AR42J cells were used between passages 7 and 13. For each group, including the controls, cells were suspended in 2 ml DMEM, which contained FBS and antibiotics, and added to 24-well plates (triplicates, density of 8 × 104 cells/well). Cells were incubated under standard cell culture conditions. After reaching 70% subconfluence, the medium was replaced by 500 μl fresh DMEM. For the calpain-2 knockdown, 0.002 nmol calpain-2 siRNA was diluted in 100 μl DMEM and mixed with 3 μl and 1.5 μl HPF, respectively. While the solution incubated at room temperature for 10 min, the transfection complexes were formed and subsequently were added drop-wise onto the cells, resulting in a final siRNA concentration of 3.33 nM. Control cells were incubated with control siRNA (scrRNA) at the same concentration and dilution, but without gene silencing effect on calpain-2 or with DMEM only. Thereafter, the cells were incubated under standard cell culture conditions. In order to induce cell differentiation, 100 nM dexamethasone were added to all samples 6 h after transfection. After further 48 h, a part of the siRNA-treated cells and untreated control cells were harvested for Western blot analysis to investigate the calpain-2 knockdown at the protein level. Additionally, cells were used for apoptosis experiments. siRNA-treated cells and control cells were exposed to H2O2 (final concentration: 250 μM). The medium was removed and replaced by DMEM 1 h after H2O2 addition. Cells without H2O2 exposure served as control. Then, the cells were incubated for further 5 h. Full paper
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