Figure 5

Presence of CPN and siGLO Red small RNAs in protoplasts. (A) Dot plots of BY-2 protoplasts cultured with medium only, 200 nM siGLO Red, 10 μM CPNs, 10 μM CPNs + 200 nM siGLO Red, 25 μM CPNs, or 25 μM CPNs+200 nM siGLO Red. CPN fluorescence is detected with FITC filter (x axis) and siGLO Red fluorescence (y axis) is detected with PE filter in protoplasts cultured for 24 h. Each dot represents a single event with emissions frequency that is the combination of the fluorophores. The gated population in the lower left quadrant represents the majority of nonfluorescent cells. The upper right quadrant represents the majority of events that contain both green and red fluorescence due to CPNs and siGLO Red small RNAs. The upper left quadrant represent events that are positive only for siGLO Red small RNAs and the lower right quadrant represent events that are positive for only CPNs. Highly fluorescent protoplasts are located furthest along the x- and y- axes. (B) Bar graph reports the average of 10 replicate experiments using FACs to record the number of green fluorescent events inside protoplasts, as an indication of the internalization of CPNs (lower right quadrant of dot plots). Samples were treated with 0, 10, or 25 μM CPNs and then incubated for 2 and 24 h. Between 18-37% of protoplasts produce positive events via cytometric analyses. (C) Bar graph reports the average and stand deviations of 10 replicate experiments, recording the number of events reporting internalization of both CPNs and siGLO Red (upper right quadrant of the dot plots). Between 27 and 42% of recorded events are positive for both CPNs and siGLO Red RNAs when they are co-delivered to protoplasts.