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. 2020 Jun 4;133(11):jcs240838. doi: 10.1242/jcs.240838

Fig. 4.

Fig. 4.

poc5Δ cells are viable and display overproduced BBs but fewer cilia. (A) Schematic outlining the homologous recombination strategy for generating a complete micronuclear knockout of TtPOC5, where the TtPOC5 ORF is replaced with a codon-optimized NEO2 (coNEO2) cassette for drug selection. The red arrowheads indicate the primers used to amplify PCR products specific to WT POC5 and coNEO2. (B) PCR confirming success of the knockout strategy, with only wild-type (WT) cells containing the TtPOC5 ORF and only poc5Δ cells containing the coNEO2 cassette. Sizes of select marker (M) bands are displayed in kilobase pairs. (C) Growth curves for WT and poc5Δ cells grown at either 30°C or 37°C for 8 h in SPP medium. Cell density (cells/ml) measurements are gathered at the initiation of the experiment (cultures starting at 0.5×105 cells/ml), and 4 and 8 h after initiation. Results are mean±s.d. for n=3 analyzed samples per strain and incubation temperature. (D) Representative WT and poc5Δ cells stained with an antibody against Cen1 (labels BBs) after incubation at either room temperature (RT), 30°C or 37°C. Loss of TtPoc5 does not disrupt the organization/orientation of cortical row BBs. The bottom panel shows a representative poc5Δ rescue (Rescue) cell at 30°C with no CdCl2 induction (N.I.) due to ‘leakiness’ of the MTT1 promoter, where TtPOC5 is reincorporated in poc5Δ cells through transformed MTT1pr-GFP-Poc5. Scale bars: 10 µm. (E) BB density quantification (average BBs/10 µm) for WT and poc5Δ cells at RT, 30°C and 37°C. Significant BB overproduction is observed in poc5Δ cells at all tested growth temperatures and is rescued with MTT1pr-GFP–Poc5 to near WT levels at 30°C. Results are mean±s.e.m. for n=300 total counts (five counts per cell across 20 cells, in triplicate) per condition. ***P<0.001 (Student's t-test). (F) Representative 10 µm sections of cortical rows labeled with an antibody against polyglutamylated tubulin (labels ciliary axonemes and BBs). (G) Ciliary density quantification (average number of cilia per 10 µm) for WT and poc5Δ cells at 30°C reveals significantly reduced ciliary density in poc5Δ cells that is rescued with TtPOC5 reincorporation. Results are mean±s.e.m. for n=100 total counts per condition. ***P<0.001; n.s., not significant (Student's t-test).