Figure S1.
Characterization of WDR62 knock-in and knockout cell lines.(A–C) Characterization of WD62 knock-in HeLa cell line. (A) Schematic illustration of primer sets and the expected PCR products. PCR genotyping (B) and Western blotting analysis (C) showing that the WDR62-GFP-Strep knock-in HeLa cell line used in this study is heterozygous. BSD, blasticidin S deaminase. (D) Western blotting with the indicated antibodies in control, katanin p80 knockout (KO), and WDR62 knockout HeLa cells. (E) Sequencing results of WDR62 knockout HeLa cell line used in this study. 1 nucleotide (nt) insertion will result in p.V65VfsX13. sgRNA, single-guide RNA. (F) Frequency of multipolarity in control or the indicated knockout HeLa cells in prometaphase or metaphase. Control, n = 224 cells; p80 knockout, n = 189; WDR62 knockout, n = 211. (G–I) Quantification of mitotic index (G), NuMA total intensity (H), and γ-tubulin total intensity (I) in control, p80 knockout, and WDR62 knockout HeLa cells. For mitotic index, n = 3 experiments, control, 1,363 cells; p80 knockout, 1,708 cells; WDR62 knockout, 1,385 cells. For NuMA intensity, n = 70 spindle poles in all conditions. For γ-tubulin intensity, n = 74 centrosomes in all conditions. (J) Western blotting with the indicated antibodies in control, katanin p80 knockout, and WDR62 knockout U2OS stable cell lines expressing PA-GFP–α-tubulin. Both knockouts are from a mixed population of cells after transient transfection with PX459 bearing single guide RNA) followed by drug selection, rather than from single cell cloning. (K and M) Immunofluorescence staining for KIF2A, γ-tubulin, and DAPI (K) and quantification of KIF2A intensity at spindle poles (M) in control and WDR62 knockout HeLa cells. n = 50 spindles poles for both conditions. (L and N) Immunofluorescence staining for γ-tubulin and DAPI (L) and quantification of KIF2C intensity at centrosomes (N) in control and WDR62 knockout KIF2C-GFP-Strep knock-in HeLa cells. The WDR62 knockout is from a mixed population of cells after transient transfection with PX459 bearing single guide RNA followed by drug selection, rather than from single cell cloning. n = 50 spindles poles for both conditions. (O and P) Western blotting (O) and PCR genotyping (P) showing that the KIF2C-GFP-Strep knock-in HeLa cell line used in L is homozygous. Red asterisk denotes a nonspecific band detected by the KIF2C antibody. (Q) Western blotting with the indicated antibodies in control and WDR62 knockout KIF2C-GFP-Strep knock-in HeLa cells. The WDR62 knockout is from a mixed population of cells after transient transfection with PX459 bearing single guide RNA followed by drug selection, rather than from single cell cloning. Scale bars, 2 µm. Data represent mean ± SD.

Characterization of WDR62 knock-in and knockout cell lines. (A–C) Characterization of WD62 knock-in HeLa cell line. (A) Schematic illustration of primer sets and the expected PCR products. PCR genotyping (B) and Western blotting analysis (C) showing that the WDR62-GFP-Strep knock-in HeLa cell line used in this study is heterozygous. BSD, blasticidin S deaminase. (D) Western blotting with the indicated antibodies in control, katanin p80 knockout (KO), and WDR62 knockout HeLa cells. (E) Sequencing results of WDR62 knockout HeLa cell line used in this study. 1 nucleotide (nt) insertion will result in p.V65VfsX13. sgRNA, single-guide RNA. (F) Frequency of multipolarity in control or the indicated knockout HeLa cells in prometaphase or metaphase. Control, n = 224 cells; p80 knockout, n = 189; WDR62 knockout, n = 211. (G–I) Quantification of mitotic index (G), NuMA total intensity (H), and γ-tubulin total intensity (I) in control, p80 knockout, and WDR62 knockout HeLa cells. For mitotic index, n = 3 experiments, control, 1,363 cells; p80 knockout, 1,708 cells; WDR62 knockout, 1,385 cells. For NuMA intensity, n = 70 spindle poles in all conditions. For γ-tubulin intensity, n = 74 centrosomes in all conditions. (J) Western blotting with the indicated antibodies in control, katanin p80 knockout, and WDR62 knockout U2OS stable cell lines expressing PA-GFP–α-tubulin. Both knockouts are from a mixed population of cells after transient transfection with PX459 bearing single guide RNA) followed by drug selection, rather than from single cell cloning. (K and M) Immunofluorescence staining for KIF2A, γ-tubulin, and DAPI (K) and quantification of KIF2A intensity at spindle poles (M) in control and WDR62 knockout HeLa cells. n = 50 spindles poles for both conditions. (L and N) Immunofluorescence staining for γ-tubulin and DAPI (L) and quantification of KIF2C intensity at centrosomes (N) in control and WDR62 knockout KIF2C-GFP-Strep knock-in HeLa cells. The WDR62 knockout is from a mixed population of cells after transient transfection with PX459 bearing single guide RNA followed by drug selection, rather than from single cell cloning. n = 50 spindles poles for both conditions. (O and P) Western blotting (O) and PCR genotyping (P) showing that the KIF2C-GFP-Strep knock-in HeLa cell line used in L is homozygous. Red asterisk denotes a nonspecific band detected by the KIF2C antibody. (Q) Western blotting with the indicated antibodies in control and WDR62 knockout KIF2C-GFP-Strep knock-in HeLa cells. The WDR62 knockout is from a mixed population of cells after transient transfection with PX459 bearing single guide RNA followed by drug selection, rather than from single cell cloning. Scale bars, 2 µm. Data represent mean ± SD.

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