Nepa21 type 2

To manipulate the cellular levels of miR-3646, 1×10⁶ cells were mixed with miR-3646 mimics at a final concentration of 10nM or antisense inhibitors at 50nM in 100ul culture medium without serum and penicillin-streptomycin. The mixtures were added into pulse cuvettes, and electroporated by Super Electroporator NEPA 21 Type II (poring pulse: pulse voltage, 125V; pulse length, 5ms; pulse interval, 50ms; NEPAGENE, Chiba, Japan). Then, the mixtures (include 5×10⁵ cells) were rapidly plated onto 6-well plate with 2ml of complete medium per well. The transfection efficiency was determined by green fluorescence.
After transfection, the cells were plated at a density of 8×10⁴ /ml into 96-well culture plate with 100μl of complete medium per well and cultured for 24h. The cells were treated with serial dilutions of Doc for 48h. For the drug cytotoxic assays, 20μl of a 5mg/ml solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT; Sigma, Germany) was added to the cells and incubated for 4h, and then 150μl of dimethyl sulfoxide (DMSO, Amresco, America) was added, and thoroughly mixed to ensure cell lysis. CliniBio128 (ASYS-Hitech, Austria) was used to measure the absorbance at 490nm. Three independent experiments were performed, and the IC50 value of Doc was calculated using SPSS 16.0.

Jurkat cells were resuspended in Opti-MEM (1.11 × 10⁷ cells/µL, 90 µL). An mRNA solution was diluted in Opti-MEM (0.2 µg/µL, 10 µL). The mRNA solution was then added to the cell suspension and set to an electroporator NEPA21 Type II (Nepa Gene Co., Ltd., Chiba, Japan). The electroporation was conducted at a Pp (poring pulse) of 175 V; a Pp duration of 2.5 ms; a Pp interval of 50 ms; a Pp frequency of 2 times; a Pp decay rate of 10%; a Tp (Transfer pulse) of ±20 V; a Tp duration of 50 ms; a Tp interval of 50 ms; a Tp frequency of 5 times for ± Voltage; a Tp decay rate of 40%. Immediately after the electric pulses, the cells were collected by adding 300 µL of culture media. The cells were incubated for 1 h at 37 °C in the CO₂ incubator. After the recovery culture, the cells were collected by centrifugation (4 °C, 500× g, 3 min), and then resuspended at a concentration of 1 × 10⁵ cells/mL in culture media containing serum. The cells were transferred to 3.5 cm dishes (1 × 10⁵ cells/mL, 2 mL).