Figure 5. Notch Signaling is not involved in the GSI effect in combination with VCR. (A) Western blot analysis on expression of activated forms of Notch receptors (ICN1-ICN3) in HeLa cells with or without DAPT treatment. Beta-actin served as a loading control. (B) HeLa cells expressing shRNAs against Notch1, Notch2, Notch3, and scramble (N1, N2, N3, and C) were treated with 10 μM DAPT and/or VCR (10 nM or 20 nM) for 24 h. Cell cycle distribution was determined based on DNA content of PI stained populations. (C) Western blot analysis on expression of ICN1–3, and Hes1 after silencing Notch receptors using shRNAs. Beta-actin served as a loading control. (D) HeLa cells were transduced with a retroviral vector expressing ICN1, ICN2, ICN3, or GFP and treated with 20 nM VCR and/or 10 μM DAPT for 48 h. Cell viability was measured by WST-1 assays. Relative cell viability is presented as O.D at 450 nm. Results are presented as mean ± SEM of triplicate assays. The statistical significance of differences was determined by t test; *P < 0.05; **P < 0.01; ***P < 0.001.