Fig 2: Aromatase promoters pI.f/I.4 in U-87 MG glioblastoma cells are stimulated upon Ang II exposure. (A,B) Upper panel, schematic map of the pI.f and pI.4 aromatase constructs. U-87 MG glioblastoma cells were transiently transfected with the reported constructs and treated with vehicle (-) or Ang II 5 µM, alone or in combination with LOS 5 µM for 24 h. (C,D) U-87 MG glioblastoma cells were treated in the presence of vehicle (-) or Ang II 5 µM for 3 h. Nuclear extracts were incubated with a biotinylated oligonucleotide containing the AP-1 or AP-1 mutant site in the aromatase promoters pI.f (left panel) or with a biotinylated oligonucleotide containing the STAT3 or STAT3 mutant site in the aromatase promoters pI.4 (right panel) and subjected to DNA affinity precipitation assay. Specifically bound proteins were subjected to Western blotting analysis. The specificity of the binding was tested by loading the unbound fraction (Negative Control). U-87 MG glioblastoma cells nuclear extracts were used as positive control. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (-) for Ang II treatment. (E,F) U-87 MG glioblastoma cells were treated in the presence of vehicle (-) or Ang II 5 µM for 3 h, then cross-linked with formaldehyde and lysed. The precleared chromatin was immunoprecipitated with anti-c-Jun or anti-RNA Pol II (left panel) or with anti-STAT3 and anti-RNA Pol II (the right panel). The 5'flanking sequence of the CYP19A1 gene was detected by real-time PCR with specific primers to amplify aromatase promoter sequence, including the AP-1 and STAT3 sites. Input DNA was amplified as loading controls. Data are expressed as means ± SD of three different experiments, each performed in triplicate. * p < 0.05; *** p < 0.001.

Fig 3: Effects of Ang II and LOS on aromatase expression and activity in U-87 MG and T98G glioblastoma cells. (A,B) Real-time RT-PCR and immunoblotting assay for CYP19A1 mRNA and protein expression in U-87 MG and T98G glioblastoma cells, treated with vehicle (-) or the Ang II 0.1, 5, and 10 µM for 24 h. GAPDH was used as a loading control. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (-) for Ang II treatment. (C,D) CYP19A1 mRNA and protein expression in U-87 MG and T98G glioblastoma cells treated with vehicle (-) and Ang II 5 µM alone or in combination with LOS 5 µM for 24 h. The histograms represent the mean average ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary unit and expressed as fold change over vehicle (-) for Ang II treatment or fold change over Ang II for Ang II in combination with LOS, 5 µM. GAPDH was used as a loading control. (E,G) Aromatase activity in U-87 MG and T98G glioblastoma cells treated with vehicle (-) and Ang II 5 µM alone or in combination with LOS 5 µM for 24 h. (F,H) ELISA for Estradiol secretion in U-87 MG and T98G glioblastoma cells treated with vehicle (-) and Ang II 5 µM alone or in combination with LOS 5 µM for 24 h. Data represent the mean ± SD of three different experiments, each performed in triplicate. ** p < 0.01; *** p < 0.001.

Fig 4: The morphology and gene expression in gonads at E10 chicken after AI treatment. (A–C) Gonads on top of the mesonephros in male, female, and AI-treated female chicken, respectively. (R) indicates right gonad, (L) indicates left gonad. (D–F) HE staining of gonads in male, female, and AI-treated female, respectively. In all HE staining images, the left gonad was selected. Scale bar as is shown in pictures, thickness = 6 µm. (G–L) Q-PCR expression of DMRT1, SOX9, AMH, CYP19A1, FOXL2, and miR-2954, respectively. Error bars show standard error of the mean (SEM) from at least three biological replicates in each group. Asterisks indicate statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.0001. (M1–M3,N1–N3,O1–O3,P1–P3) Immunofluorescence results of DMRT1, SOX9, FOXL2, and CYP19A1 in control and AI-treated gonads. (Q1–Q3) In situ hybridization of miR-2954 in male, female, and AI-treated female. Blue fluorescence shows DAPI, which marks the nucleus; red fluorescence indicates expression of sex markers or miR-2954. Scale bar = 200 µm, thickness = 10 µm.

Fig 5: Aromatase and clinical outcomes in GBM patients. Kaplan–Meier survival analysis relating CYP19A1 levels and overall survival (OS) in GBM patients (TCGA dataset).