Journal: Alcoholism, clinical and experimental research

Article Title: Alcohol-mediated missplicing of Mcl-1 pre-mRNA is involved in neurotoxicity

doi: 10.1111/acer.13474

Figure Lengend Snippet: A. PHFN cells were plated in 6-well tissue culture dishes and transfected with pCDNA3.1-MCL-1L expression plasmid (Addgene#25375). At 24 h post-transfections, cells were exposed to 50 mM EtOH in incubators with “compensation system” to prevent EtOH evaporation. Cellular viability was determined by MTT assay at 24h post-exposure to EtOH. Bar graph represents three independent experiments. B. MCL-1L stable cells are resistant to toxicity associated with EtOH exposure. SH-SY5Y cells were transfected with pCDNA3.1 vector and pCDNA3.1-MCL-1L expression plasmid and treated with G418 for the selection of transfected cells over un-transfected cells. G418 resistant cells were seeded in 96-well tissue culture dishes as 1 cell/5 wells (limiting dilution) for the establishment of sub-cell line clones. All the clones were analyzed by Western blot for stable over-expression of MCL-1L gene. The control cells (originated from pCDNA3.1 control vector) and MCL-1L stable cells (originated from pCDNA3.1-MCL-1L) were plated in 6-well tissue culture dishes and exposed to 50 mM EtOH for 24h. MTT assay was performed to determine cellular viability. Bar graph represents three independent experiments.

Article Snippet: Stable cell lines SH-SY5Y cells (1 × 10 5 cells/35 mm tissue culture dish) were either transfected with pcDNA3.1 vector alone (Invitrogen) or pcDNA3.1-hMCL-1L (2 μg/plate) using lipofectamine 2000 method according to the manufacturer’s recommendations (Invitrogen).

Techniques: Transfection, Expressing, Plasmid Preparation, Evaporation, MTT Assay, Selection, Clone Assay, Western Blot, Over Expression