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. 2019 Oct 14;9(25):7628–7647. doi: 10.7150/thno.36277

Figure 4.

Figure 4

HOXA11 suppressed apoptosis and anoikis of gastric cancer cells and activated Stat3 signaling pathways. (A) Representative images of Tunel assays in peritoneal foci derived from NCI-N87-Vector and NCI-N87-HOXA11 cell groups. The scale bar, from left to right, 400 μm, 100× magnification; 200 μm, 200× magnification; 100 μm, 400× magnification. Each experiment was performed in triplicate. (B) The protein expression of PARP, Cleaved PARP, Bcl2, Bax, Survivin, Caspase3, and Cleaved caspase3 in NCI-N87-Vector, NCI-N87-HOXA11, SGC-7901-Vector, SGC-7901-HOXA11, MGC-803-Control, MGC-803-shHOXA11 #1 and MGC-803-shHOXA11 #2 cells treated with Doxorubicin (1 μm) for 24 hours or not were analyzed using western blot with the indicated antibodies. GAPDH was used as the internal protein loading control. Each experiment was performed in triplicate. (C) The apoptosis of indicated cells were analyzed by flow cytometry via labeling with APC-Annexin-V and 7-AAD. Representative images were shown. (D) The anoikis of indicated cells were analyzed by flow cytometry via labeling with APC-Annexin-V and 7-AAD. Representative images were shown. (E & F) Histograms shown the apoptosis and anoikis level of indicated cells respectively. Results were shown as mean ± SEM of three independent experiments, each experiment was performed in triplicate. *, P<0.05; **, P<0.01; ***, P<0.001 (Student t test and the analysis of variance test). (G) Immunohistochemistry assays shown the expression of phosphorylation (Tyr705) of Stat3 in peritoneal foci derived from NCI-N87-Vector and NCI-N87-HOXA11 cell groups. The scale bar, from left to right, 400 μm, 100× magnification; 200 μm, 200× magnification; 100 μm, 400× magnification. Each experiment was performed in triplicate. (H) The protein expression of phosphorylation (Tyr705) of Stat3 and Stat3 in NCI-N87-Vector, NCI-N87-HOXA11, SGC-7901-Vector, SGC-7901-HOXA11, MGC-803-Control, MGC-803-shHOXA11 #1 and MGC-803-shHOXA11 #2 cells were analyzed using western blot with the indicated antibodies. GAPDH was used as the internal protein loading control. Each experiment was performed in triplicate. (I) HOXA11 overexpression increased Stat3 nuclear accumulation in indicated cells, as confirmed by western blot analysis. GAPDH and Histone H3 were used as loading controls. Each experiment was performed in triplicate.