Figure 1.

Claudin-3 (Cldn3) and claudin-4 (Cldn4) expression and distribution analysis and sensitivity toward recombinant CPE (recCPE) in human PC cell line panel. (A) Quantitative real time PCR (qRT-PCR, graph upper panel) and Western blot analyses (two lower panels for claudin and β-actin, Figure S5) for Cldn3 (left) and Cldn4 (right). The human colorectal cancer cell line HT-29 served as positive control and the human melanoma cell line SK-MEL-5 as negative control with no Cldn3/4 expression. Data are represented as mean values ± S.D. (n = 3). (B) Representative immunohistochemistry of Cldn3 (left) and Cldn4 (right) in respective pancreatic cancer cell lines, demonstrating different distributions of Cldn3/4 (brown). (C) Western blot analysis of cell fractions from four PC cell lines using Cldn3/4, and Lamin B1, showing whole cell lysates, cytoplasmic, membrane and nuclear localization (Lamin B1 marker nuclear fraction, Figure S5). Representative immunofluorescence images (right panel) of respective cell lines, confirming diverse Cldn3 (yellow) and Cldn4 (red) expression in different cell compartments (scale bar = 25 µm). DAPI and actin staining was used for nuclei and cytoplasm, respectively. (D) Sensitivity toward recCPE at 72 h post treatment using MTT assay and compared to untreated/solvent treated controls with strong cytotoxic effects in all PC cell lines and positive control line HT-29. SK-MEL-5 negative control cells remained unaffected. MTT data are represented as mean values ± S.D. (n = 6), expressed as mean percentage of untreated control. (ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).