Lidocaine

PEM (detached by 15 min incubation at 37°C with 15 mM lidocaine (Sigma-Aldrich) plus 5 mM EDTA in PBS), purified BM-DC or unfractionated splenic cells (2 × 10⁵ leukocytes) were pre-incubated for 30 min on ice in 0.1 ml of FCS-RPMI containing 40% mouse serum and 50 μg/ml of non-immune mouse IgG2a (BD Biosciences), in order to decrease non-specific binding. Subsequently, 0.1 ml solutions of receptor-specific or control mAb were added, to give final mAb concentrations of 10 μg/ml, and the incubation was continued for 50 min. Unbound mAb were removed by washing with 2 ml of ice-cold PBS, and the cells were incubated for another 50 min with 5 μg/ml of PE-conjugated secondary Ab in 0.2 ml FCS-RPMI. In order to enable identification of DC and macrophages, splenic cells were subjected to additional incubation with 5 μg/ml of allophycocyanin-conjugated anti-CD11c (clone N418) or F4/80 mAb (eBioscience), respectively. Following washing twice, binding of fluorescently-labelled Ab to cells was assessed by flow cytometry.
The surface expression of MHC-II, CD40 and CD86 was also determined by flow cytometry, by direct labelling of these molecules with 5 μg/ml of PE-conjugated MHC-II-, CD40-, or CD86-specific or isotype-matched control mAb obtained from BD Biosciences or eBioscience.

The local anesthetic agents lidocaine, bupivacaine, benzocaine, and procaine and the chemotherapeutic agents carboplatin and paclitaxel were purchased as salts from Sigma-Aldrich (St. Louis, MO, USA). The sodium channel inhibitors zonisamide and rufinamide were purchased as salts from Selleck Chemicals (Houston, TX, USA).
Stock solutions of all drugs were prepared by dissolving the drugs in sterile water, except for the stock solutions of benzocaine and paclitaxel, which were prepared by dissolving the drugs in ethanol. The stock solutions were aliquoted and stored at −20 °C. The stock solution concentrations were based on the manufacturer stated limits of solubility of the compounds. Immediately prior to use, final test concentrations were achieved by making a serial dilution of stock solutions with standard growth medium.

MDA-MB-231 and MCF-7 breast cancer cell lines were provided by the cell resource center of the Chinese Academy of Medical Sciences (Beijing, China). MDA-MB-231 cells were grown in DMEM/F12 (HyClone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA). MCF-7 breast cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Gibco, Rockville, MD, USA). Cells were cultured at 37 °C in 5% CO₂ to 80%–90% confluence and were subjected to DMEM/F12 or DMEM supplemented with 2% FBS, containing 0.01, 0.02, 0.1, 0.5 or 1 mM lidocaine (Sigma–Aldrich, St. Louis, MO, USA), 10 μM 5-aza-2'-deoxycytidine (DAC) (Sigma–Aldrich) or (and) 0.2 μM cisplatin (Sigma–Aldrich for various hours for the DNA methylation sequencing, cell viability or cell apoptosis assay, and cell colony forming assay. DAC or cisplatin was utilized to as a positive demethylation agent or as an apoptosis inducer. To abrogate the expression of RARβ2 or RASSF1A, siRNA–RARβ2 (5'-CAGC UGAG UUGG ACGA UCU-3'), siRNA–RASSF1A (5'-GAC CUC UGU GGC GAC UUCA-3') or siRNA control (5'-AGCG AATT AGCT TGCC GTG-3') was synthesized by GenePharma Technology (Shanghai, China) and was transfected by lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) with a concentration of 50 nM according to the manufacurer’s guidance.