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. 2015 Nov 28;7(2):1675–1686. doi: 10.18632/oncotarget.6423

Figure 5. Evaluation of the e8-siRNA#1 off-target effect.

Figure 5

(A) Sequence alignment between Hipk2 e8siRNA#1 and Nr1D2 in the homologous region. (B) CSC1 cells were transfected with the indicated siRNAs. At 96 hrs post-transfection, Hipk2-FL and Nr1D2 expression were monitored by RT-PCR. (C) Hap-1 parental (Hipk2-WT) and Hipk2-KO cells were transfected with e8-siRNA#1 or its scrambled version (Scr-siRNA) and collected after 72 hrs. Cells were counted to assess viability and analyzed by both RT-PCR and WB. Cleaved CASPASE 3 (clvd CASP3) was used as apoptotic marker. Deletion of exon 3 in Hipk2-KO cells was confirmed by specific primers (Hipk2 e3). (D) U2OS cells were transfected with GFP-tagged constructs for HIPK2-FL (GFP-FL) and HIPK2-Δe8 (GFP-Δ8) isoforms. HIPK2 expressing cells were identified by GFP expression. BrdU positive, proliferating cells, were detected by immunofluorescence with an anti-BrdU antibody. Representative fluorescent images are reported in the right panels. Bars, 10 μm.