WDR62 loss increases spindle microtubule stability. (A) Immunofluorescence images of cold-treated metaphase RPE1 cells, stained with α-tubulin antibodies and DAPI. Cells were categorized into three representative classes based on k-fiber integrity in 3D. (B and C) Quantification of cold-stable assay in metaphase RPE1 cells treated with siCTRL or siWDR62 (B) or metaphase HeLa WDR62^+/+ or WDR62^−/− cells (C); stack bars indicate mean percentages of class 1, 2, and 3 spindles as depicted in A: N = 3 independent experiments, n = 184–188 cells per condition (B) or n = 207–215 cells (C); error bars represent mean ± SEM; ****, P < 0.0001, χ² test. (D) Immunofluorescence images of RPE1 cells expressing eGFP or WDR62-eGFP treated with siCTRL or siWDR62 and stained with pericentrin antibodies and DAPI. (E) Quantification of cold-stable assay in RPE1 eGFP or WDR62-eGFP metaphase cells treated with siCTRL or siWDR62 as in B and C: N = 3, n = 183–206 cells; ****, P < 0.0001, χ² test. (F) Time-lapse sequences of MG132-arrested RPE1 metaphase cells treated with siCTRL or siWDR62, labeled with SiR-tubulin, and treated with 200 ng/ml nocodazole at t = 0. (G) Quantification of the spindle microtubule decay over time in RPE1 metaphase cells treated with siCTRL or siWDR62: N = 3, n = 55–58 cells; thick lines represent mean; thin lines represent single experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001, repeated two-tailed paired t test. (H) Immunofluorescence images of metaphase HeLa cells stained with centrin-1 antibodies and DAPI. Cells were categorized according to centriole numbers. (I and J) Quantification of centriole numbers in metaphase WDR62^+/+ or WDR62^−/− HeLa cells (I) or RPE1 cells treated with siCTRL or siWDR62 (J): stack bars indicate mean percentage of cells with corresponding centrioles numbers; error bars represent mean ± SEM; N = 1, n = 200 cells; χ² test. Scale bars = 5 µm; 2.5 µm (inset).