Fig 1: Oxygen-dependent regulation of migration by estrogen in HT-29 cells(A) Wound healing assay in HT-29 cells under normoxic (20.9% O2) and hypoxic (2% O2) conditions. Representative images are shown of the scratches at time 0 and following 24h. Dashed lines indicate wound margins. Graph shows the % wound closure under each condition. Cells were treated with 10nM estradiol (E2), 1µM G1 (a GPER-selective agonist) or 5µM G15 (a GPER-selective antagonist). Mean ± SEM, n=6, **P<0.01, ***P<0.001, ****P<0.0001. (B) Boyden chamber migration assay in HT-29 cells under normoxic and hypoxic conditions. Representative images are shown. Cells were treated with E2, G1, G15, G15+E2 or G15+G1. Mean ± SEM, n=6, *P<0.01 E2 and G1 treatment compared to Ctl (Veh only), †P<0.01 G15+E2 compared to E2 treatment, ‡P<0.01 G15+E2 compared to G15 treatment, §P<0.01 G15+G1 compared to G1 treatment, ***P<0.0001 G15 compared to E2 and G1, †††P<0.001 G15+E2 treatment compared to E2 treatment, §§§P<0.0001 G15+G1 compared to G1 treatment.
Fig 2: Semi-quantitative analysis of western blotting showing that levels of GPR30 were significantly lower in HTR8/SVneo cells that had been treated with E2 or G1 in conditions of H/R (black bar) as compared with normoxic conditions (white bar). GPR30, G-protein coupled receptor 30; E2, 17ß-estrogen; H/R, hypoxia/reoxygenation.
Fig 3: (A) Western blotting and (B) semi-quantitative analysis indicating that levels of GPR30 were significantly increased in HTR8/SVneo cells that had been treated with E2 or G1 in normal conditions. The increased levels of GPR30 induced by E2 were inhibited by G15. (C) Western blotting and (D) semi-quantitative analysis indicated that levels of GPR30 were significantly increased in HTR8/SVneo cells that had been treated with E2 or G1 under conditions of H/R. The increased levels of GPR30 induced by E2 were inhibited by G15. GPR30, G-protein coupled receptor 30; E2, 17ß-estrogen; H/R, hypoxia/reoxygenation; DMSO, dimethyl sulfoxide.
Fig 4: Mitotic laggards and whole chromosomal instability depend on estrogen-activated GPER1 function.(A) HCT116 cells were transiently transfected with SCRAMBLED (Scr) or GPER1-specific siRNA (GPER) after treatment with DMSO, 10 nM 17ß-estradiol (E2), bisphenol A (BPA), diethylstilbestrol (DES), or 5 nM nocodazole (Noc) and synchronization in the anaphase of mitosis as described in (9). The quantification of the amount of anaphase lagging chromosomes is shown (mean ± s.d., n = 4 with a total of 400 cells). ANOVA was used to calculate the P-value of DMSO + GPER1 siRNA. Wald’s z-statistics computed by the R function glmmTMB was used to calculate the P-value of the remaining treatments. ns, not significant; *P < 0.05 and **P < 0.01. (B) CCD 841 CoN cells pretreated with G15 for 30 min after additional treatment as in (A) for 48 h (median ± s.d., n = 3 with a total of 300 cells). ANOVA was used to calculate the P-value of DMSO + GPER1 siRNA and all G15 treatments. The bootstrap procedure was used to calculate the P-value of the remaining treatments. ns, not significant; *P < 0.05; **P < 0.01; and ***P < 0.001. (C) HCT116 single-cell clones stably expressing SCRAMBLED (Scr) or shRNAs targeting GPER1 (GPER) were grown for 30 generations in DMSO, 10 nM E2, BPA, DES, or 5 nM Noc. The graph shows the amount of cells harboring an aneuploid karyotype with chromosome numbers deviating from the modal (modal number = 45). Data were collected from 50 cells per clone (n = 1 biological replicate). (D) CCD 841 CoN cells pretreated with G15 for 30 min before additional exposure to DMSO, 10 nM E2, BPA, DES, or 5 nM Noc for 30 generations. The graph shows the amount of cells (N = 50) harboring an aneuploid karyotype with chromosome numbers deviating from the modal (modal number = 46); (n = 1 biological replicate). (E) Chromosome number variability/whole chromosomal instability of HCT116 cell clones depicted from (C). For each cell clone, the distribution of individual chromosome numbers was determined from 50 metaphase spreads. (F) Chromosome number variability/whole chromosomal instability of CCD 841 CoN cells depicted from (D). For each condition, the distribution of individual chromosome numbers was determined from 50 metaphase spreads. A detailed description of statistics is provided in the Materials and Methods section.P-values are available for this figure.
Fig 5: Oxygen-dependent regulation of wound healing and migration by estradiol in DLD-1 cells(A) Wound healing assay in DLD-1 cells under normoxic (20.9% O2) and hypoxic (2% O2) conditions. Representative images are shown of the scratches at time 0 and following 24h. Dashed lines indicate wound margins. Graph shows the % wound closure under each condition. Cells were treated with 10nM estradiol (E2), 1µM G1 (a GPER-selective agonist) or 5µM G15 (a GPER-selective antagonist). Mean ± SEM, n=6, ***P<0.001, ****P<0.0001. (B) Boyden chamber migration assay in DLD-1 cells under normoxic and hypoxic conditions. Representative images are shown. Cells were treated with E2, G1, G15, G15+E2 or G15+G1. Mean ± SEM, n=6, *P<0.01 E2 and G1 treatment compared to Ctl (Veh only), †P<0.01 G15 compared to G1 and E2 treatment, ‡P<0.01 G15+E2 compared to G15 treatment, §P<0.01 G15+E2 compared to E2 and G1 treatment, #P<0.01 G15+G1 compared to G15, ***P<0.0001 G15 compared to E2 and G1, †††P<0.001 G15+E2 treatment compared to E2 or G1 treatments, ‡‡‡P<0.0001 G15+G1 compared to E2 or G1 treatments.
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