Figure 1.

Effect and Toxicity of PPMOs and OECs in Cell Culture. (A–D) PMO, PPMO and OEC dose responses. (A) HeLaEGFP654 cells were incubated overnight with various concentrations of splice correcting PPMO654 or with a control PPMO having a 5 base mismatch (PPMO-MM). The cells were rinsed and further incubated and then processed for EGFP fluorescence and viability as described in Methods. (B) Cells were incubated with 0.5μM PPMO654, PPMO-MM or PMO654 and further incubated with various concentrations of OEC UNC7938. After rinsing and further incubation, EGFP fluorescence and viability were determined. (C) The concentration of PPMO654 was varied plus or minus subsequent treatment with 10 μM OEC UNC2383. (D) Subsequent to treatment with 0.5 μM PPMO654, cells were treated with various concentrations of OEC UNC2383. Data in A-D are expressed as mean ± SD, n = 4 (error bars fall within symbols). (E) PPMO intracellular distribution. HeLaEGFP654 cells were incubated with lissamine-PPMO654 overnight followed by vehicle or 10 μM OEC UNC7938 for 1 h and then cells were observed by microscopy. The upper images are phase contrast while the lower images are superposition of fluorescence from lissamine-PPMO (red) and Hoechst nuclear stain (blue). Lissamine-PPMO654 accumulated in endosomal compartments but after OEC treatment the PPMO re-localized into the nuclei, bar = 10 μm.