Fig. 6. RNAi-mediated depletion of diaphanous increases the motility of mitochondria in BG2-C2 cells. Cell lines with fluorescently labeled mitochondria, peroxisomes and lysosomes were incubated in culture medium containing 10 µg per ml of dsRNA complementary to diaphanous mRNA. The content of target protein was estimated before treatment and after 3 and 7 days of incubation. The organelle motility in these cells was analyzed after 7 days of incubation. (A) Western blot analysis of diaphanous before and after incubation of cells with dsRNA for 3 and 7 days, respectively. Left panel shows results of serial dilutions of control cell extract used for calibration of the western blot. KHC, kinesin heavy chain. (B) Fluorescent image of cells with labeled mitochondria after 7 days of incubation with dsRNA. Bars, 10 µm. (C) Quantification of mitochondrial motility in cells after RNAi. The control cells were treated the same way as those exposed to RNAi with the only exception that dsRNA was omitted. Values are the mean percentage of movements exceeding 0.2 µm/second from all movements ± s.e.m.; P<0.05; n = number of cells and, in brackets, number of organelle movements. (D) The motility of lysosomes and peroxisomes labeled with EGFP-tagged probes (see Materials and Methods) was analyzed as described for mitochondria. Average travel distances and velocities were measured as described in the Materials and Methods, and fast movements were determined as in Fig. 2. Values are the mean percentage ± s.e.m.; P<0.05; n, number of cells and, in brackets, number of organelle movements.