Summary of microarray analysis result.

<p>Gene-lists were created using genes with p-values≤0.05 and fold changes either ≥2 or ≥1.5 relative to intact islets.</p

Schematic diagram of the workflow.

<p>Frozen sections of the intact human pancreas, freshly isolated islets (d0) and cultured islets (d3) were processed for LCM. The beta-cells were identified by their intrinsic autofluorescence and captured by LCM, followed by RNA extraction, amplification and labeling. Labeled RNA was hybridized to Human WG-6 Expression Arrays (Illumina) and scanned by BeadArray Reader. Expression data were analyzed by Flexarray, DAVID and IPA and validated by immunostaining.</p

Differentially expressed pancreas-specific transcription factors.

<p>Knock out phenotype, ANOVA p-values and fold changes of differentially expressed pancreas-specific transcription factors.</p>a<p>KO phenotype : knock out phenotype.</p>b<p>knock out study is not available, function has been established by other methods.</p

NFKB target genes from top 15 upregulated genes and differentially expressed cytokines and chemokines.

<p>(A) Analysis of the top fifteen upregulated genes from both groups (d0 and d3) revealed that 6/15 genes have NFKB binding sites and, (B) among 39 differentially expressed cytokines/chemokines from d3-islets 22 genes have NFKB binding sites.</p

IL-8 expression in isolated islets.

<p>Immunofluorescence staining was performed for IL8 in frozen sections from pancreas (a–h), d0-islets (i–p) and d3-islets (q–x) and costaining was performed with insulin (c, k, s) or glucagon (g, o, w). IL-8 expression increases after islet isolation and culture. Most of IL-8 positive cells also costain with insulin but not glucagon. Scale bar (10 micron) on merged image represents scale for all images on its left side.</p

Hierarchical clustering of differentially expressed genes in fresh (d0) and cultured islets (d3).

<p>The expression data for intact islets and isolated islets (d0-islets & d3-islets) were analyzed and top 359 genes with p<0.05 and fold change >2.5 were used to create cluster map.</p

Principal Component analysis (PCA) plot of variability in whole genome expression from intact or isolated islets.

<p>Samples were distributed by their similarity in expression data using dimensionality reduction. Pancreatic intact islets (green dots) cluster at right whereas samples from d3-islets (red dots) cluster at left, apart from other groups. The freshly isolated islet samples (yellow dots) cluster at middle and partially intermix with other groups. Principal component 1 and Principal component 2 together accounted for 27% variation.</p

Function, ANOVA p-values and fold changes of the top ten genes upregulated in freshly isolated (d0) and 3-days cultured islets (d3).

a<p>Genes common between top 10 upregulated genes from d0-islets and d3-islets.</p

Statistically significant altered Biological Processes in islets.

<p>The differentially expressed genes (p<0.05, fold change>2) from d0-islets and d3-islets were classified into functional groups using DAVID.</p><p>GO Term: Gene Ontology Term.</p>a<p>: Fold enrichment: the overall enrichment score of the biological annotation term based on the associated genes.</p

Validation of microarray data by quantitative real-time PCR.

<p>Quantitative real-time PCR was performed for IL-8, ID2, SOX4 and SOX9. Fold-changes represent expression level of genes in isolated islets relative to intact islets. Mean fold changes derived from quantitative PCR (white bars) were plotted along with the corresponding mean fold changes from microarray analysis (solid bars). Error bars represent standard deviation. * represents t-test p-values<0.05. For microarray standard deviation and t-test was performed on the expression values obtained after normalization with Lumi. The mean fold changes determined by quantitative PCR were in concordance with the mean fold changes from microarray data.</p