Nm mechlorethamine hydrochloride

NM (Mechlorethamine hydrochloride) (Cat# 122564), DEX (Cat #D1756), (2-Hydroxypropyl)-β-cycloDEXtrin (2HBC; Cat #H107), TWEEN® 80 (Cat #P4780), (Hydroxypropyl)-methyl cellulose (HMC; Cat #423238), and sodium chloride (Cat #S3014), were obtained from Sigma. The primary antibody used in immunohistochemistry (IHC) for COX-2 (cat#160112) was procured from Cayman chemicals, Ann Arbor, MI. The primary antibody for VEGF (Cat#ab28775) was obtained from Abcam, Cambridge, MA. The primary antibody used as negative control, mouse IgG antibody, was procured from N-Universal, DAKO.

JB6 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) and cultured in minimal essential medium (MEM; Gibco BRL, Grand Island, NY) containing 5% heat-inactivated fetal bovine serum (FBS) and 25 µg/mL gentamycin. Cells were grown under standard culture conditions at 37 °C in a humidified 5% CO₂ incubator. NM (mechlorethamine hydrochloride; 98%) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO) and the stock solution of NM was prepared fresh in dimethyl sulfoxide (DMSO) for the exposures. For the inhibitor studies, NU7026 and BO2 were obtained from Sigma and EMD Millipore (Billerica, MA), respectively, and the stocks were prepared in DMSO. Cells were treated with either 10 µM NU7026 1 h before NM exposure, or with 12.5 µM BO2 along with NM exposure and desired assays were carried out 4 to 48 h following these treatments. Unless stated otherwise, the final concentration of DMSO in the culture medium during treatments did not exceed 0.1% (v/v). All NM preparations were carried out in a continuously operated chemical and biological safety hood, and exposures were initiated under a safety laminar hood using all required and approved personal protective equipment.

Hematoxylin and eosin stains and NM (mechlorethamine hydrochloride; purity, 98%) were obtained from Sigma-Aldrich Chemicals Co. (St. Louis, MO). Paraffin wax for tissue block preparation was obtained from Triangle Biomedical Sciences (Durham, NC). DeadEnd^™ Colorimetric TUNEL (TdT-mediated dUTP Nick-End Labeling) system was purchased from Promega (Madison, WI) and Fluoro MPO^™ Fluorescent MPO Detection Kit was obtained from Cell Technology (Mountain View, CA). Primary antibodies for H2A.X and MMP-9, COX-2, and iNOS were obtained from Cell Signaling (Beverly, MA, USA), Cayman Chemicals (Ann Arbor, MI, USA), and Abcam (Cambridge, MA), respectively. We used β actin antibody from Sigma-Aldrich (St. Louis, MO).