Rabbit anti claudin 4

To detect γH2AX and pericentrin, HMECs were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in a 1xPBS-0.5% Triton- X100 for 20 minutes. To detect γH2AX and claudin-4, cells were fixed with ice-cold methanol for 10 minutes. Cells were incubated for 1 hour with blocking solution (1xPBS-0.1% Tween20-3% FBS) before applying primary antibodies mouse anti-γH2AX (Ser139) (clone JBW301, Millipore, Madrid, Spain), rabbit anti-pericentrin (Abcam, Cambridge, UK) or rabbit anti-claudin-4 (Abcam) at 1:1000, 1:2000 and 1:250 final concentrations respectively. Secondary antibodies anti-mouse Cy3 (Jackson ImmunoResearch Inc., Cambridge, UK) and anti-rabbit A488 (Thermo Fisher Scientific, Waltham, MA, USA) were applied at a final concentration of 1:800 and 1:500 (claudin-4) or 1:1000 (pericentrin) respectively. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) at a final concentration of 0.25 μg/ml. For image acquisition an Olympus BX61 epifluorescent microscope equipped with a CV-M4+CL camera (JAI, Grosswallstadt, Germany) and Cytovision software (Applied Imaging, Newcastle, UK) were used.

For immunofluorescent detection of protein markers under a fluorescence microscope, cells were plated into chamber slides, fixed with paraformaldehyde 4 % for 10 minutes permeabilized for 15 minutes in 1 × PBS 0.5 % Triton X100 solution, rinsed twice with 1 × PBS and, blocked in PBS 0.1 % Tween20 2 % fetal calf serum for 1 h at room temperature. Antibodies used were rabbit anti-Claudin-4 (Abcam, Cambridge, UK, Ref 1504), mouse anti-CD-10 (Abcam, Ref. 10323), rabbit anti-cytokeratin K-14 (Covance, Madrid, Spain, Cat# PRB-155P-100) and rat anti-cytokeratin K-19 (Troma III, Iowa, USA) and rabbit anti-human TERT (Rockland, Limerick, PA, USA). Staining for antibodies against Claudin-4, CD-10, cytokeratin K14 and cytokeratin K19 was evaluated under an optical epifluorescence microscope with specific filters for each of the fluorochromes used, and images were obtained using Isis Fluorescence Imaging software (MetaSystems GmbH, Altussheim, Germany). Mouse anti-CD-10 (BioLegend, San Diego, CA, USA) and mouse anti-CD-227 (BD Pharmigen, Franklin Lakes, New Jersey, USA) were detected by flow cytometry.

Proteins were extracted with CHAPS lysis buffer, quantified with NanoDrop 2000 (Thermo Fisher Scientific, Barcelona, Spain), denatured at 70 °C, separated on a 10 % SDS-PAGE gel (Novex) and transferred on a nitrocellulose membrane. Antibodies used were rabbit anti-Claudin-4 (1:1000, Abcam, Ref 1504), mouse anti-p16^INK4a (1:1000, Neomarkers, Freemont, CA, USA), mouse anti-p53 (1:1000, Santa Cruz Biotechnologies, Heidelberg, Germany), rabbit anti-phospho S15 p53 (1:1000, Invitrogen) and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Abcam) diluted on 1 × PBS-3 % BSA 0.1 % Tween20. Anti-mouse and anti-rabbit horseradish peroxidase (HRP) conjugate was used as secondary antibody (1:2000, Millipore, Madrid, Spain). Chemiluminescent detection of antibodies was performed using HRP solution and luminol (Immobilion Western kit, Millipore).