Sinofection

Expression vectors were introduced into the NCI-H1299 cells by Sinofection (Sino Biological, Beijing, China). After 6 hours, the transfection mixture was removed and replaced by medium with serum. Colony forming assays were carried out after incubation for 72 hours. Cells were treated with DSF and DSF/Cu in the same manner as described above.

The expression vector ALCAM cDNA ORF clone pCMV3-C-GFPSpark (Sino Biological, Beijing, China) was used for ALCAM overexpression. D341 cells were transfected with the ALCAM cDNA plasmid using a lipofection method and Sinofection (Sino Biological). pCMV3-C-GFPSpark Control Vector (Sino Biological) was used as negative control. Overexpression of ALCAM was confirmed by qPCR after hygromycin B selection.

HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37 °C with 5% CO₂. Cells suspended in D-MEM containing 10% FBS were plated on 35-mm dishes 1 day before transfection. The attached cells were transfected with ~1.5 µg of the endotoxin-free plasmid in 350 µl of Opti-MEM I (Gibco) containng 3.5 µl Sinofection (Sino Biological). After incubation for 5 h at 37 °C with 5% CO₂, the media was replaced with D-MEM containing 10% FBS (500 µl). And cell culture for 2 days, the culture media was removed and the drug diluted in Dulbecco’s modified Eagle’s medium was added into the stable transfected cells for incubation 30 min. The cells were washed two times with PBS (PH7.4) and lysed in 500 µl PBS by re-freezing before centrifugation at 2000 rpm for 20 min. The amount of intracellular cAMP extracted from harvested supernatant was determined using ELISA reagent (Huijia Biotech) according to the manufacturer’s instructions. Each measurement was performed in duplicate and a minimum of three independent assays were carried out for each compound and concentration tested.