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. 2016 Nov 25;8(1):1190–1203. doi: 10.18632/oncotarget.13595

Figure 5. HG impairs HIPK2 apoptotic activity.

Figure 5

A. Clonogenic survival assay of HCT116 transiently transfected with HIPK2-GFP, K1182-GFP and empty-GFP expression vectors. After transfection cells were switched to HG condition and death-resistant cells were stained with crystal violet 1 week later. B. RKO cells were transfected with HIPK2-GFP or K1182-GFP expression vectors and 24 h later cultured in HG for 24 h. Twenty-four hours later, cells were in part fixed and stained with PI for subG1 evaluation (upper panel) or lysed and analyzed by western immunoblotting to assess p-Ser46 (lower panel). Data are presented as mean ± S.D. *P < 0.001. C. RKO cells were transiently co-transfected with p53AIP1-luc reporter and HIPK2-GFP or K1182-GFP expression vectors. Twenty-four hours after transfection culture medium was changed with HG medium. Results, normalized to β-gal activity are the mean of three independent experiments ± S.D. performed in duplicate. *P = 0.001 D. RKO cells were transfected with HIPK2-GFP or K1182-GFP expression vectors and, after transfection, switched to either HG condition for 24 h or FTY720 treatment for 16 h, before being assayed for semi-quantitative RT-PCR of p53 target genes. 28S was used as a control for efficiency of RNA extraction and transcription. Densitometry was performed with ImageJ software and relative band intensity normalized to 28s and quantified with respect to controls set to 1.0.