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. 2017 Jan 5;7:40187. doi: 10.1038/srep40187

Figure 1. AMBN induces apoptosis and sensitivity to doxorubicin through the Src-Stat3 pathway in osteosarcoma cells.

Figure 1

(A) The expression of AMBN at the protein level and AMBN, RUNX2, ALP, and OCN at the mRNA level among human osteosarcoma cell lines is shown. (B) The expression of AMBN, RUNX2, ALP, and OCN at the mRNA level in control and AMBN-stable 143B-Luc cells was examined. (C) Cell growth was counted on days 0, 1, 3, and 5 (N = 3). (D) Cell cycle distributions were analyzed by PI staining and FACS, and quantified (N = 3). (E) The apoptotic cell ratio was analyzed by annexin-V/7-AAD staining and FACS, (F) and quantified (N = 3). (G) Control and AMBN-stable 143B-Luc cells were treated with doxorubicin at indicated concentration for 48 hours, then the cells attached on the dishes were counted and quantified (N = 3). (H) After the treatment with DMSO and doxorubicin (0.5 μg/mL) for 12 h, the apoptotic cell ratio was analyzed by annexin-V/7-AAD staining and FACS, (I) and quantified (N = 3). (J) The expression of pY416-Src and total-Src in control and AMBN-stable 143B-Luc cells after attachment on the culture dish (0, 30, 60 minutes) was examined. Relative expression of pY416-Src (Src activation) is shown. (K) Empty vector and Src-Y527F plasmid were transfected into control and AMBN-stable 143B-Luc cells, the expression of pY416-Src, total-Src, pY705-Stat3, total-Stat3 was examined. Relative expression of pY705-Stat3 (Stat3 activation) is shown. (L) Empty vector and Src-Y527F plasmid were transfected into AMBN-stable 143B-Luc cells, and the expression of pY416-Src, total-Src, and cleaved caspase-3 was evaluated (upper panel). The apoptotic cell ratio was analyzed by annexin-V/7-AAD staining and FACS, and the representative results are summarized (lower graph) (N = 3). (M) After the transfection of empty and Src-Y527F into control and AMBN-stable 143B-Luc cells, these cells were treated with DMSO and doxorubicin (0.5 μg/mL) for 24 h. The expression of cleaved caspase-3 was examined. Mean ± SEM (C,D,F,G,I,L); ***P < 0.001; **P < 0.01; *P < 0.05.