Figure 2.

A test of RNAi screen efficacy: identifying genes involved in cell-cycle progression. (a) Gene identity and phenotypic annotation for RNAi phenotypes identifying predicted cell-cycle regulators. The 'Profile' column provides a summary of the phenotypic profiles distinguishing sets of genes involved in specific stages of the cell cycle. The 'Classification' column gives a single predicted functional category assigned to each targeted gene on the basis of primary sequence and/or known functional data. The 'FlyBase ID' and 'Gene name' columns are information as annotated at FlyBase [12]. The 'Predicted function' column provides detail on the putative molecular function of each specific gene. 'Cell type' refers to whether the phenotype was observed in Kc₁₆₇ (Kc) and/or S2R⁺ (S2R) cells. Profile I: RNAi phenotypes resulting in an increase in cell size, uniform or disorganized microtubules, irregular cell shapes and decreased cell numbers identified genes involved in cell-cycle progression through G1 to S and G2 to M stages. Phenotypes were further distinguished on the basis of levels of F-actin accumulation and DNA morphology. Profile II: RNAi phenotypes resulting in aberrant morphology or increased frequency of microtubule-based mitotic spindles identified genes involved in mitosis. Profile III: RNAi phenotypes observed in S2R⁺ cells identified additional genes with putative roles in cell cycle/mitosis progression. (b-g) Kc₁₆₇ cells stained for F-actin (red), α-tubulin (green), DNA (blue), imaged using automated microscopy and scored visually. (b) Control. (c, d) Profile I: Dp and string RNAi resulting in big cells. (e, f) Profile II: fizzy and polo RNAi resulting in increased frequency of cells with mitotic spindles. (g) Cdk5 RNAi resulting in smaller cells and disorganized microtubules (and increased spindles in S2R⁺ cells; not shown). Scale bar, 30 μm.