br Figure Optimization of CX concentration for cytotoxicity and apoptosis

Figure 2. Optimization of CX concentration for cytotoxicity and apoptosis induced by CX treatment in A549RT-eto cells.
active caspase 3 and 7 in A549RT-eto GSK126 (Fig. 2C), indicating that CX induced not only extrinsic but also intrinsic apoptosis.
(A) A549RT-eto cells were treated with CX (20–0.16 μg/mL) for 72 h, and then, the percentage of cell viability was measured by MTT. IC50 values were calculated by the MTT assay. Results were averaged from triplicate wells, and the error bars indicate S.D. (**p < 0.001; A549 vs A549RT-eto). (B) A549RT-eto cells were treated with CX (0, 0.1, 2, and 5 μg/mL) for 24 h. After treatment, the morphological changes of cells and cell viability were observed under an optical mi-croscope (100× and 400× ). (C) The cell lysates from A549RT-eto treated with CX were prepared and separated on 12% SDS-PAGE gel. The expressions of pre-caspase 8, 9, cleaved PARP, NF-κB, and P-gp proteins were detected by immunoblotting with the corresponding antibodies.
2.3. CX decreases migration, invasion and spheroid formation of A549RT-eto cells
To evaluate the effects of CX on the migration of A549RT-eto cells, we performed wound-healing assay. For this experiment, the cells were co-incubated with non-cytotoxicity doses (0 and 1.5 μg/mL) of CX for 24 h. The treatment with CX (1.5 μg/mL) for 24 h significantly de-creased the cell migration rates in A549RT-eto cells (Fig. 3A). We also examined cell invasion capacity using a three-dimensional matrigel-coated filter after A549RT-eto cells were treated with CX. As shown in Fig. 3B, the treatment of A549RT-eto cells with CX significantly de-creased the cell invasion rates (approximately two-fold) compared with mock treatment.
Because CX inhibited cell migration and invasion, we next examined the inside of cells during the inhibition of EMT by measuring the levels of relevant proteins. Because it is well known that EMT is characterized by the down-regulation of E-cadherin and up-regulation of N-cadherin, CX treatment increased the expression of E-cadherin as well as 
inhibited the expression of N-cadherin and vimentin. Therefore, CX can potentially inhibit EMT by modulating the expression of E-cadherin, N-cadherin, and vimentin (Fig. 3C).
CSCs play a major role in cancer initiation, progression, metastasis, and drug resistance.19 Therefore, it is a challenge to search for antic-ancer drugs to overcome CSCs. We thus examined whether CX treat-ment can interrupt A549RT-eto sphere formation because the sphere formation assay is considered a useful method to evaluate self-renewal of CSCs in vitro. As shown in Fig. 3D, CX treatment inhibited the size and number of spheroid formation compared with mock treatment. The expression of transcription factors or co-activators responsible for stemness such as Oct4, Bmi1, Nanog, and Sox2 was measured. As shown in Fig. 3E, CX treatment reduced the protein levels of Oct4, Bmi1, Nanog, and Sox2 in a dose-dependent manner. These results suggest that CX possessed an inhibitory capability on CSC phenotypes of A549RT-eto cells.
(A) A549RT-eto cells were treated with CX (1.5 μg/mL) for 24 h before the cell scratch assay was performed to evaluate the migration ability of the treated cells and (B) a transwell assay was used to evaluate the effect of CX on the invasion of the cells. The data are representative of three experiments and statistical analysis of invested cells (***P < 0.001 compared with the control group). (C) The cell lysates from A549RT-eto treated with CX were prepared and separated on 12% SDS-PAGE gel. The expressions of E-cadherin, N-cadherin, vimentin, and NF-κB proteins were detected by immunoblotting with the corre-sponding antibodies. (D) A549RT-eto cells were treated with CX (1.5 μg/mL) for 24 h before the cells were trypsinized and cultured in non-adherent 24-well flat-bottom plates for 6 d. Spheroid formation was observed at intervals over 48 h (bar = 50 μm). The number of spheroids was counted at day 6. The data are representative of three experiments (***P < 0.001 compared with the control group). (E) The cell lysates from A549RT-eto treated with CX were prepared and se-parated on 12% SDS-PAGE gel. The expressions of Oct4, Bmi1, Nanog,