Estradiol

eSF were treated with ovarian steroids as described [57 (link)]. This treatment protocol allows eSF to differentiate appropriately in response to estradiol and progesterone [15 , 57 (link)], including proper induction of decidualization, the progesterone-driven differentiation of eSF during the secretory phase in preparation for implantation. Briefly, cells were treated with 0.1% ethanol as vehicle control, 10 nM estradiol (Sigma-Aldrich), or 10 nM estradiol and 1 μM progesterone (Sigma-Aldrich) in SCM made with 2% instead of 10% FBS. Cells were treated for 14 d, with feeding every other day. Decidualization of cells in the presence of estradiol and progesterone was confirmed by a human IGF-binding protein 1 (IGFBP1) ELISA kit (Alpha Diagnostics International and Abcam). Samples treated with estradiol and progesterone effectively decidualized as assessed by secretion of IGFBP-1 (level > 500 ng/100K cells in 0.5 ml of culture) [15 ].

To determine the effect of phalloidin on shoot induction, calli were cultured on SIM containing various concentrations of phalloidin (prepared in DMSO as a 20 mmol/L stock; Sigma) for 20 days. The regeneration frequencies of shoots under treatments of various concentrations of phalloidin were statistically analyzed, and 90 samples for each treatment were collected. Means and standard deviations were calculated (shown as ‘mean ± SD’ in Table 1). Callus cultured on SIM containing 5 μmol/L phalloidin for 0, 4, 8, 12, and 16 days were individually harvested to isolate total RNA for qRT-PCR analysis.
To induce transcription of artificial microRNAs, the calli were cultured on SIM with 10 μmol/L estradiol (prepared in DMSO as a 10 mmol/L stock; Sigma) for 20 days and estradiol was added every 2 days. Total RNA was isolated from the callus on SIM with estradiol at 12 days and used for qRT-PCR to determine the expression of ADF genes.

MCF-7 and MCF-10A cells (60% confluence) were cultured as described above in T25 flasks with the corresponding culture media in the absence or in the presence of either 20 nM estradiol (Sigma) or 20 nM estradiol plus 2 μM tamoxifen (Sigma), for 12, 24 or 48 h. After incubation, cells were harvested after treatment with 0.1% trypsin (Gibco) for 5 min and processed for immunoblotting.

For inducible expression of BSCTV C4 in Arabidopsis, the pER8-C4 plasmid described previously was used (Lai et al., 2009 (link)). To generate the C8S mutation versions of this plasmid, a 5′-primer harboring the mutation site was used in PCR amplification. The constructs were transformed into Agrobacterium EHA105, which was then used to transform wild-type Arabidopsis (Columbia) by the floral-dip method (Clough and Bent, 1998 (link)). The seeds were germinated on media including DMSO or 2 µM estradiol (Sigma), or germinated on regular MS medium for 7d and transferred onto media with or without 2 µM estradiol for an additional 3 weeks.

To assess tumorigenic potential of cells after PLK1 inhibition, SUM149PT cells were treated in vitro for 3 days with rigosertib or DMSO in estradiol-free cell culture medium. Subsequently, 1 × 10⁶ SUM149PT cells were resuspended in 100 µL matrigel (Corning, cat# 356237) and PBS (Gibco, cat# 20012-019) mixed (1:1) and injected into the fourth mammary fat pads of 8 to 12-week-old female NSG mice. Mice were supplemented with estradiol for the whole duration of the experiments in the drinking water, with 8 µg/mL estradiol (Sigma, cat# E8875; stock diluted in ethanol) as previously described [63 (link), 64 (link)]. For the limiting dilution experiment, mice were not supplemented with estradiol. SUM149PT cells were treated in vitro for 3 days with rigosertib or DMSO in estradiol-free cell culture medium. Subsequently, 100 000, 10 000 or 1 000 SUM149PT cells were resuspended in 100 µL matrigel and PBS mixed (1:1) and injected into the fourth mammary fat pads of 8 to 12-week-old female NSG mice. The frequencies of TICs were calculated and statistically compared using the Extreme Limiting Dilution Analysis (ELDA) online tool [65 (link)]. Tumour volumes were monitored as described above.