Mouse α tubulin

Cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature (RT) and permeabilized in methanol for 5 min at -20°C. Cells were then blocked with PBS containing 4% BSA for 30 min and incubated with the following primary antibodies diluted in the blocking solution overnight at 4°C: rabbit ß-tubulin (Abcam, Cambridge, UK ) 1:200; mouse α-tubulin (Abcam) 1:1000; mouse acetylated α-tubulin (Abcam) 1:500. Cells were then rinsed in PBS several times before incubation with secondary antibodies, goat anti rabbit conjugated with Alexafluor 633 (1:2000), goat anti mouse conjugated with Alexafluor 488 (1:2000), (Life Technologies, CA, USA) for 30 min at RT. After extensive washing, coverslips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories Burlingame, CA, USA) and then observed at a Leica TCS SP5 confocal microscope (Mannheim, Germany).

The cells were treated identically to the flow cytometry measurement, except for the p21 staining, where the cells were grown on coverslips. For β-galactosidase staining the cells were stained according to the manufacturer’s protocol (Sigma-Aldrich, Taufkirchen, Germany). In short, the washed cells were fixed, washed again, and the cells were incubated overnight in the staining solution at 37 °C. Images were acquired by an inverse Leica DMILLED (Leica, Wetzlar, Germany) microscope. To assess the expression of the p21 and α tubulin proteins an immune staining was performed. The cells were washed and the primary rabbit monoclonal antibodies p21 (Cell signaling Danvers, MA, USA, #2947, 1:200) and mouse α tubulin (Abcam, Cambridge, UK, #ab7291, 1:1000) were incubated overnight at 4 °C. Coverslips were washed and secondary green fluorescence anti-rabbit antibodies Alexa488 and red anti-mouse antibodies Alexa594 (Thermo Fisher Scientific, Waltham, MA, USA) were incubated at 37 °C for 2 h. The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield (Vector Laboratories, Peterborough, UK). The images were acquired with the Zeiss Axio Imager Z2 fluorescence microscope at 400× magnification (Zeiss, Oberkochen, Germany). Overlays were created using image processing software (Biomas Version 4.1 07/2018, MSAB, Germany).