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. 2019 Oct 1;10(55):5727–5729. doi: 10.18632/oncotarget.27230

Figure 6. Aloe-emodin inhibited ILK signaling pathways in HER-2-overexpressing breast cancer cells.

Figure 6

(A) SkBr3 cells were treated with various concentrations of AE for 48 h. Cell lysates were immunoblotted with anti-ILK and anti-phospho-ILKThr173 antibody. (B) SkBr3 cells were treated with 20 μM AE or 300 nM ILK inhibitor cpd-22 for 48 h. Cell lysates were immunoblotted with anti-ILK, anti-phospho-ILKThr173, anti-HER-2, anti-YB-1, and anti-Twist antibodies. (C) SkBr3 cells were treated with various concentrations of AE for 48 h. Cell lysates were immunoblotted with anti-phospho-AktSer473 and anti-phospho-mTORSer2448 antibodies. (D) SkBr3 cells were transfected with constitutively active Akt and treated with 20 μM AE for 48 h. Cell lysates were immunoblotted with anti-phospho-AktSer473, anti-phospho-mTORSer2448, anti-HER-2, anti-YB-1, and anti-Twist antibodies. β-Actin was used as the loading control. (E) SkBr3 cells were then harvested and lysed for the detection of phospho-GSK3βSer9 and β-Actin. (F) SkBr3 cells were treated with 20 μM AE or 1 μM phospho-GSK3βSer9 inhibitor SB216763 for 48 h. Cell lysates were immunoblotted with anti-phospho-GSK3βSer9, anti-HER-2, anti-YB-1, and anti-Twist antibodies. (G) SkBr3 cells were treated with 40 μM AE for 24 h. Following cell fractionation, Twist and YB-1 content in the cytoplasmic or nuclear fraction was determined through Western blotting. PARP was used as the nuclear marker. β-Tubulin was used as the cytoplasmic marker.