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  • br A A RT eto cells

    2020-08-28


    (A) A549RT-eto CHIR 99021 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, r> Figure 3. CX treatment decrease CSC-like phenotypes of A549RTeto cells.
    and Sox2 proteins were detected by immunoblotting with the corre-sponding antibodies.
    2.4. NF-κB suppression accelerates CX-induced apoptotic cell death
    Because the enhanced levels and activity of NF-κB protein in A549RT-eto cells were found, whether NF-κB was involved in the re-sistance to etoposide in A549RT-eto cells was studied. We treated A549RT-eto cells with NF-κB siRNA and examined cell viability with MTT assays. As shown in Fig. 4A, NF-κB siRNA treatment or CX treat-ment (1 μg/mL) alone slightly induced the death of A549RT-eto cells. However, the combined treatment with CX and NF-κB siRNA ac-celerated the apoptosis in A549RT-eto cells, which was confirmed by the observation of the cleavage of PARP and pro-caspase 8 and 9 (Fig. 4B). Moreover, we examined the protein levels of NF-κB and P-gp after treatment with NF-κB siRNA alone, CX alone, or NF-κB siRNA plus CX. As shown in Fig. 4B, NF-κB siRNA alone decreased the expression levels of NF-κB and P-gp in the cells, and CX treatment alone drastically diminished the protein levels. In comparison, the combined treatment more significantly reduced the protein levels of NF-κB and P-gp. Therefore, NF-κB was involved in MDR in A549 cells by the up-reg-ulation of P-gp, resulting in the resistance to etoposide.
    (A) A549RT-eto cells were treated with CX (1.0 μg/mL) for 24 h after transfection with a control or NF-κB siRNA (100 nM). Cell viability was observed under an optical microscope and relatively measured using the MTT assay. The data were calculated as the percentage of relative cell viability and expressed as the mean of three experiments (control siRNA + CX vs NF-κB siRNA + CX; ***p < 0.001). (B) Cell lysates from the treated A549RT-eto cells 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.
    2.5. NF-κB expression is associated with CSC-like phenotypes in A549RT-eto cells
    Because CX treatment inhibited NF-κB expression and activity as well as CSC-like phenotypes (Figs. 3 and 4), a question was raised as to whether NF-κB was involved in CSC-like phenotypes. Thus, siRNA was introduced against NF-κB. The treatment with NF-κB siRNA inhibited cell invasion into the lower well through a matrigel-coated membrane in A549RT-eto cells (Fig. 5A). Supporting this result, we observed that the knockdown of NF-κB decreased the E-cadherin levels and increased the N-cadherin or vimentin levels (Fig. 5B). In addition, the knockdown of NF-κB inhibited the spheroid size and number in A549RT-eto cells (Fig. 5C). Moreover, the silencing diminished the protein levels of Bmi1, Sox2, Oct4, Nanog, and Klf4 (Fig. 5D), all of which are tran-scription factors or co-activators responsible for stemness. Therefore, NF-κB was involved in CSC-like phenotypes such as EMT and stemness. Taken together, CX treatment inhibited EMT and stemness through the down-regulation of NF-κB.
    (A) A549RT-eto cells were transfected with a control or NF-κB siRNA (100 nM), and a transwell assay was used to evaluate the effect of NF-κB suppression on the invasion of the cells. The data are re-presentative of three experiments (***P < 0.001 compared with the control group). The cell lysates from A549RT-eto were prepared and separated on 12% SDS-PAGE gel. “C” indicates control. (B) The ex-pressions of E-cadherin, N-cadherin, vimentin, and NF-κB proteins were detected by immunoblotting with the corresponding antibodies. (C) The transfected cells were trypsinized and cultured in non-adherent 24-well flat-bottom plates for time-lapse recording over 6 days. The spheroid formation was observed at intervals over 48 h (bar = 50 μm). The