Estrogen Signaling Pathways in JKT-1 Cells

JKT-1 cells were seeded in six-well plates (0.6 × 10⁶ cells/well). After 48 h, the JKT-1 cells were washed and oestrogen starved overnight in phenol red-free DMEM supplemented with 1% charcoal-stripped FBS. We then added E2 (Sigma-Aldrich^®, Saint Louis, MO, USA), freshly prepared E2-BSA (Sigma-Aldrich^®, Saint Louis, MO, USA) devoid of free E2, which is removed by filtration, ICI-182,780 (fulvestrant; Falsodex^®, AstraZeneca, Wilmington, DE, USA), G1 (Calbiochem^®, Merck KGaA, Darmstadt, Germany), G15 (kindly supplied by Eric R. Prossnitz) [42 (link)], or ethanol (as a vehicle control) at 10⁻⁹ M concentration [15 (link),43 (link)], and incubated them for 24 h. We harvested the cells using trypsin and counted them using the Vi-CELL software (Beckman Coulter, Fullerton, CA, USA). Results are expressed as percentages of variation compared with the control.