Establishment of chemoresistant PDAC cell clones.

<p>Generation of chemoresistant PDAC cell clones by repeated pulsatile treatment over 3 days with constant sublethal concentrations of 0.4μM (A) or 0.06μM (D) gemcitabine followed by recovery-periods and quantification of cell viability by MTT assay as well as apoptosis assay in parental vs. chemoresistant PANC-1 (A, B, C) and MIA-PaCa-2 (D, E, F) cell clones.</p

Protein target expression.

<p>Protein expression of MRP-1, mutant p53 (mt p53 R273H and mt p53 R248W), CDK1, Bcl-2, and actin (loading control) in parental (PANC-1, MIA-PaCa-2) and gemcitabine resistant PDAC cell clones (PANC-1-GR, MIA-PaCa-2-GR) by Western blot.</p

MicroRNA profiling.

<p>Procedure and results of miR expression profiling by GeneChip microarray and qRT-PCR validation in parental and gemcitabine resistant PANC-1 and MIA-PaCa-2 cell clones.</p

Protein densitometry.

<p>Adjusted relative density of MRP-1, mutant p53 (mt p53 R273H and mt p53 R248W), CDK1, Bcl-2, and their loading controls measured by ImageJ densitometry software. Asterisks indicates to a significant difference of p<0.05, respectively.</p

Intrinsic drug sensitivity of parental PDAC cell lines.

<p>Relative cell viability of five human PDAC cell lines following 72 h exposure to ascending concentrations of gemcitabine by MTT cytotoxicity assay.</p

In-silico target analysis.

<p>Overview of selected and highly predicted gene targets of dysregulated miRs in chemoresistant PDAC cell clones. (N.A., not available)</p

Morphologic changes in parental and chemoresistant PANC-1 and MIA-PaCa-2.

<p>Representative attached epithelial cells with spindle-shaped cells in untreated MIA-PaCa-2 (A) and PANC-1 cells (B) compared to more plump rounded morphology and enhanced formation of pseudopodia in their chemoresistant cell clones (C, D).</p

Interaction of mutant TP53 and microRNA signaling pathways in chemoresistant pancreatic cancer.

<p>Target analysis was performed by Ingenuity Pathway Analysis and DIANA-mirPath.</p

MicroRNA microarray validation.

<p>MiR microarray validation with qRT-PCR for significantly dysregulated miRs in PANC-1-GR (A) and MIA-PaCa-2-GR cell clones (B). Fold change from miR microarray is demostrated by log2 values (left y-axis, dark grey bars). Fold change from qRT-PCR was determined using the 2^-ΔΔCt method (right y-axis, light grey bars). Error bars represent the standard deviation of mean.</p

MicroRNA heat mapping.

<p>Hierarchical cluster heat maps of significantly dysregulated miRs in PANC-1-GR late and early cell passage (A) as well as in PANC-1-GR and MIA-PaCa-2-GR late cell passage (B). Each row shows the relative expression level for a single miR and each column shows the expression level for a single sample; fold change greater than ±2 (p<0.05). The red or green color indicates low or high expression, respectively.</p