A21445

Cells grown on glass slides or coverslips were fixed with PTEMF (0.2% Triton X-100, 0.02 M PIPES (pH 6.8), 0.01 M EGTA, 1 mM MgCl₂, 4% formaldehyde). After blocking with 3% BSA, cells were incubated with primary antibodies according to suppliers’ instructions: CREST (Antibodies Incorporated, 15-234-0001), γH2aX (Millipore, 05-636), RPA70 (Abcam, ab79398). Secondary antibodies used were goat anti-mouse AlexaFluor 488 (A11017, Invitrogen), goat anti-rabbit AF594, AF488 (A11012, A11008, Invitrogen), and goat anti-human AF647 (109-606-088-JIR, Stratech or A21445, Invitrogen). DNA was stained with DAPI (Roche) and coverslips mounted in Vectashield (Vector H-1000, Vector Laboratories). EdU incorporation and staining was achieved using the Click-It kit (Life Technologies), following the manufacturer’s instructions.

Cells grown on glass slides or coverslips were fixed with PTEMF (0.2% Triton X-100, 0.02 M PIPES [pH 6.8], 0.01 M EGTA, 1 mM MgCl₂, and 4% formaldehyde). After blocking with 3% BSA, cells were incubated with primary antibodies according to suppliers’ instructions: beta-tubulin (ab6046; Abcam), Centrin 3 (ab54531; Abcam), CREST (15-234-0001; Antibodies Incorporated), and CENP-E (ab5093; Abcam). Secondary antibodies used were goat anti-mouse Alexa Fluor 488 (A11017; Invitrogen), goat anti-rabbit AF594 and AF488 (A11012 and A11008; Invitrogen), and goat anti-human AF647 (109-606-088-JIR [Stratech] or A21445 [Invitrogen]). DNA was stained with DAPI (Roche), and coverslips were mounted in Vectashield (Vector H-1000; Vector Laboratories).

Cells were fixed in cold methanol for 2 minutes at -20°C. Cells were washed in TBST (0.05% Triton-X 100 in TBS) and blocked with 2% BSA in TBST. Primary and secondary antibodies were diluted in TBST + 2% BSA and incubated for one hour at room temperature (primary antibodies) or 50 minutes at room temperature (secondary antibodies). DNA was labeled with 1 μg/ml Hoechst 33342 prior to mounting on slides with ProLong Gold Antifade Mountant (Thermo Fisher P36934). The following primary antibodies were used: mouse anti-α-tubulin DM1α conjugated to Alexa Fluor 488 (1:100, Cell Signaling Technologies 8058S), mouse anti-centrin clone 20H5 (1:200, Sigma-Aldrich 04–1624), and human anti-centromere protein CREST antibody (1:25, Antibodies Incorporated 15–234). Normal mouse IgG (1:100, Santa Cruz Biotechnology sc-2025) was used as a block before incubating in pre-conjugated mouse anti-α-tubulin DM1α Alexa Fluor 488. The following secondary antibodies were used: goat anti-mouse conjugated to Alexa Fluor 488 and 568 (1:400, Invitrogen A11001 and A11004) and goat anti-human conjugated to Alexa Fluor 647 (1:400, Invitrogen A21445).

Primary antibodies: γH2A.X (Millipore, Merck 05–636, 1:1000), γH2A.X (Cell Signalling Technology, #9718, 1:1000), human CREST serum (AntibodiesInc, SKU 15–234, 1:800), R-loops (S9.6, Absolute Antibody, Ab01137-23.0, 1:400), TRF2 (Abcam, ab13579, 1:500), BRCA1 (Santa-Cruz Biotech, sc-6954 (D-9), 1:250), 53BP1 (Merck, MAB3802, 1:1000), 53BP1 (Abcam,ab36823, 1:1500), DAXX (Cell Signaling Technology, #4533, 1:25), a-tubulin (Proteintech, 66031-1-Ig, 1:750), ATRX (Abcam, ab97508, 1:500). Secondary antibodies: anti-mouse AlexaFluor 488 (Invitrogen, A11001, 1:500), anti-mouse AlexaFluor 555 (Invitrogen, A21422, 1:500), anti-mouse AlexaFluor 647 (Invitrogen, A-21235, 1:500), anti-rabbit AlexaFluor 488 (Invitrogen, A-11008, 1:500), anti-rabbit AlexaFluor 555 (Invitrogen, A21428, 1:500), anti-rabbit AlexaFluor 647 (Invitrogen, A21244, 1:500), anti-human AlexaFluor 647 (Invitrogen, A-21445, 1:500).

Mouse tumors were frozen in OCT (TissueTek) and frozen sections cut. HEK293T cells were grown on top of poly-l-lysine–coated cover slides (MilliporeSigma) and transfected using a human or murine FSHR expression vector. Slides were then fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Sections were blocked using 5% normal goat serum followed by staining with D2AP11 antibody, then AF647-conjugated secondary antibodies specific for human (A-21445, Invitrogen) or mouse (A-21235, Invitrogen) IgG. Slides were viewed using a Leica TCS SP-5 confocal microscope and Leica LAS-X software (immunocytochemistry) or a Nikon ECLIPSE 80i microscope and NIS-Element Imaging (immunohistochemistry). For immunohistochemistry analysis of tissue microarray (TMA) slides (US Biomax), they were deparaffinized and rehydrated, followed by antigen retrieval, blocking with 5% normal goat serum, and staining with D2AP11 antibody and then biotinylated anti-mouse secondary antibody (BA-9200, Vector Laboratories). Then the TMA slides were incubated with peroxidase solution (Vector Laboratories) followed by DAB substrate and counterstained with hematoxylin (Leica). The slides were viewed and imaged using a Nikon NIS-Element Imaging system (×20, scale: 500 μm).

Primary antibodies used: mouse anti-AIM-1 (1:100; 611082, BD Biosciences, Dublin, Ireland), rabbit anti-pSer7CENP-A (1:200; 04-792, Millipore, Cork, Ireland), anti-centromere (1:100; 15-234, Antibodies Incorporated, CA, USA), mouse anti-PLK1 (1:200 for IF; ab17057, Abcam, Cambridge, UK), mouse anti-PLK1 (1:500 for WB; sc-56948, Santa Cruz, Heidelberg, Germany), rabbit anti-Histone H3 pSer10 (1:50; 06-570, Millipore), rabbit anti-GAPDH (1:1000; 14C10 Cell Signaling, MA, USA), mouse anti-HEC1 (1:500 for IF, 1:1000 for WB; clone 9G2.23, Genetex, Eching, Germany), rabbit anti-ZWINT-1 (1:100 for IF, 1:1000 for WB; IHC-00095, Bethyl, TX, USA), mouse anti-pT210-PLK1 (1:300 for IF, 1:1000 for WB; 39068, Abcam), pT3H3 (1:400 for IF 1:1000 for WB; 9714S, Cell Signaling), pMPM2 (1:1000; 05-368, Millipore), anti-mouse Cyclin B (1:1000; SC-245, Santa Cruz). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse (1:300; A11001, Life Technologies, Dublin, Ireland), Alexa Fluor 546 goat anti-rabbit (1:300; A11010, Life Technologies), Alexa Fluor 647 goat anti-human (1:300; A21445, Life Technologies), goat anti-rabbit Alexa Fluor 488 (1:50 for FACS; A11008, Life Technologies), IRDye^® antibodies (1:10,000; LI-COR Biosciences, NE, USA).