br Corresponding author br E
E-mail address: [email protected] (W. Li). 1 These authors contributed equally to this work.
2. Methods & materials
2.1. Cell culture and transfection
MCF7 and T47D cells were further transfected with shRNA CDCA8 and shRNA control (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's recommendation.
RNA was extracted with TRIzol (Life Technologies, Mulgrave, VIC, Australia) as the manufacturer indicated. iScript RT Supermix (Bio-Rad, Gladesville, NSW, Australia) was used to synthesize complementary DNA, and the transcripts were quantified with SYBR Green Mix (Invitrogen, Carlsbad, CA, USA) on a ViiA7 machine (Applied Biosystems, Thermo Fisher Scientific, Rockford, IL, USA). The reaction procedures were set up as 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Comparative CT method was utilized to detect the relative gene expression, which would calculate the change in Ct value between interest gene and the reference gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of designed primer were supplied in supplementary Table 1.
Cell lysates were loaded and separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which were further trans-ferred onto polyvinylidene difluoride membranes. 5% nonfat dry milk was utilized to block non-specific binding, then the membranes were incubated with CDCA-8, cyclin-dependent kinase inhibitor 1a (P21), cyclin-dependent kinase inhibitor 1b (P27), Cyclin D1 (CCND1), and B-cell leukemia/lymphoma 2 (BCL2) primary SB 431542 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a 1:1000 dilution (4 °C, overnight). Then, peroxidase-conjugated secondary antibodies (1:1000, 1 h, room temperature) were used to incubate the membranes, which was further developed with an ECL system (GE Healthcare Life Sciences, Chalfont, Buckinghamshire, UK). The relative intensity was calculated by correcting for β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) with NIH-Image J1.51p 22 software.
2.4. Cell proliferation assay
MCF7 and T47D cells (1 × 105) were cultured in 6-well plates in phenol red-free media containing 5% charcoal-stripped FBS with or without E2 (10 nmol/L) and the cell number was determined at the indicated days with the Z1 particle counter (Beckman Coulter, Inc., Brea, CA, USA).
2.5. Colony formation assay
Transfected or untreated MCF7 and T47D cells (1 × 103) were cultured on a six-well plate. After two weeks, 4% paraformaldehyde was used to fix the forming colonies and crystal violet was utilized to stain the colony. Gene 703 (2019) 1–6
2.6. Cell cycle and apoptosis assay
The cells were fixed with 70% ethanol overnight, re-suspended with phosphate buﬀer saline (PBS), and incubated with propidium iodide (PI) (Life Technologies, Mulgrave, VIC, Australia) (10 mg/mL) for 30 min at 4 °C in the dark. The DNA content of cells was analyzed with a flow cytometer (excitation wave 488 nm; emission wave 610 nm) with LYSIS software (Becton Dickinson, San Jose, CA, USA). Apoptosis was detected with the Annexin-V FITC Apoptotic Detection Kit I (Becton Dickinson, San Jose, CA, USA).
Antigen retrieval was performed with 1 mM ethylenediaminete-traacetic acid (EDTA) in 10 mM Tris buﬀer (pH 8.5) at 120 °C for 3 min and nonspecific blocking was applied with 4% normal goat serum in PBS for 1 h. The 2-μm sections of breast carcinoma tissues (with the patient's consent) were incubated with CDCA8 antibody (1:150; Invitrogen, Carlsbad, CA, USA). Avidin: biotinylated enzyme complex, biotinylated secondary goat antibody, and 3,3′-diaminobenzidine sub-strate were utilized to develop signals (Zhongshan Goldenbridge Biotechnology, Guangzhou, Guangdong, China). The slides were further counterstained with hematoxylin and finally mounted with poly-vinylpyrrolidone mounting medium (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) and observed.
2.8. Statistical analysis
Data were shown as the mean ± standard deviation (S.D.). Student's t-test was performed to compare the diﬀerence between two groups, while the comparisons between multiple groups were per-formed with one-way analysis of variance. The association between CDCA8 expression and patient's survival was investigated by Kaplan-Meier analysis. p < 0.05 was considered to be statistically significant.
3.1. Up-regulated CDCA8 in E2-stimulated breast cancer cells
MCF7 and T47D cells were cultured with or without E2, and it clearly demonstrated that E2 stimulation could up-regulate CDCA8 expression at both the mRNA (Fig. 1A, p < 0.01) and protein levels (Fig. 1B) after 12 h and 24 h of incubation. All of these indicated that the residual amounts of E2 could induce the activation of CDCA8.