Figure 3: Dose dependence of vtRNA1-1 levels on the impact of apoptosis resistance.

(a) Northern blot analyses demonstrate an shRNA-mediated knockdown of vtRNA1-1 levels in BL41 cells of ∼50%. See also Supplementary Fig. 6. (b) Annexin V staining revealed a reduced apoptosis resistance in the vtRNA1-1 knockdown BL41 cell line after staurosporine (Stau) treatment. The knockdown has been performed in two biological replicates whereas the mean and standard deviations of four technical replicates are shown. (c) Northern blot analysis showed an ∼60% reduction of endogenous vtRNA1-1 levels in HeLa cells upon transfection of an anti-sense oligonucleotide analogue (ASO). Administration of a control oligonucleotide (ASO_ctr) strand had no effect. (−) depict HeLa cells that were treated as the transfected samples, but in the absence of any oligonucleotides. Quantifications of band intensities are shown below the blot and represent the mean and standard deviation of three independent transfection experiments. The 5.8S rRNA served as internal loading control. (d) Reduced endogenous vtRNA1-1 levels result in an increased susceptibility to cell death in HeLa cells. The mean and standard deviations of three independent transfection experiments are shown. (e,f) Analogous to c and d, dose-dependent effects on apoptosis, resistance as a function of vtRNA1-1 concentration were monitored in the human breast cancer cell line HS578T. (g) In the presence of the NF-κB inhibitor (inh.) IKK VII, northern blot analysis showed a decreased vtRNA1-1 expression level in HeLa and HS578T cells. The 5.8S rRNA serves as internal loading control. The endogenous levels of vtRNA1-1 in HeLa and HS578T cells was taken as 1.0 and compared with cells treated with a NF-κB inhibitor. In b, d and f, significant differences were determined using the two-tailed unpaired Student’s t-test (***P<0.001, **P<0.01). pos., positive.