Studied cancer cells were fixed in ice-cold methanol (176845000; Thermo Fisher Scientific, Waltham, MA) and stained with Centrin 1 and γ-tubulin or α-tubulin antibody along with Hoechst staining. Cells were mounted with Pro-Long Gold antifade reagent (P36934; Invitrogen) and scored for chromosome rings and for the presence of multipolar anaphases using a ZEISS Axiocam 506 color microscope with
the LSM 900 Airyscan 2 Confocal system (Carl Zeiss Microscopy LLC, White Plains, NY). Primary antibodies recognized: Centrin 1 (12794–1-AP, 1:1000, Proteintech, Rosemont, IL), α-tubulin (T6199; 1:1000, Sigma-Aldrich, St. Louis, MO) or γ-tubulin (T5326; 1:1000, Sigma-Aldrich, St. Louis, MO), respectively. Secondary antibodies were Texas red anti-murine IgG (H+L) (TI-2000; 1:1000, Vector Laboratories, Newark, CA) and goat anti-rabbit IgG (H+L) Cross-Adsorbed Alexa Fluor™ 488 (A11008;1:1000, Thermo Fisher Scientific, Waltham, MA), respectively. Hoechst 33,342 (62249, Thermo Fisher Scientific, 1:10000) stained for DNA. Pro-Long Gold anti-fade reagent preserved immunofluorescence. Each assay was performed in triplicate. Independent triplicate biologic replicate experiments were done.
the LSM 900 Airyscan 2 Confocal system (Carl Zeiss Microscopy LLC, White Plains, NY). Primary antibodies recognized: Centrin 1 (12794–1-AP, 1:1000, Proteintech, Rosemont, IL), α-tubulin (T6199; 1:1000, Sigma-Aldrich, St. Louis, MO) or γ-tubulin (T5326; 1:1000, Sigma-Aldrich, St. Louis, MO), respectively. Secondary antibodies were Texas red anti-murine IgG (H+L) (TI-2000; 1:1000, Vector Laboratories, Newark, CA) and goat anti-rabbit IgG (H+L) Cross-Adsorbed Alexa Fluor™ 488 (A11008;1:1000, Thermo Fisher Scientific, Waltham, MA), respectively. Hoechst 33,342 (62249, Thermo Fisher Scientific, 1:10000) stained for DNA. Pro-Long Gold anti-fade reagent preserved immunofluorescence. Each assay was performed in triplicate. Independent triplicate biologic replicate experiments were done.