Fig. 4. shRNA-mediated depletion of ChlR1 results in mitotic delay. (A) RT-PCR results with ChlR1-specific (top panel) or actin-specific (lower panel) primers from DNase-treated RNA purified from RPE1-TetR-ChlR1 cells with shRNA expression uninduced or induced at 48 and 72 hours with 2 µg/ml doxycycline. (B) ChlR1 (upper panel) and -tubulin (lower panel) protein levels in RPE1-TetR-EGFP cells or RPE1-TetR-ChlR1 cells with uninduced and induced shRNA for 24 hours with 2 µg/ml doxycycline. (C) RPE1-TetR-ChlR1 cells were grown on coverslips and ChlR1 shRNA was uninduced or induced for 24 hours with 2 µg/ml doxycycline before cells were pre-extracted with 0.1% Triton X-100 and fixed in methanol. Mitotic cells were stained for -tubulin (red) by indirect immunofluorescence and for DNA (blue) using Hoechst no. 33568. More than100 cells were counted per experiment and the mean percentage of mitotic cells for three independent experiements determined and expressed as the mitotic index (± s.d.). (D) More than 50 mitotic cells per experiment were assessed for specific mitotic stage and the percentage of total mitoses for three independent experiments calculated (± s.d.). An increase in pro-metaphase cells with concomitant decrease in other mitotic stages was observed following depletion of ChlR1. Shown is the mean and standard deviation of at least three independent experiments. (E) Representative confocal images of RPE1-TetR-ChlR1 cells with ChlR1 shRNA induced for 24 hours; -tubulin was stained red and DNA stained blue. Bar, 8 µm.