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. 2018 Jun 21;2018:8987568. doi: 10.1155/2018/8987568

Figure 3.

Figure 3

β-Tubulin profile and TUBB3 knockdown in parental and eribulin-resistant cell lines. (a) The β-tubulin profile (TUBB1, TUBB3, TUBB4, and TUBB5) was investigated by real-time quantitative PCR in each cell line. Values represent mean ± SD. ∗ P < 0.05, versus parental cell line. (b) Overexpression of TUBB3 was confirmed by Western blotting. GAPDH was used for internal normalization. (c) The expression of TUBB3 after transfection of siRNA was analyzed by real-time quantitative PCR and Western blotting. Values represent mean ± SD. ∗ P < 0.05. (d) The sensitivity to eribulin after transfection of siRNA was assessed by determination of cell viability and cell cycle analysis. Left panel: the cell viability assay was performed after transfection of siRNA and 48-hour incubation with eribulin. Right panel: cell cycle analysis was performed after transfection of siRNA and 12-hour incubation with eribulin. Values represent mean ± SD. ∗ P < 0.05, versus resistant cell line transfected with siControl. (e) Cell cycle analysis of the resistant cell line 4 weeks after incubation without eribulin. The left panel represents analysis of the resistant cell line. The right panel represents analysis of the revertant cell line which was incubated without eribulin for 4 weeks. There was no significant difference in each cell cycle, so chemoresistance to eribulin was maintained 4 weeks after incubation without eribulin. (f) The doubling time of three cell lines, parental, resistant, and revertant, was measured by using CellTiter-Glo. The solid line represents the parental cell line, the dotted line represents the resistant cell line, and the broken line represents revertant used in Figure 3(e). Values represent mean ± SD.