Fig. 6

BC200 overexpression rescues the knock-down phenotype. (a) HeLa cells were transfected with expression vectors containing BC200 under control of the endogenous promoter (WT_BC200), BC200 under control of the U6 promoter (U6_BC200) as well as an siRNA resistant sequence mutant (U6_BC200 siMUT). BC200 was detected by denaturing TBE gel electrophoresis followed by northern blotting with DIG-labelled LNA probes targeting either the 3′ or 5′ ends of the RNA. Total RNA was detected in an identical gel run in parallel that was stained with SYBR gold. (b) HeLa cells were transfected with the indicated expression vectors for 24 h. 24 h post plasmid transfection cells were split an reverse transfected with BC200 siRNA or LNA GapmeR. Expression of endogenous and exogenous BC200 was assessed by qPCR with specific primers 48 h post transfection of the BC200 siRNA or LNA GapmeR. (c) Cell viability was measured by MTT assay 48 h post transfection of the BC200 siRNA or LNA GapmeR. Data represents the mean of 8 biological replicates +/− standard deviation. * indicates statistically significant deviation (p < 0.05) from Empty Vector transfected with BC200 siRNA. # indicates statistically significant deviation (p < 0.05) from empty vector transfected with BC200 LNA GapmeR. P-values were calculated by an unpaired two-tailed t-test. (d) The region of BC200 targeted by the LNA GapmeR and siRNA is shown in the context of the primary sequence and proposed secondary structure of BC200. The sequence scrambled in U6_BC200 siMUT (163-185) is shown alongside the wild-type sequence