNMT1 immunostained cells. Note that the cluster of cytokine-stimulated and virus-transduced cells overlap.</p