MSLN overexpression protects PC cells from TNF-α induced cell viability decrease and apoptosis. (A). MIA-MSLN and control cells viability assay with or without TNF-α treatment. About 3000 cells were plated in 96 well plates, serum starved for 24 h and treated with TNF-α at 20 ng/ml for 96 h after which viability was measured by MTT. Relative increase in viability was measured by dividing viability at a time point by viability of same cells at day 0 (day after plating) and is plotted along Y axis. Data plotted show mean of triplicate wells. Bars denote s.d. of triplicate data. *, # denote p < 0.05, **, ## denote p < 0.01, compared with controls by using student t test. (B). MIA-MSLN cells are resistant to TNF-α induced apoptosis using TUNEL assay. Cells were treated ± 20 ng/ml of TNF-α for 72 h and apoptosis was measured by TUNEL assay. Representative flow histograms show percentage of dUTP-FITC-positive apoptotic cells. Lower panel shows mean number of TUNEL positive cells (duplicate wells). (C). siRNA-silencing MSLN renders MIA-MSLN cells become TNF-α-sensitive measured by TUNEL assay. MIA-V/MIA-MSLN ells were plated in 6 well plates, transfected with siRNA, 24 h after transfection media was replaced by growth media, cells were serum starved and treated with TNF-α at 20 ng/ml for 72 h after which cells were collected, fixed and tested for DNA strand breaks by TUNEL assay. Bars denote s.d. of duplicate data. *, # denote p < 0.05, **, ## denote p < 0.01, compared with controls by using student t test.