Isis v5

Immunodetection of γH2AX was performed on the same samples previously analyzed for TUNEL or sorted after Annexin-V/PI staining. Pericentrin detection was performed on newly obtained samples of irradiated lymphoblasts allowed to attach onto poly-L-lysine slides. Cells were fixed for 15 min in 4% paraformaldehyde and permeabilized in 1xPBS-0.5% Triton-X100 solution for 15 min. After 30 minutes of blocking with 0.1% Tween20 and 5% FBS, mouse anti-γH2AX (Ser139) (Upstate/Millipore, MA, USA) or rabbit anti-pericentrin (Abcam, UK) was applied at a 1 : 1000 concentration and allowed to incubate for 1 hour at room temperature. Anti-mouse Cy3 (Amersham Biosciences/GE Healthcare, NJ, USA) and anti-rabbit A488 (Invitrogen/Molecular Probes, OR, USA) secondary antibodies were applied at 1 : 1000 final concentration for 45 minutes at room temperature, followed by extensive washing. Before analysis, Vectashield Mounting Medium for fluorescence (Vector Laboratories Inc., CA, USA) supplemented with DAPI was applied. Slides were analyzed using an Olympus BX41TF epifluorescence microscope equipped with an Olympus U-TVIX digital camera using the Isis v5.4.9 software (MetaSystems, Germany).

The TUNEL assay was performed following the manufacturer's instructions (In Situ Cell Death Detection Kit, Fluorescein, Roche, Switzerland). Briefly, lymphoblasts were centrifuged, washed with 1xPBS, and dropped on poly-L-lysine coated slides. Cells were then fixed with 2% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton-X100 and 0.1% sodium citrate in 1xPBS for 5 min in ice. The TUNEL mix was applied to the cells following the manufacturer's instructions and allowed to be incubated at 37°C for 40 minutes. Before analysis, Vectashield Mounting Medium for fluorescence (Vector Laboratories Inc., CA, USA) supplemented with 4′,6-diamino-2-phenylindole (DAPI) was applied. TUNEL analysis was performed with an Olympus BX41TF epifluorescence microscope equipped with an Olympus U-TVIX digital camera using the Isis v5.4.9 software (MetaSystems, Germany).

Additional verification that the primary keratinocyte cultures EK1547 and EK100 were of pure equine origin and not mixed with other cell lines was done by FISH. We used horse bacterial artificial chromosome (BAC) clones (CHORI-241: http://bacpac.chori.org/equine241.htm) containing select chromosome-specific markers (Table 2). BAC DNA was isolated by Plasmid Midiprep kit (Qiagen), labeled with biotin-16-dUTP or digoxigenin-11-dUTP using Biotin- or DIG-Nick Translation Mix (Roche, Basel, Switzerland), and hybridized to metaphase chromosomes. Hybridizations and signal detection were carried out according to standard protocol [25 (link)]. The results were examined with Zeiss Axioplan2 fluorescence microscope and at least ten images were captured and analyzed for each experiment using Isis V5.2 (MetaSystems GmbH) software.Table 2

Horse chromosome specific BAC clones used for FISH

BAC ID	Horse chromosome	Known gene content	Reference	Label and detection
049H16	9	LCORL	Staiger et al. 2016 [35 (link)]	Biotin-FITC
076H13	29	CREM	Ghosh et al. 2014 [36 (link)]	Digoxigenin-Rhodamine