γh2afx

Immunohistochemistry was performed on 4 μ-sections of paraffin-fixed embedded tissue. Antigen retrieval was performed with citrate buffer (pH 6) using a Microwave Tender Cooker (Nordic Ware) and a 700 W microwave (15 min 700 W/15 min 350 W). Endogenous peroxidase activity was inhibited with 0.3% H₂O₂ in methanol (25 min) and after that the sections were permeabilized and blocked simultaneously with PBS + 1%BSA + 0,1% TX-100, during 30 min. The slides were then washed once with PBS for 5 min. Endogenous biotin, biotin receptors and avidin binding sites were blocked by using the avidin/biotin blocking kit from Vector Laboratories (cat.#SP-2001). The slides were washed with PBS and incubated ON, in a humidity chamber, with the primary antibody diluted in blocking solution (PBS + 1%BSA). Incubations with the secondary biotinylated antibodies and development were performed with the kits MP-7402 (ImmPRESS) and SK-4605 (ImmPACT VIP HRP substrate) from Vector Laboratories, following their instructions. Methyl green (cat.#M8884-5G; Sigma-Aldrich) was used as counterstaining and xylene substitute mountant (cat.#1,900,231; Thermofisher) as mounting medium. Primary antibody used was γH2AFX (1:1000, cat.#05–636 Millipore). Damaged cells were counted from representative immunohistochemical fields for each experimental condition.

Cell monolayers were washed with ice-cold PBS and lysed in triple-detergent lysis buffer [50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 100 μg/ml PMSF, 2 μg/ml pepstatin, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and phosphatase inhibitors cocktail 2 or 3 (cat.#P5726, P0044, Sigma-Aldrich)]. SDS-PAGE and immunoblotting were performed under standard conditions. Basically, samples in Laemmli buffer (30 μg/lane) were separated through 8% or 12% gels and transferred to nitrocellulose membranes for 90 min at RT in the presence of 20% methanol and 0,1% SDS. Membranes were blocked with 3% BSA in PBS-Tween 0,05% (PBST-BSA) and incubated O/N at 4 °C with specific antibodies diluted in PBST-BSA. Antibodies used were: γH2AFX (cat.#05–636, Millipore), CDKN1A (cat.#sc-6246, Santa Cruz Biotech), CDKN2A (cat.#04–239, Millipore), CDKN2A (Millipore, cat.#PA5–20379), ACTA2 (cat.#MA5–11547, Thermoscientific), VIMENTIN (cat.#MA1–10459, Thermoscientific), Vitamin D receptor (VDR) (cat.#12550S, Cell Signaling), Tata box-binding protein (TBP) (cat.#818, abcam), tubulin (TUBA1A) (cat.#T9026, Sigma-Aldrich) and actin β (ACTB) (cat.#8224, abcam).