Figure 1. Identification of an estradiol-induced prostaglandin biosynthesis signature in cells and preclinical models. (a) ELT3 cells were treated with 10 nM estradiol for 24 h. Cellular metabolites were profiled by mass spectrometry (n = 5; Metabolon LC-MS/MS). Box plots of PGE2 (6.0–59.6 ng), PGD2 (1.3–7.2 ng), and 6-keto PGF1α (2.5–18.3 ng) are shown. Data show the mean of five sets of independent samples. **, P < 0.01; Welch’s t tests and Wilcoxon rank sum tests. (b) Immunoblot analysis of ELT3 cells treated with estradiol for 2 or 24 h. β-Actin was used as a loading control. (c and d) Secreted PGE2 levels were measured in conditioned media collected from cells treated with estradiol or control at the indicated times. Data were normalized to control group (n = 3; ELISA). Results are representative of six sets of independent samples per group from three experiments. (e and f) LAM patient–derived cells were serum-starved for 24 h, and then treated with estradiol for 0.25, 0.5, 2, 4, 6, 8, or 24 h. Immunoblot analyses of tuberin, phospho-Akt S473, phospho-MAPK (T202/Y204), and phospho-S6 (S235/236). (g and h) Immunoblot analyses of COX-2, phospho-S6 (S235/236), phospho-MAPK (T202/Y204), and phospho-Akt S473. (i and j) LAM patient–derived cells treated with 50 µM PD98059 or 5 µM PI-103 for 1 h, and then treated with 10 nM estradiol for 24 h. Immunoblot analyses of COX-2, phospho-S6 (S235/236), phospho-MAPK (T202/Y204), and phospho-Akt S473. (e–j) Results are representative of three different experiments. (k) Levels of phospho-MAPK (Thr202/Tyr204) and phospho-S6 (Ser235/236) from xenograft tumors from placebo or estradiol-treated CB17-scid mice were measured by immunoblotting. (l) Cellular metabolites extracted from xenograft tumors from estradiol or placebo-treated mice were profiled (n = 8; Metabolon LC-MS/MS). Box plots of PGE2 (23.3–133 ng), PGD2 (6.8–76.96 ng), and 6-keto PGF1α (17.5–167.3 ng) are shown. (k and l) Results are representative of eight mice per group. *, P < 0.05; **, P < 0.01; Welch’s t tests and Wilcoxon rank sum tests. (m) Urinary PGE2 and creatinine from placebo or estradiol-implanted ovariectomized female mice bearing xenograft tumors was measured five-week after cell inoculation (ELISA). PGE2 levels were normalized to creatinine. Results are representative of four mice per group. *, P < 0.05; Student’s t test.
Figure 1.

Identification of an estradiol-induced prostaglandin biosynthesis signature in cells and preclinical models. (a) ELT3 cells were treated with 10 nM estradiol for 24 h. Cellular metabolites were profiled by mass spectrometry (n = 5; Metabolon LC-MS/MS). Box plots of PGE2 (6.0–59.6 ng), PGD2 (1.3–7.2 ng), and 6-keto PGF1α (2.5–18.3 ng) are shown. Data show the mean of five sets of independent samples. **, P < 0.01; Welch’s t tests and Wilcoxon rank sum tests. (b) Immunoblot analysis of ELT3 cells treated with estradiol for 2 or 24 h. β-Actin was used as a loading control. (c and d) Secreted PGE2 levels were measured in conditioned media collected from cells treated with estradiol or control at the indicated times. Data were normalized to control group (n = 3; ELISA). Results are representative of six sets of independent samples per group from three experiments. (e and f) LAM patient–derived cells were serum-starved for 24 h, and then treated with estradiol for 0.25, 0.5, 2, 4, 6, 8, or 24 h. Immunoblot analyses of tuberin, phospho-Akt S473, phospho-MAPK (T202/Y204), and phospho-S6 (S235/236). (g and h) Immunoblot analyses of COX-2, phospho-S6 (S235/236), phospho-MAPK (T202/Y204), and phospho-Akt S473. (i and j) LAM patient–derived cells treated with 50 µM PD98059 or 5 µM PI-103 for 1 h, and then treated with 10 nM estradiol for 24 h. Immunoblot analyses of COX-2, phospho-S6 (S235/236), phospho-MAPK (T202/Y204), and phospho-Akt S473. (e–j) Results are representative of three different experiments. (k) Levels of phospho-MAPK (Thr202/Tyr204) and phospho-S6 (Ser235/236) from xenograft tumors from placebo or estradiol-treated CB17-scid mice were measured by immunoblotting. (l) Cellular metabolites extracted from xenograft tumors from estradiol or placebo-treated mice were profiled (n = 8; Metabolon LC-MS/MS). Box plots of PGE2 (23.3–133 ng), PGD2 (6.8–76.96 ng), and 6-keto PGF1α (17.5–167.3 ng) are shown. (k and l) Results are representative of eight mice per group. *, P < 0.05; **, P < 0.01; Welch’s t tests and Wilcoxon rank sum tests. (m) Urinary PGE2 and creatinine from placebo or estradiol-implanted ovariectomized female mice bearing xenograft tumors was measured five-week after cell inoculation (ELISA). PGE2 levels were normalized to creatinine. Results are representative of four mice per group. *, P < 0.05; Student’s t test.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal