Figure S4.
Characterization of TPX2- or Aurora A–mAID-mClover knock-in cell lines.(A) Schematic representation of the strategy to construct conditional AID mutant of TPX2 or Aurora A via CRISPR/Cas9 as well as primer sets for genotyping and the expected PCR products. Tet-on 3G, Tet-On 3G transactivator; pPGK, Phosphoglycerate kinase 1 promoter; pTRE3GS, TRE3GS promoter; KI, knock-in; Puro, puromycin resistance gene; Hygro, hygromycin resistance gene. Parental cell lines were first generated by introducing 3xHA-OsTIR1 at the AAVS1 locus. Subsequently, the mAID-mClover cassette was introduced into parental cells after the last codon of TPX2 or Aurora A. (B and C) The homozygous TPX2- or Aurora A (AurA)–mAID-mClover knock-in HeLa cell lines were confirmed by Western blotting with indicated antibodies (B) and PCR genotyping (C). (D) Western blotting analysis of indicated protein levels in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells without or with doxycycline (Dox) and IAA treatment. For all conditions, cells were synchronized in mitosis with STLC. (E and F) Images showing indicated spindle defects in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells treated with doxycycline and IAA. (G) Quantification of mitotic index in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells without or with doxycycline and IAA treatment, n = 3 experiments. For TPX2-mAID-mClover, control cells, 1,113 cells; doxycycline and IAA treatment, 838 cells. For Aurora A–mAID-mClover, control, 1,126 cells; doxycycline and IAA treatment, 1,142 cells. (H and I) Percentage of cells with indicated spindle phenotypes in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells in prometaphase or metaphase without or with doxycycline and IAA treatment. For TPX2-mAID-mClover, control, n = 221 cells; doxycycline and IAA treatment, n = 244 cells. For Aurora A–mAID-mClover, control, n = 231 cells; doxycycline and IAA treatment, n = 220 cells. (J) Quantification of the length of bipolar spindle in Aurora A–mAID-mClover knock-in HeLa cells without or with doxycycline and IAA treatment. For both conditions, n = 40 cells. (K and L) Immunofluorescence staining and quantification of Aurora A intensity at spindle poles in TPX2-mAID-mClover knock-in HeLa cells without or with doxycycline and IAA treatment. For both conditions, n = 100 spindle poles. (M–P) Immunofluorescence staining and quantification of Aurora A and TPX2 intensities at spindle poles in control or the indicated knockout (KO) HeLa cells. For all conditions, n = 100 spindle poles. Scale bars, 2 µm. Data represent mean ± SD. ***, P < 0.001; two-tailed t test.

Characterization of TPX2- or Aurora A–mAID-mClover knock-in cell lines. (A) Schematic representation of the strategy to construct conditional AID mutant of TPX2 or Aurora A via CRISPR/Cas9 as well as primer sets for genotyping and the expected PCR products. Tet-on 3G, Tet-On 3G transactivator; pPGK, Phosphoglycerate kinase 1 promoter; pTRE3GS, TRE3GS promoter; KI, knock-in; Puro, puromycin resistance gene; Hygro, hygromycin resistance gene. Parental cell lines were first generated by introducing 3xHA-OsTIR1 at the AAVS1 locus. Subsequently, the mAID-mClover cassette was introduced into parental cells after the last codon of TPX2 or Aurora A. (B and C) The homozygous TPX2- or Aurora A (AurA)–mAID-mClover knock-in HeLa cell lines were confirmed by Western blotting with indicated antibodies (B) and PCR genotyping (C). (D) Western blotting analysis of indicated protein levels in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells without or with doxycycline (Dox) and IAA treatment. For all conditions, cells were synchronized in mitosis with STLC. (E and F) Images showing indicated spindle defects in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells treated with doxycycline and IAA. (G) Quantification of mitotic index in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells without or with doxycycline and IAA treatment, n = 3 experiments. For TPX2-mAID-mClover, control cells, 1,113 cells; doxycycline and IAA treatment, 838 cells. For Aurora A–mAID-mClover, control, 1,126 cells; doxycycline and IAA treatment, 1,142 cells. (H and I) Percentage of cells with indicated spindle phenotypes in TPX2- or Aurora A–mAID-mClover knock-in HeLa cells in prometaphase or metaphase without or with doxycycline and IAA treatment. For TPX2-mAID-mClover, control, n = 221 cells; doxycycline and IAA treatment, n = 244 cells. For Aurora A–mAID-mClover, control, n = 231 cells; doxycycline and IAA treatment, n = 220 cells. (J) Quantification of the length of bipolar spindle in Aurora A–mAID-mClover knock-in HeLa cells without or with doxycycline and IAA treatment. For both conditions, n = 40 cells. (K and L) Immunofluorescence staining and quantification of Aurora A intensity at spindle poles in TPX2-mAID-mClover knock-in HeLa cells without or with doxycycline and IAA treatment. For both conditions, n = 100 spindle poles. (M–P) Immunofluorescence staining and quantification of Aurora A and TPX2 intensities at spindle poles in control or the indicated knockout (KO) HeLa cells. For all conditions, n = 100 spindle poles. Scale bars, 2 µm. Data represent mean ± SD. ***, P < 0.001; two-tailed t test.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal