Tcs sp2 aobs uv

Cells were seeded onto glass coverslips in media with 1000 mg/L of glucose, fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 5 min. The coverslips were incubated with blocking solution (PBS + 0.1% Triton X-100 + 3% BSA) for 1 h and then incubated for 6–7 h with anti-MYBBP1A antibody (1:50, Abcam #ab89121). The coverslips were washed with PBS + 0.1% Triton X-100 and incubated for 6-7 h with the second antibody, anti-c-MYB (1:100, Abcam #ab117635) or anti-UBF (1:50, Novus biological #NBP1-82545, Centennial, CO, USA). Secondary antibodies anti-mouse Alexa Fluor 546 (1:250, Thermo Fisher #A21123, Eugene, OR, USA) and anti-rabbit Alexa Fluor 488 (1:250, Thermo Fisher #A11008) were used. The nuclei were stained with DAPI, and the coverslips were mounted with Prolong Gold Antifade (Life Technologies, Carlsbad, CA, USA). A confocal ultraspectral microscope (Leica TCS-SP2-AOBS-UV, Wetzlar, Germany) that allowed sequential scanning of emission channels was used for image detection.

Cells were seeded on glass cover slips and cultured for 16 h. Then, 10 μM palbociclib was added. After 48 h, cover slips were fixed in 4% paraformaldehyde for 5 min at room temperature, washed twice with PBS, permeabilized in 0.5% Triton X-100 in PBS for 5 min and washed twice more with PBS. The samples were incubated in blocking solution (PBS containing 3% bovine serum albumin) at 37°C for 1 hour, followed by incubation for 2 hours at room temperature in an anti-53BP1 antibody (Novus Biologicals, NB100-304) diluted 1:100. After washing with PBS, the cells were incubated in a species-specific Alexa 488-conjugated secondary antibody diluted 1:500 in blocking buffer for 1 hour at room temperature in the dark. The nuclei were counterstained with DAPI, and the slides were mounted using Prolong Gold Antifade reagent (Life Technologies). The samples were visualized under a confocal ultra-spectral microscope (Leica TCS-SP2-AOBS-UV) via sequential scanning of the emission channels. The mean fluorescence intensity was measured for a minimum of 300 cells per condition using Leica confocal imaging software. The plotted values represent the means (±SD) of each condition. Statistical significance was calculated using Student's t-test.