Semiquantitative analysis for these markers was evaluated by image analysis Romidepsin buy software, Image-Pro Plus (Media Cybernetics, Silver Spring, MD) (33). Three randomly chosen fields were calculated for statistics. Overexpression of AANAT in Mz-ChA-1 Cells The expression vector pCMV6-XL containing human AANAT cDNA was purchased from Origene (Rockville, MD); the control vector was pCMV6-XL. The 624-bp ORF of AANAT containing a COOH-terminal MYC/DDK tag was cloned into the pCMV6-XL entry, and the expression protein was 23.2 kDa. The transfection and the selections of clones were performed as described (27). The plasmid (10 ��g) was transfected by nucleofection (27) into Mz-ChA-1 cells, according to the manufacturer’s instructions. Mz-ChA-1 cells (1 �� 106 cells) per reaction were resuspended in 100 ��l of nucleofector solution (Lonza, Basel, Switzerland).

Ten micrograms of AANAT cDNA plasmid DNA (Origene) were mixed with 100 ��l of cell suspension and transferred into a cuvette. The cuvette was inserted into the nucleofector (Lonza), and cells were pulsed with program U-017. After pulse, the cells were rinsed and transferred to a six-well plate. Culture medium was replaced 24 h after nucleofection. Stable overexpressing AANAT Mz-ChA-1 cells were selected based on neomycin resistance, and individual colonies were ring-cloned. The overexpression of AANAT in Mz-ChA-1 cells was verified by real-time PCR analysis (15).

In Mz-ChA-1 cells (stably overexpressing AANAT or the control vector), we measured 1) cell proliferation by real-time PCR analysis for PCNA (15) and alamarBlue cell proliferation assay (Invitrogen, Rockville, MD) (50), according to the manufacturer’s instructions; 2) cell apoptosis with annexin V-FITC apoptosis detection kit (ab14085) (BD Biosciences) by FACS analysis (57); and 3) MT1/MT2 expression by FACS (38). For the alamarBlue cell proliferation assay (50), 5,000 cells per well were seeded in 96-well plates. Subsequently, cells were incubated for 0, 24, and 48 h after the cells were attached. Briefly, 10 ��l of medium alamarBlue reagent were added to 100 ��l of cell culture and incubated in 37��C for 2 h before detecting the fluorescence intensity under the fluorescence excitation of 540 nm and the fluorescence emission Cilengitide of 590 nm. Data were expressed as the fold change of different time points compared with 0 h. For the annexin V-FITC apoptosis detection kit I (BD Biosciences) (57), cells were harvested and processed according to the manufacturer’s instructions. Unstained cells, stained cells with propidium iodide (PI; without annexin V), and stained cells with annexin V (without PI) were used as controls to divide the events into four parts.