Fig. 4.

Ago2 improves shRNA-mediated cell death specifically in sensitive cells. (A and B) PC-9 cells contain a small deletion in EGFR leading to constitutionally active EGFR and making PC-9 cells exquisitely sensitive to loss of EGFR signaling (18), whereas NCI-H1299 cells are not sensitive to EGFR knockdown. Both cell lines were lentivirally infected with different shEGFR constructs containing a Puromycin resistance cassette combined with an empty pWPI lentivirus or expression constructs for tagged or untagged Ago2, selected by Puromycin and analyzed for cell density by Syto60 staining. Depicted is the mean growth inhibition (+SEM) as compared to cells infected with shEGFP and empty pWPI. Without Ago2 expression, only shEGFR-4705 induced cell death in these cells mirroring the results from protein knockdown. With ectopic Ago2, all shEGFR constructs targeting EGFR in PC-9 cells were capable of inducing cell death. As controls, shEGFP or shEGFR-2467 (located within deletion) infected cells were viable whereas noninfected cells died in Puromycin selection. Ago2 expression had very little effects on controls. NCI-H1299 cells did not show any sensitivity to the shRNAs, neither without nor with additional Ago2.