PMC

Evaluation of untarget effects. Cells were transfected with 20 nmol/liter or 80 nmol/liter siRNAs of two different siRNA sets against Akt-1, ErbB2, and MEK1, respectively. Potential effects were measured for both sets on p38, IGF-1R, ER-α, and CDK2 expression, as well as on mRNA level (Left) and on protein level (Right).

Evaluation of different siRNA sets on specific and untarget effects. Cells were transfected with 20 nmol/liter or 80 nmol/liter siRNAs of two different siRNA sets, respectively, each containing siRNAs against a single target region within the corresponding genes. The effects of both sets were measured on mRNA level (Left) by using qRT-PCR, as well as on protein level (Right) by RPPA. The stars on top of the bars show where the reduction was presumed for the given set-up.

Simultaneous knockdown of three different proteins (Akt-1, ErbB2, and MEK1) using different siRNA concentrations. (A) Western blot showing the reduction at the protein level after RNAi (48 h) targeting Akt-1, ErbB2, and MEK1, alone or in the indicated combinations. For each gene, three different concentrations of siRNA pools [20 nmol/liter (Left), 40 nmol/liter (Center), and 80 nmol/liter (Right)] were tested. Red stars indicate samples where a reduction of protein was expected after transfection with the corresponding siRNAs. (B) Determination of protein knockdown after RNAi by RPPA. Signal intensities were normalized to 1 for the non-targeted control protein actin, or for the respective target proteins (ErbB2, Akt-1, or MEK1) in the case of nontransfected cells. For each gene, three different concentrations of siRNA pools [20 nmol/liter (Left), 40 nmol/liter (Center), and 80 nmol/liter (Right)] were tested. Gray bars indicate samples where a reduction of the protein level was expected whereas the bars in white show samples where no such reduction was presumed. (C) Cytotoxicity of siRNA transfections was determined for each concentration of siRNA by cell counting. MOCK (10 μl) is for transfections applied in 10 μl of transfection reagent, and MOCK (20 μl) is for the 20-μl set-up. Each sample was counted four times in three independent experiments, and the averages and standard deviations are shown.

Systematic pathway analysis by combinatorial use of siRNAs. (A) Effect of protein knockdown by combinatorial RNAi strategy on the invasion of HCC1954 cells. Cells were transfected with 20 nmol/liter siRNA pools for single, double, and triple knockdown of the respective proteins (Akt-1, ErbB2, MEK1) in 10 μl of transfection reagent. The cells that had invaded were detached from the membrane by trypsinization, centrifuged, and then resuspended in PBS. The number of invaded cells for each condition was determined by flow cytometry. Error bars were calculated from triplicate experiments. (B) Scheme of the ERK1/2 and PI3-kinase pathway (red boxes), and assumed cross-talk effects via other pathways (green boxes, intermittent arrows). ErbB2 activates two pathways in parallel: the Akt-1 pathway influences cell invasion, whereas the ERK1/2 pathway regulates proliferation. For the HCC1954 cells, we showed that, indeed, the Akt-1 pathway is dominant for modulating the invasion capacity as compared with the ERK1/2 pathway. Our result for ErbB2 knockdown suggests that other receptors activate Akt-1 because the invasion capacity is not reduced. The double knockdown of ErbB2 and MEK1 implies that the activation of Akt-1 through other receptors results in a reduction of cell invasion. Our data for the triple knockdown reveal parallel mechanisms that attenuate the invasion potential of HCC1954 cells when both the PI3-kinase and ERK1/2 are blocked.