Fig. 3

Specificity of CPE in the isogenic cell lines SK-Mel5 and SK-Mel5 Claudin-3-YFP a Western blot analysis for claudin-3-YFP gene expression in Sk-Mel5 Cldn-3-YFP cells and no claudin-3 expression in Sk-Mel5 cells. b Representative immunofluorescence of Sk-Mel5, and Sk-Mel5 Cldn-3-YFP cells. Claudin-3 negative cells were counterstained with Phalloidin (red). Nuclei were counterstained with Hoechst33343. Scale bar: 10 μm. The image demonstrates specific claudin-3 expression (yellow) of Sk-Mel5 Cldn3-YFP cells. c Isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP cells were treated with recCPE for 72 h at indicated concentrations. recCPE toxicity was determined by MTT cytotoxicity assay. Decreased cell viability mediated by recCPE cytotoxicity is demonstrated in Sk-Mel5 Cldn-3-YFP cells, SK-Mel5 cells remained unaffected. All assays were performed in three independent experiments and expressed as mean percent of untreated control. Bars: SD. Level of significance was calculated by 2way-ANOVA; * P < 0.05, **; P < 0.001, *** P < 0.0001. The assay revealed highest specificity of recCPE mediated cytotoxicity by binding to claudin-3. d Cytotoxicity of optCPE-GFP gene transfer in isogenic Sk-Mel5 pair and proof of claudin specificity. Isogenic cells were transfected with empty vector (e.v.) expressing or optCPE-GFP expressing construct. MTT assay was performed 72 h after transfection. Controls are transfection reagent and e.v. transfected cells. All assays were performed in three independent experiments and expressed as mean percent of untreated control. Bars: SD. Level of significance was calculated by 1way-ANOVA, *** P < 0.0001. The assay demonstrates high cytotoxicity for optCPE-GFP expressing Sk-Mel5 Cldn3-YFP cells, which acts selectively on claudin-3 cells, leaving claudin-negative Sk-Mel5 cells unaffected. e Representative images for co-localization of Cldn-3-YFP and CPE. Upper panel represents Sk-Mel5 Cldn-3-YFP cells, incubated with recCPE (250 ng ml ^-1) for 30 min and counterstained with specific CPE antibody and Alexa555 labeled secondary antibody; Cldn-3-YFP (green), recCPE (red) and co-localization of CPE/Cldn-3-YFP (yellow) is shown. Nuclei were counterstained with Hoechst 33343 (blue). Scale bar: 10 μm. Lower panel demonstrates Sk-Mel5 Cldn-3-YFP cells transfected with optCPE–GFP and fixed 24 h later. Expressed Cldn-3-YFP (green), optCPE (red) and their co-localization (yellow) were detected (indicated by arrows). CPE was mainly located at the cell membrane. Counterstaining of nuclei with Hoechst 33343 (blue). Scale bar: 20 μm. These images confirm co-localization and specificity of CPE binding to claudin-3 as well as CPE accumulation within the cytoplasm. f Time-dependent cell death analysis after external application of recCPE (150 ng ml ^-1). Sk-Mel5 Cldn-3-YFP cells were imaged for 45 min after recCPE addition. This process continued with swelling of treated cells, cell blebbing and size reduction of nuclei. Cell nuclei were stained with Hoechst33343 (blue). Scale bar: 25 μm