Figure 4.

Functional discovery based on genetic interactions. (A) Top panels shows the western blot of total cell lysates expressing both wild-type (TTC31-V5) and shRNA-resistant (TTC31-shR-V5) TTC31 protein tagged with V5 in PTTG1^−/− and PTTG1^+/+ cell lines. The bottom panel shows the effect of two independent shRNAs in TTC31-V5 and TTC31-shR-V5. TTC31 was knocked down by infecting HCT116 cells with two independent hairpins targeting TTC31, followed by a western blot with anti-V5 antibody to confirm on-target knockdown. (B) Rescue experiment showing the on-target effect of sh2-TTC31 as evaluated by the percentage inhibition in growth WT-TTC31-expressing cells relative to shR-TTC31-expressing cells. (C) Immunofluorescence microscopy images of PTTG1^+/+ cells expressing control shLacZ (top) or sh2-TTC31 (bottom). Centrosomes are indicated by pericentrin (red) and NEDD1 (green) staining, and the nucleus was detected with DAPI (blue). Arrowheads indicate lagging chromosomes. (D and E) Quantification of cells with abnormal spindle morphology and cells with congression defects was performed by analyzing at least 100 cells (n=3). **P-value<0.01 calculated using χ²-test. Scale bar, 4 μm. (F) Time-lapse imaging of HeLa cells stably expressing the centrosome marker NEDD1-GFP. Cells were either infected with the negative control shRNA targeting LacZ or with two independent shRNAs against TTC31 (sh1- or sh2-TTC31). Frames taken at the indicated time points (h:min) relative to entry into mitosis are shown. Scale bar, 10 μm. (G) Localization of centrin structures in PTTG1^+/+ cells expressing shLacZ control or one of two independent shRNAs against TTC31 (sh1- or sh2-TTC31). DNA and centrosomes were labeled as in C. Representative prometaphase, metaphase and anaphase cells with supernumerary centriole phenotypes are shown. Insets are fourfold magnifications of centrosomal regions. Scale bar, 10 μm. (H) Top panels shows the western blot of total cell lysates expressing two isoforms of ZC3H13 in PTTG1^+/+ cells. ZC3H13 was knocked down by infecting HCT116 cells with two independent hairpins (sh1- and sh2-ZC3H13) and followed by western blot with an anti-ZC3H13 antibody (Abcam). The bottom panel shows the negative genetic interaction of two independent shRNAs as assessed by proliferation. (I) Western blot analysis of whole-cell lysates showing the cell cycle-dependant regulation of ZC3H13 in PTTG1^−/− and PTTG1^+/+ cell lines. The first four lanes represent the time after release from double thymidine block and the last two lanes from thymidine-nocodazole block. The levels of different cyclins fluctuate across different phases of cell cycle and are used as controls. (J) Immunofluorescence microscopy images of PTTG1^+/+ cells expressing a negative control shRNA, shRFP (top), or sh2-ZC3H13 (bottom). NEDD1 antibody (green) was used to stain for centrosomes and phosphohistone H3 antibody (red) to stain for mitotic cells. Nuclei were stained with Hoechst stain (blue). The distances between the poles are shown for representative cells. (K) Quantitation of chromosome congression defects in control as well as cells following ZC3H13 knockdown from two independent experiments. A minimum of 30 cells were counted in each case. *P<0.01, Student’s t-test. (L) Immunofluorescence microscopy of spindle checkpoint proteins CENP-E and BUBR1 in PTTG1^+/+ cells expressing the control shRFP (top) or sh2-ZC3H13 (bottom). Cells were stained with BUBR1 antibody (green) and CENP-E antibodies (red). Nuclei were stained with Hoechst stain (blue). (M and N) Quantitation of localization defect of spindle checkpoint proteins, measured as the sum of mean fluorescence intensity of BUBR1 or CENP-E in each cell. The aggregate results from two independently infected cell populations are shown with either shControl, sh1-ZC3H13 or sh2-ZC3H13. Thirty to 50 cells were counted in each case. **P<0.001, **P<0.01 by Student’s t-test.