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. 2016 Jan 1;129(1):155–165. doi: 10.1242/jcs.178293

Fig. 2.

Fig. 2.

Activation of apoptosis in xenograft tumors and cancer cells with suppressed levels of NAF-1. (A) Left, immunohistochemistry (IHC) analysis using an antibody against activated caspase-3 showing a higher number of active-caspase-3-positive cells in NAF-1(−) tumors. Active-caspase-3-positive cells are marked by white arrowheads. Right, quantification of staining of active caspase-3. (B) Left, IHC analysis using an antibody against γH2AX showing a higher number of γH2AX-positive cells in NAF-1(−) tumors. Right, quantification of γH2AX staining. For A,B, cells were counted in ten high-power fields (×40) for each section obtained from five mice in each group; ***P<0.001 (t-test). (C) Left, activation of apoptosis in NAF-1(−) MDA-MB-231 cells observed by using annexin-V staining; ***P<0.001 (t-test). Right, representative apoptotic NAF-1(−) cells are indicated by the white arrowhead. Nuclei are counterstained with Hoechst 33342. (D) NAF-1(−) MDA-MB-231 cells show increased caspase-3 enzymatic activity, as measured by using a colormetric activity assay; *P<0.05 (t-test). (E) Western blot analysis showing the accumulation of activated caspase-3 and caspase-7 in MDA-MB-231 and MCF-7 cells in which NAF-1 expression had been suppressed. Protein expression was calculated as a percentage of that of control from three different experiments. **P<0.01 (n=3; t-test). Error bars represent mean±s.d. WT, wild type.