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. 2016 Nov 4;8(12):18773–18791. doi: 10.18632/oncotarget.13095

Figure 6.

Figure 6

(A–D) Influences on Bcl2 family members as well as mitochondrial potential and caspase-3 activity. Immunoblot detecting Bcl2, Bcl-xL, Bak1 and Bax (A) as well as procaspase-3 and PARP cleavage (B) after miRNA transfection. SKOV3 cells were seeded in a 96-Wellplate with a total volume of 100 μl 24 h before treatment. 25 μM etoposide, 8 μM carboplatin or 0.25 nM paclitaxel were used as a positive control as well as a transfection with a functional death control siRNA (DT). A non-targeting miRNA (NT) was used as negative control. 72 h after transfection with a final concentration of 50 nM miRNA mimics and 0.4 μl ScreenFect®A cells were harvested followed by SDS-PAGE and Immunoblotting with the respective antibodies. To correlate the blot intensity ß-Actin was used as loading control. The densitometry analyses were done using Fusion Software. Influence of mmu-miR-96-5p, -1912-5p, -147b-5p and -3073a-3p on mitochondrial potential (ΔΨm) in SKOV3 cells (C) and caspase-3/−7 activity in ovarian cancer cell lines (D). The miRNA mimics were individually transfected into SKOV3, OVCAR3, TOV21G, TOV112D, A2780 and A2780-cis cells (conditions of the experiment as described in Figure 1). For controls cells were treated with either 8 μM carboplatin or 0.25 nM paclitaxel. To compare the effect of the transfected miRNAs a non-targeting control (NT) was used. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test [n = 3; Mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001].