Figure 6.

Silencing of FOXO1 or overexpression of CCND1 counteracts the role of Pae on 4-OHT resistance in BC. MCF-7/4-OHT cells were treated with si-FOXO1 with Scr as a control, while T47D/4-OHT cells were transfected with CCND1 with pcDNA3.1 as a control. (A) The expression of FOXO1 or CCND1 mRNA in drug-resistant cells assayed by RT-qPCR; (B) the IC₅₀ values of 4-OHT on MCF-7/4-OHT cells with FOXO1 knockdown or T47D/4-OHT cells overexpressing CCND1 in the presence of 100 μg/mL Pae examined by CCK-8 assays; (C) the apoptosis levels of MCF-7/4-OHT cells with FOXO1 knockdown or T47D/4-OHT cells overexpressing CCND1 in the presence of 100 μg/mL Pae detected by flow cytometry under treatment of 4 μM and 8 μM 4-OHT, respectively; (D) the apoptosis levels of MCF-7/4-OHT cells with FOXO1 knockdown or T47D/4-OHT cells overexpressing CCND1 in the presence of 100 μg/mL Pae assessed by Caspase-3 activity kit; (E) the cytotoxicity of MCF-7/4-OHT cells with FOXO1 knockdown or T47D/4-OHT cells overexpressing CCND1 in the presence of 100 μg/mL Pae determined by a LDH kit; (F) colony formation of MCF-7/4-OHT cells with FOXO1 knockdown or T47D/4-OHT cells overexpressing CCND1 in the presence of 100 μg/mL Pae tested by colony formation assays; (G) number of S-phase cells transfected with si-FOXO1 or CCND1 in the presence of 100 μg/mL Pae detected by EdU assays. Data are displayed in the form of mean ± SD. All experiments were repeated at least three times. In panel (B) two-way ANOVA along with Tukey’s multiple comparison was applied, while in the rest panels, one-way ANOVA along with Tukey’s multiple comparison was used. *p < 0.05, **p < 0.01.

Abbreviations: FOXO1, forkhead box O1; CCND1, cyclin D1; Pae, paeoniflorin; BC, breast cancer; miR, microRNA; 4-OHT, 4-hydroxytamoxifen; CCK-8, cell counting kit-8; LDH, lactose dehydrogenase; EdU, 5-ethynyl-2ʹ-deoxyuridine; SD, standard deviation; ANOVA, analysis of variance.