X treme gene hp dna transfection reagents

HEK cells were transfected with 1 µg of the minigene-containing pSpliceExpress vectors using the XtremeGene HP DNA transfection reagents (Roche), according to the manufacturer’s instructions. After 48 hours, total RNA was isolated using Qiazol and the RNeasy micro kit with on-column DNA digestion using the RNase-free DNase set (all Qiagen). Next, total RNA was reversely transcribed using the iScript Advanced cDNA synthesis kit (BioRad) according to the manufacturer’s instructions. Finally, cDNA was purified using MinElute PCR purification columns (Qiagen) and subjected to Sanger sequencing for analysis of alternative splicing events.

Dulbecco's modified Eagle's medium (DMEM, LM001-05), fetal bovine serum (FBS, S001-07) and Roswell Park Memorial Institute (RPMI, LM011-01) 1640 medium were purchased from Welgene (GD, Korea). 3-methyladenine (3-MA, 5142-23-4), Z-Leu-Leu-Leu-al (MG132, C221) and cycloheximide (CHX, 01810) were purchased from Sigma-Aldrich (St Louis, MO, USA). X-tremeGENE HP DNA transfection reagents (06366236001) were purchased from Roche (Mannheim, Germany). DC Protein Assay Kit II (500-0113, 0114 and 0115) was purchased from Bio-Rad (Hercules, CA, USA). ECL western blotting detection reagents (RPN2209) were purchased from GE Healthcare (Buckinghamshire, UK). Antibodies for CNOT2 (34214, 1:2000 dilution), p62/SQSTM1 (5114, 1:2000 dilution), Myc-taq (2276, 1:2000 dilution), LC3B (3868, 1:1000 dilution) and ATG5 (8540, 1:2000 dilution) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for OctA-taq (Flag, SC-807, 1:2000 dilution), HA-taq (SC-7392, 1:2000 dilution), immunoglobulin G (IgG, SC-69786), actin (SC-47778, 1:5000 dilution), 4′, 6′-diamidino-2′-phenylindole dilactate (DAPI, 28718-90-3) and Protein A/G plus-agarose (SC-2003) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Other chemical reagents were obtained from Sigma-Aldrich.

The open reading frames of the Smad6, Smad7, Bambi and Fst mRNAs (NCBI reference sequences NM_005585.4, NM_001042660.1, NM_012342.2 and NM_006350.3, respectively) were amplified from a cDNA library of the HepG2 cell line and inserted into the pCMV‐3tag‐3A vector (Stratagene). All constructs and their protein products were confirmed using DNA sequencing and Western blot analysis, respectively. Huh7 cells, a human hepatoma cell line, were plated in 12‐well plates and cultured at 37°C in 5% CO₂ with 1 mL/well DMEM (Gibco) containing 15% (v/v) heat‐inactivated foetal bovine serum (Gibco). The cells were then transfected with the respective plasmid using X‐tremeGENE HP DNA transfection reagents (Roche). Where indicated, 36 hours after transfection, human recombinant BMP6 (R&D systems) was added to the wells to a final concentration of 10 ng/mL. After incubating for an additional 12 hours, the cells were collected for the following analyses.

H1299 (ATCC^® CRL-5803D™; human non-small cell lung carcinoma) cells were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 supplemented with 10 % FBS. ATG5 ^+/+ and ATG5^−/− MEF and HEK293 QBI (human embryonic kidney 293; ATCC^® CRL-1573™) cells provided by Prof. J Ha (Kyung Hee University, Seoul, Korea) were maintained in DMEM supplemented with 10 % FBS. HEK293 QBI cell line is a superior subclone of HEK293 QBI cells, harboring the E1A and E1B regions of the adenoviral genome, and complementing the deletion of the E1 region in the recombinant Adenovirus (Qbiogene, Livingstone, UK). All cells were maintained in the growing medium in a humidified 5 % CO₂ atmosphere at 37°C. DNA transfection was performed using the X-tremeGENE HP DNA transfection reagents (Roche, Quebec, Canada).