Sonifer

At the end of the 5-day exposure to f-SWCNTs, bone marrow was excised under
anesthesia. The bone marrow was flushed in ice-cold physiological saline. A 10% homogenate
of each tissue was prepared separately in 0.05 M phosphate buffer (pH 7.4) containing 0.1
mM EDTA using a motor driven Teflon-pestle homogenizer (Fischer), followed by sonication
(Branson Sonifer), and centrifugation at 500 × g for 10 min at 4° C. The
supernatant was aspirated and centrifuged at 2000 × g for 60 min at 4° C.
The cellular fraction obtained after centrifugation was called ‘homogenate’
and used for the assays.

Human keratinocytes were treated with varying concentrations of arsenite, zinc or both compounds for 24 h. Chromatin preparation and collection protocols were followed as previously described (King et al., 2012 (link)). Following collection, chromatin suspension was sonicated on ice (90 s) in immunoprecipitation lysis buffer (0.01 M Tris-HCl, pH 8.0, 0.14 M NaCl, 1% Triton X-100, 0.1% deoxycholate, 1% SDS) using a Branson Sonifer (output, 3; duty, 40%; pulsed). The samples were isolated by centrifugation (13,000 rpm at 4°C for 15 min) with the supernatant containing cross- linked chromatin. A 50 μl aliquot of the supernatant was used to determine DNA concentration using the DNeasy blood and tissue kit (Qiagen). For each sample, an equal amount of cross- linked chromatin (40–50 μg) was immunoprecipitated with 0.2 μg of specific antibody (XPA or PARP-1) overnight at 4°C. The immunocomplexes were adsorbed onto protein A Dynabeads (Invitrogen) for 3 h at 4°C. The samples were washed five times in ChIP wash buffer (Santa Cruz), resuspended in loading buffer, and boiled for 10 min at 95 °C before loading onto a 10% polyacrylamide gel followed by western blotting.