Ab16123

Cell lysates were obtained using RIPA (20 mM Tris-HCl pH = 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). A total of 60–120 μg protein were fractionated by SDS–PAGE and transferred to PVDF membranes (GE Healthcare Life Sciences, Buckinghamshire, UK). These were blocked in 5% nonfat dry milk in TBS-T (20 mM Tris-HCl pH = 7, 5, 150 nM NaCl, 0,1% Tween20) for one hour and immunoblotted. Blots were developed using ECL Detection Reagent (GE Healthcare Life Sciences), images acquired on ImageQuant LAS 4000 (GE Healthcare Life Sciences) and analyzed using Image J 1.52p (National Institute of Health, Bethesda, MD, USA). Primary antibodies were used against HPV-18 E6 (AVC #1006, Arbor Vita Corporation, Fremont, CA, USA), p53 (sc-126, Santa Cruz, Dallas, TX, USA), pRb (ab24, abcam), p16INK4a (ab16123, abcam, Cambridge, UK), p21 (ab7960, abcam), PCNA (ab29, abcam, Cambridge, UK) and α-tubulin (T9026, Sigma-Aldrich, St. Louis, MO, USA).

MSCs seeded on coverslips or frozen sections of the above described tissue samples were fixed with paraformaldehyde for 15 min and subsequently permeabilized with 1% Triton X-100 for 20 min. Then, the slides were blocked with 10% goat serum for 30 min and incubated with anti-p-H2A.X (1:400, Cell Signaling Technology, 9718T), anti-CD105 (1:500, Abcam, ab231774), anti-p53 (1:4,000, Cell Signaling Technology, 2524S), anti-p21 (1:250, Invitrogen, 33-7000), or anti-p16 (1: 1000, Abcam, ab16123) antibodies at 4 °C overnight. Then, the slides were stained with fluorophore-labeled secondary antibodies (1:500, Cell Signaling Technology, 4409S and 4412S), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. The slides were observed with a laser scanning confocal microscope (Nikon Eclipse Ni-E) or a fluorescence microscope according to the manufacturer’s instructions.