Figure 2.

Fluorescence analysis of splicing product in the FMv2 transfected cells. (A) Fluorescence images of FMv2 transfected HeLa cells. The FMv2 transfected HeLa cells were scanned for fluorescence by a fluorescence microscope. Representative fluorescence images of HeLa cells are shown. Both red (mCherry) and green (eGFP) signals were clearly detected (top and middle, respectively). The merged figure of red- and green images (merge) produced yellow color (bottom). Scale bars: 100 µm. (B) Histograms of fluorescence positive cells. By scanning three wells for each fluorescence, the fluorescence intensity of each cell was obtained. Then, the fluorescence strength (FS) was calculated by the following formula; FS = cell fluorescence intensity (luminance)/cell area (µm²). Histograms of FS of red and green fluorescence were constructed in three independent experiments with FS separated into 0.1 FS/bin and are shown. (C) Splicing efficiency of FMv2. All histograms of red- and green-positive cells were accumulated, and the accumulated histograms are shown. The number of red- and green-positive cells was 2168 and 2096, respectively. The exon skipping index was calculated to be 96.7. (D) RT-PCR analysis of splicing product. Splicing product of FMv2 transfected HeLa cells was analyzed by RT-PCR amplification. The electropherogram of the amplified product is shown. One band was visualized at the position corresponding to 1533 bp (FMv2). Whilst no band was visible in the lane of non-transfected cells (NT). (E) FMv2 was transfected into HeLa cells and fluorescence was analyzed at one-hour interval for 24 hrs. The time course of numbers of cell with red-(mCherry) or green-(eGFP) positive signal is shown. Signal-positive cells started to appear at 4 h.