Journal: Toxicological Sciences

Article Title: Acetaminophen Modulates the Expression of Steroidogenesis-Associated Genes and Estradiol Levels in Human Placental JEG-3 Cells

doi: 10.1093/toxsci/kfaa160

Figure Lengend Snippet: Gene Expression in Placental JEG-3 Cells After Acetaminophen Treatment

Article Snippet: For CYP19A1, the HRP-linked antibody (Cell signaling, catalog #7076) was used at a 1:1000 dilution.

Techniques: Gene Expression

Journal: Toxicological Sciences

Article Title: Acetaminophen Modulates the Expression of Steroidogenesis-Associated Genes and Estradiol Levels in Human Placental JEG-3 Cells

doi: 10.1093/toxsci/kfaa160

Figure Lengend Snippet: CYP19A1 protein expression in relation to acetaminophen exposure in placental JEG-3 cells. JEG-3 cells were cultured and treated with acetaminophen (0.1, 1, or 5 mM) for 24 h and compared with the vehicle control (0.5% DMSO) using a Western blot. A, Representative image of a Western blot where GAPDH was used as a loading control. The relative optical density (OD) was expressed relative to control (0.5% DMSO treated) cells. B, Densitometry results are shown as the percent change in protein expression relative to control. Each endpoint represents the mean ± SD of n = 4 independent experiments. Asterisks (*) represents statistically significant differences (p < .05) compared with controls.

Article Snippet: For CYP19A1, the HRP-linked antibody (Cell signaling, catalog #7076) was used at a 1:1000 dilution.

Techniques: Expressing, Cell Culture, Control, Western Blot

Journal: Annals of Translational Medicine

Article Title: RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells

doi: 10.21037/atm-22-393

Figure Lengend Snippet: RAF1 involved in estradiol secretions by activating ERK phosphorylation. The primary ovarian GCs were treated with RAF709, 5 nM for 6 h then induced by FSH (100 ng/mL) for 24 h. (A-D) The protein expression ratios of FSHR, RAF1, ERK-P, and CYP19A1 were respectively detected by WB, and the protein ratios were analyzed in treatment groups relative to the GAPDH protein abundance. (E) Estradiol content was measured in each treatment group by RIA. The values are the means ± SEM of three independent experiments. Different letters indicate a significant difference between the compared groups (P<0.05). Different letters (a, b, c, and d) between two bars show a significant difference (a versus b, b versus c, and c versus d: P <0.05).

Article Snippet: All reagents and antibodies were commercially available, and included RAF709 (HY-100510, MCE); anti-Raf1 (ab137435, Abcam); FSHR(1:1,000; sc-13935, Santa); GAPDH (1:2,000; Am4300, Ambion); CYP19A1 antibody (1:2,000; BA3704, Boster); P-ERK antibody (1:1,000; CST); goat anti-rabbit IgG (1:5,000, ZB-2301; Zhongshan, Beijing, China); ECL Western blotting substrate (32209; Thermo Scientific, Waltham, MA); DMEM/F12 (D2906; Sigma); fetal bovine serum (FBS, Gibco); streptomycin (Sigma); corn oil (Sigma); FSH (Boleide, Beijing, China).

Techniques: Phospho-proteomics, Expressing, Quantitative Proteomics

Journal: Annals of Translational Medicine

Article Title: RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells

doi: 10.21037/atm-22-393

Figure Lengend Snippet: RAF1 acts as a downstream molecule to mediate the FSH signaling pathway to stimulate E2 synthesis and secretion in vivo. Mice were given a single dose of FSH (10 IU/mouse) by intraperitoneal injection, and after 24 h, were injected with RAF709. After 24 h, blood samples of mice were collected for E2 content. Vegetable oils were used as RAF709 reference substance and PBS served as a comparison with FSH for first injections. (A-D) FSHR, RAF1, ERK-P, and CYP19A1 protein expression in each treatment group detected by WB. Data were analyzed by GraphPad Prism version 5. (E) Estradiol content in mouse serum was measured in each treatment group by RIA. The same letters indicate the difference is not significant, and different letters indicate the difference is significant (P<0.05). The values are the means ± SEM of three independent experiments. Different letters (a, b, c, and d) between two bars show a significant difference (a versus b, b versus c, and c versus d: P <0.05).

Article Snippet: All reagents and antibodies were commercially available, and included RAF709 (HY-100510, MCE); anti-Raf1 (ab137435, Abcam); FSHR(1:1,000; sc-13935, Santa); GAPDH (1:2,000; Am4300, Ambion); CYP19A1 antibody (1:2,000; BA3704, Boster); P-ERK antibody (1:1,000; CST); goat anti-rabbit IgG (1:5,000, ZB-2301; Zhongshan, Beijing, China); ECL Western blotting substrate (32209; Thermo Scientific, Waltham, MA); DMEM/F12 (D2906; Sigma); fetal bovine serum (FBS, Gibco); streptomycin (Sigma); corn oil (Sigma); FSH (Boleide, Beijing, China).

Techniques: In Vivo, Injection, Comparison, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase

doi: 10.3390/ijms25031440

Figure Lengend Snippet: Measurement of enzymatic activity of recombinant CYP19A1 wild-type and mutant proteins. In two sets of experiments, 3 H-testosterone and 3 H-androstenedione were converted to 3 H-estradiol and 3 H-estrone, respectively, under standardized reaction conditions. After a reaction time of 30 min, the products were separated via HPLC and the 3 H activity was measured in the collected fractions. The X -axis shows the mean values of the 3 H activity in the androgen substrates and in the estrogen products of three experiments, each as a percentage of the input 3 H activity. Error bars indicate standard errors of the mean.

Article Snippet: The anti-CYP19A1 mouse monoclonal antibody raised against polypeptide AA376-390 of human CYP19A1 (clone H4, SM2222P, Acris Antibodies, Herford, Germany) and the anti-ACTB mouse monoclonal antibody against chicken beta-actin (sc-47778, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) served as the primary antibodies.

Techniques: Activity Assay, Recombinant, Mutagenesis