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SUPPLEMENTARY TABLE LEGENDS
Supplementary table 1: List of 24 probe-sets specifically and significantly upregulated 2-fold between untreated control muscle (CN) and treated muscle after
electrotransfer with saline only (ET). All probe-sets were significantly different with a
Benjamini and Hochberg, p<0.05 (Benj.). Probe-set ID is Affymetrix ID number,
whereas gene ontology refers to the simplified system used in Figure 1 and explained
above. Additional references are also shown for some genes.
Supplementary table 2: List of 22 probe-sets significantly down-regulated between
untreated control muscle (CN) and treated muscle after electrotransfer with saline
only (ET). See legend for Suppl. Table 1 for more details.
Supplementary table 3: List of common tissue genes corresponding to 46 probe-sets
significantly up-regulated between untreated control muscle (CN) and muscle treated
with either saline (ET) or plasmid (pET). See legend for Suppl. Table 1 for more
details.
Supplementary table 4: List of 5 probe-sets significantly down-regulated between
untreated control muscle (CN) and muscle treated with either saline (ET) or plasmid
(pET). See legend for Suppl. Table 1 for more details.
Supplementary table 5: List of 421 probe-sets specifically and significantly upregulated 2-fold between untreated control muscle (CN) and treated muscle after
electrotransfer with non-coding plasmid pCMV (pET). This list forms the key innate
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immune response to plasmid DNA. See legend for Suppl. Table 1 for more details.
Supplementary table 6: List of 35 probe-sets specifically and significantly downregulated 2-fold between untreated control muscle (CN) and treated muscle after
electrotransfer with non-coding plasmid pCMV (pET). See legend for Suppl. Table 1
for more details.
Supplementary table 7: List of 18 probe-sets associated with pancreatic protease
function designated as outliers. Probe sets were specifically and significantly upregulated 2-fold between untreated control muscle (CN) and muscle after
electrotransfer with saline (ET). See legend for Suppl. Table 1 for more details.
Supplementary table 8: Significant enrichment of immunological GO terms for 421
probe-sets up-regulated after electrotransfer with non-coding plasmid DNA. To
simplify representation, only key nodes of the GO biological process tree
(http://www.geneontology.org/) associated with immunological function are shown
(see Results). Columns show official GO term reference ID; term name; corrected P
value (cut off <0.01); number of genes associated with each term from the list of 70
up-regulated ET probe-sets; number of genes from the 33884 genes in the genome
annotated to the term; percentage of genes from the input list and/or the genome
annotated to this term; and list of annotated gene symbols from input list.
Supplementary table 9: Significant enrichment of non-immunological GO terms for
421 probe-sets up-regulated after electrotransfer with non-coding plasmid DNA.
Column descriptions as in Suppl. table 8.
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Supplementary table 10. Values show mean % of cells relative to total CD45+ve cells
± STD for each of the time points and treatments shown at left. The number of mice
used per time point and sample is also shown. The n=1 for saline treated mice at 7
days post-treatment represents a pool of 2 mice. n/a is “not applicable” due to n of 1.
Supplementary table 11: Marker gene and primer list representing key biological
processes induced by electrotransfer with saline and/or non-coding plasmid as
summarised in Figure 6.
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SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1 Clustergram analysis of arrays and probe-sets.
Individual arrays (horizontal) and probe-sets (vertical) are shown with relative
expression scale bar below. ET arrays were grouped together with CN arrays
suggesting that saline electrotransfer is a mild process since the global profiles were
not intensely different, however, pET arrays were grouped separately due to the
immune response to plasmid. The 571 up- and down-regulated probe-sets formed 8
clusters. Clusters a-f correspond to the six subdivisions in Figure 1a, where c, e and f
represent the 467 (421+46) genes up-regulated in pET muscle, with 34/46 common
probe-sets located in cluster f. Cluster e contained the most intensely up-regulated
probe-sets, with 61 of 104 (58.7%) total probe-sets related to immunological function.
We also detected an outlying cluster of 18 protease genes which showed exaggerated
up-regulation in ET, but not pET samples. Cluster H2-D1 was formed due to different
signals of H2-D1/LOC100045864 MHC class I locus probe-sets in CN samples.
Supplementary Figure 2: Infiltration of muscle after electrotransfer. H&E stained
transverse sections of formol-fixed tibialis anterior muscle at 1, 4, 7 or 14 days in
untreated controls (CN), after electrotransfer with saline-only (ET), or after
electrotransfer with the non-coding plasmid pCMV (pET). Original magnification
x20. Needle track sites are shown for comparison.
Supplementary Figure 3: Quantification and composition of muscle cellular
infiltrate after electrotransfer. (a) Total number of cells obtained from muscle cell
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lysates (which includes inflammatory cells, satellite cells and fibroblasts) after
electrotransfer with saline or pCMV and pCpG-free plasmids. (b) Quantification of
inflammatory cell subsets by FACS, including total CD45+ve cells (top left), pro- and
anti-inflammatory macrophages (top right), lymphocytes (bottom left) and
granulocytes (bottom right). Values show mean of absolute cell number obtained per
gram of muscle tissue ± STD. The number of animals used per experiment is
indicated in Suppl. Table 10. * indicates statistical significance (p <0.05) when
compared to saline at the same time point. # indicates statistical significance (p <0.05)
when comparing pCMV and pCpG-free at the same time point. No statistical
analysis was possible with saline-treated muscle at day 7 due to the low n.
Supplementary Figure 4: FACS and gene expression profiles of muscle
inflammatory infiltrate after electrotransfer . (a) FACS profiles of DAPI-ve
CD45+ve cells showing Ly6c PE (x axis) and F4/80 APC-Cy7 (y axis) signal for
different time points and treatments as indicated. Lower events were recorded at day 7
due to the overall decrease in infiltrate. The granulocyte population is shown in green
(Ly6c+ve F4/80-ve cells), pro-inflammatory macrophages in red (Ly6c+ve F4/80+ve cells)
and anti-inflammatory macrophages in blue (Ly6c-ve F4/80+ve cells). Ly6c-ve F4/80-ve
CD3+ve lymphocytes are not shown. Values indicate the proportion of each population
as a mean percentage of total CD45+ve cells ± STD. (b) qRT-PCR quantification of
different inflammatory cell markers confirming the presence of macrophages and
neutrophils, and suggesting some involvement of CD8a positive cytotoxic T cells,
CD161 (Nk1.1) expressing natural killer (NK) cells and also tissue repair
macrophages (CD206) at later stages of treatment.
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Supplementary Figure 5: Infiltration of muscle by macrophages and neutrophils
after electrotransfer. (a) Macrophage and (b) neutrophil infiltration of needle track
sites as shown in Supplementary Figure 1, confirmed by F4/80 and Ly6g staining,
respectively, at 1, 3 and 7 after electrotransfer with saline-only (ET), or non-coding
plasmids pCMV (pET) or pCpG-free (pCpG-free).
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