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. 2015 May 12;6(18):16559–16572. doi: 10.18632/oncotarget.4116

Figure 1. Notch1 and Notch2 silencing decreases cell viability and expression of Mcl-1 protein in CLL cells.

Figure 1

CLL cells were transfected with control siRNA (siCtrl), Notch1 siRNA (siNotch1), Notch2 siRNA (siNotch2) or combined siNotch1 and siNotch2 (siNotch1/2) as described in “siRNA transfection” and then cultured in complete medium for 72 hours. A., C. Western blot analysis of Notch1, Notch2, Mcl-1 and Bcl-2 expression was performed on 15 μg whole-cell lysates separated on a 7.5% SDS-PAGE. The antibodies used for Notch1 and Notch2 recognized the 120-kDa transmembrane/cytoplasmic subunit (TM) and the 100-kDa active intracellular domain (IC). Protein loading was assessed by reprobing the blots with an anti-GAPDH antibody. Vertical line inserted in Notch2 blot of CLL7 indicates a repositioned gel lane. Data of CLL7 and 18 are representative of six samples. D. The blots of Mcl-1 were subjected to densitometric analysis and densitometry units (U) were calculated relative to GAPDH. Data are the mean ± SD of six samples. *P < 0.05, **P < 0.01 (each siNotch transfection condition versus siCtrl) according to Student t test. B. Cell viability was evaluated by flow cytometric analysis of Annexin V/PI (An V/PI) staining. Viability (An V−/PI−) of siCtrl cells was set to 100%. Data are the mean ± SD of six samples. **P < 0.01 (each siNotch transfection condition versus siCtrl) according to Student t test.