E7023

mIMCD-3 cells (ATYCC CRL-2123, American Type Culture Collection) were cultured as previously described [27 (link)] in Dulbecco’s Modified Eagle Medium (F12, ThermoFisher Scientific) containing 10% fetal bovine serum (ThermoFisher Scientific) and 1% penicillin-streptomycin (ThermoFisher Scientific). Cells were incubated at 37 °C in 5% CO₂-95% air. Passages 4–6 were used. Cells were grown in 12-well plates and allowed to reach 100% confluency. Cells were serum starved for 3 h, then they were treated with 17ß-estradiol (E₂, E2758, Sigma-Aldrich Co.) or vehicle for 24 h at final concentrations of 10, 100, or 1000 nM. E₂ was dissolved in 0.1% ethanol (molecular grade, E7023, Sigma-Aldrich Co.). Values reported are means ± SE and represent results of cells from three experiments with cell lysates assayed in triplicate.

SK-Hep1 cells were seeded in 6-well plates (~1000-5000 cells per well) and subjected to the indicated treatments, with the drug being removed to terminate the treatment. Two weeks later, plates were washed in PBS, fixed with 100% methanol (Sigma-Aldrich, E7023) and stained with a filtered solution of 5% w/v crystal violet (crystal violet, Sigma-Aldrich, C3886). After washing with tap water, the colonies were counted both manually (by eye) and digitally using a ColCount automated colony counter.

The collected nasal mucosa tissues of mice were fixed in 10% neutral formalin (HT501128, Sigma-Aldrich, St. Louis, MO) for 24 h, and then embedded in paraffin (P3558, Sigma-Aldrich, St. Louis, MO). Next, the tissues were made into sections using paraffin slicer (E0972, Beyotime, Shanghai, China). Subsequently, the sections were immersed in xylenes (247642, Sigma-Aldrich, St. Louis, MO) for deparaffinization. After that, the tissue samples were placed in different gradients of ethanol prepared with 100% ethanol (E7023, Sigma-Aldrich, St. Louis, MO) and double distilled water for sample hydration. Later, the tissue sections were soaked and washed three times for 3 min using phosphate buffer saline solution (PBS; C0221A, Beyotime, Shanghai, China). After being stained thoroughly with haematoxylin solution (C0107, Beyotime, Shanghai, China) for 10 min and eosin solution (C0109, Beyotime, Shanghai, China) for 3 min, the sections were soaked with xylene twice for 4 min each time, and then the tissue samples were sealed with neutral resin. Finally, the sections were observed and photographed under an optical microscope (DMi8, Leica, Wetzlar, Germany) at a magnification of ×200.

RNA was isolated from the abdominal fat bodies using Trizol LS (ThermoFisher, #10296028). 300 μL of Trizol LS was added to fat bodies in 100 μL of PBS. The tissue was homogenized with a motorized pestle for 2 minutes before adding another 600 μL of Trizol LS (3:1 mixture of Trizol LS:PBS). The resulting solution was centrifuged at 12,000g for 10 minutes at 4°C. The aqueous supernatant layer was collected in a new tube, while carefully avoiding disturbing the other layers of the phase separated solution. RNA was extracted from the aqueous supernatant layer by vigorously shaking with 240 μL of chloroform (Fisher Scientific, #C298), again carefully avoiding other layers following phase separation. The aqueous phase was transferred to a new tube and the RNA was precipitated by incubating at room temperature with 500μL of 100% isopropanol (Sigma-Aldrich, #I9516). Following centrifugation at 12,000g for 10 minutes at 4°C, the supernatant was removed leaving only the RNA pellet. The pellet was washed with 1 mL of 75% ethanol (Sigma-Aldrich, #E7023), then air dried for 5–10 minutes before resuspension in RNase–free water.