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. 2014 Dec 9;16(12):1070–1081. doi: 10.1016/j.neo.2014.09.013

Figure 3.

Figure 3

ER-α is responsible for centrosome amplification. (A) Tax sensitivity is not altered by HIFs. Si-HIF-1α or HIF-2α did not sensitize A498 cells to Tax-induced cell death. A498 cells were transfected indicated Si-RNA for 24 hours and incubated with Tax for 72 hours. Cell viability was determined by MTT assay. (B) Elimination of ER-α can restore the sensitivity to Tax and Colcemid (Colce). Si-ER-α sensitized Tax (3 μM) or Col (2 μM)-induced cell death in VHL-deficient A498 cell. A498 cells were transfected with Si-Con (Si-control) or Si-ER-α for 24 hours. After Tax or Col treatment for 72 hours, the viability of transfected A498 cells was monitored by MTT assay. (C) ER-α overexpression induces Tax-resistance. VHL-intact ACHN cells were transfected with ER-α expression vector (upper panel) for 24 hours. MTT assay was used for cell viability measurement (lower panel). (D) pVHL and ER-α are involved only in Tax-sensitivity but not Adriamycin-induced cell death (Adr; 2 μg/ml). Comparing to Tax, Adriamycin sensitivity was not affected pVHL or ER-α status. HCT116 p53 −/− cells were transfected with indicating vectors. After Taxol treatment, the cell viability was monitored by MTT assay. (E and F) Fulvestrant (FST) can block centrosome amplification in VHL-deficient C2 cell. C2 cells were incubated with FST (2 μM) or Estrogen (Est; 1 μg/ml) for 72 hours and IF stained with γ-tubulin (green) and DAPI (blue). Reduction of centrosome was detected in FST-treated cells (E). Centrosome number was counted from about 50 cells of each condition and presented as graph (F). (G and H) The effect of FST on centrosome in A498. Suppression effect of FST on centrosome amplification in A498 was confirmed by IF staining with γ-tubulin Ab (G) and counting (H). Experimental condition was identical to above. (I) Inhibitory effect of FST on γ-tubulin expression. FST reduced Est-induced γ-tubulin expression in A498 cells. (J) Est induces γ-tubulin expression in C2V. Treatment of Est for 72 hours could induce γ-tubulin. However, due to strong background expression, additional induction of γ-tubulin in C2 was not obviously detected. (K) Est promotes centrosome amplification in VHL-intact cell lines, C2V and ACHN. Cells were incubated with Est for 72 hours and IF stained with γ-tubulin Ab (green) and DAPI (blue). (L) The different effect of Tamoxifen (Tam) and FST on Tax-resistance. Tam (2 μM), FST and Tax were treated for 72 hours in VHL-negative A498 cells. The cell viability was estimated by MTT assay.