Primary antibodies including anti-β-catenin (BD Bioscience, USA), anti-wnt1 (ab15251, Abcam, UK), anti-CyclinD1 (ab6125, Abcam, UK), MK-2206 cell line anti-c-Myc (ab32, Abcam, UK) were applied, followed by incubation with secondary antibodies (Goat Anti-rabbit IgG, ZB2301; Goat Anti-mouse IgG, ZB2305, Zhongshan Golden Bridge Biotechnology CO., LTD., China). Blots were developed by ChemiDoc XRS System (Bio-Rad, USA). Statistical analysis Student’s independent-samples t-test, one-way ANOVA,
and χ 2-test were used for statistical analysis by SPSS 10.0 software (SPSS, China, 657180). P < 0.05 was considered significant. Results The effect of CKI on the number of SP cells in vitro In Figure 1A, the P3 gate showed the SP cells with Hoechst 33342 negative/dim. SP cells accounted for approximately 2.7% of total cells. The percentage of SP population was decreased markedly by treatment E2 conjugating inhibitor with verapamil, which was consistent with the reports that verapamil could prohibit Hoechst 33342
efflux . Figure 1 Analysis of SP cells by CKI treatment. (A) MCF-7 cells were labeled with Hoechst 33342 and analyzed by flow cytometry or with the addition of Verapamil. The percentage of SP cells appeared as the Hoechst low fraction in the P3 is about 2.7%. (B) MCF-7 cells were treated with CKI (30 μl/ml, 50 μl/ml, 70 μl/ml) for 48 h, and SP cells were analyzed by flow cytometry. P3 gate is the percentage of SP cells. Data from a representative experiment (from a total of three) are shown. To determine whether the SP cell number decreased with CKI treatment, cells were treated with a range of concentrations of CKI (30, 50, 70 μl/ml) for 48 hours and then the
SP cells were analyzed by flow cytometry. The results showed that the size of the SP population was decreased by CKI treatment in a dose-dependent manner (Figure 1B). However, our previous study didn’t find the same phenomena in the cisplatin-treated cells, which were broadly used as an anti-breast cancer agent . Canonical Wnt/β-catenin pathway analysis on CKI group in vitro RT-PCR analysis was used to investigate whether CKI could Combretastatin A4 down-regulate Mirabegron the expression of the main genes of Wnt/β-catenin Pathway. Sorted SP cells were treated with CKI (70 μl/ml) for 48 h and then analyzed by Quantitative RT-PCR. The study found a dramatic decrease of β-catenin, CyclinD1, c-Myc at the mRNA level with CKI treatment (Figure 2). Figure 2 The main genes of Wnt/β-catenin pathway was down-regulated in the CKI group in vitro. Quantitative RT-PCR analysis revealed that the expression of β-catenin, CyclinD1 and c-Myc (mean ± SD) were lower in CKI group than those in the control group. Most of the differences were statistically significant (** P < 0.01,*** P < 0.001). SP cells are more tumorigenic in vivo SP (P3) and non- SP (P4) cells were isolated by flow cytometry and collected for this experiment (Figure 3A, B). Tumorigenicity assays were performed by injecting MCF-7 unsorted, SP and non-SP cells into NOD/SCID mice.