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. 2021 Aug 31;13(17):4393. doi: 10.3390/cancers13174393

Figure 2.

Figure 2

Expression of optCPE and selective cytotoxicity after in vitro gene transfer. (A) Representative immunofluorescence images showing expression of optCPE (green) 12 h after transfection in Capan-1, HUP-T3 and PA-TU-8902 cells (40-fold magnification). Cytoplasm was stained with phalloidin (red) and nuclei with DAPI (blue). The images reveal strong CPE expression, with vesicle-like cytoplasmic accumulation, localization on cell membrane and cell contact regions. (B) Kinetic of optCPE expression in Capan-1, HUP-T3, MIA PaCa-2 and PA-TU-8902 cells 24 to 72 h after transfection (Figure S6). (C) Cytotoxicity of optCPE and vector control (VC) gene transfer was analyzed by MTT assay 72 h after transfection. All transfected Cldn3/4 expressing cells showed significant optCPE toxicity. SK MEL-5 negative control cells remained unaffected, supporting selectivity of optCPE. Data are represented as mean values ± S.D. (n = 6), expressed as mean percentage of VC treated cells. (D) Rescue experiment in MIA PaCa-2 and HUP-T3 cells transfected with a pool of two specific small interfering RNAs or respective control (siCtrl), leading to efficient knockdown of Cldn3/4 at protein level (Western blot, (Figure S6). (E) Cldn-silenced cells were transfected with optCPE. MTT assay was performed 72 h later showing significantly reduced CPE cytotoxicity in MIA PaCa-2 and HUP-T3 cells compared to siCtrl treated cells, demonstrating selectivity optCPE. Data are represented as mean values ± S.D. (n = 2), expressed as mean percentage of siCldn3/4 treated cells. (ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).