Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: List of primary antibodies used in this study. According to the technical information provided by the manufacturers, the commercial primary antibodies used in the study were verified by Knockdown or Relative expression to ensure that the antibody binds to the antigen stated.

Article Snippet: Aromatase NB100-1596 (Novus Biologicals) AB_10000919 , Rabbit/Polyclonal , C-terminal portion of the human aromatase protein (between residues 400–502) , Human, Mouse, Rat, Primate, Bovine, Rabbit , 1:100 , [ ] .

Techniques: Knockdown, Expressing, Binding Assay, Recombinant

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Primary and secondary antisera used for Western blot, IFL, and IHC.

Article Snippet: Aromatase (Cyp19) anti-rabbit polyclonal IgG (E-AB-64300, Elabscience: Houston, TX, USA) , 1:2000.

Techniques: Western Blot, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of Kiss1R, Kiss1, Cyp19, GnRH , and miR expression in mediobasal hypothalamus homogenates. Representative Western blots ( n = 4) for Kiss1R, Kiss1, and Cyp19 (A) and normalization of Kiss1R (B) , Kiss1 (C) , and Cyp19 signals (D) carried out against α-tubulin. Quantitative expression ( n = 6) of GnRH (E) and miR-132-3p , Mir-145-5p , let-7a-5p , and let-7b-5p (F–I) carried out by qPCR. Data are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin / U6 and the control group used as reference sample. C, control group (placebo injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA (SA), animals first received SR141716A and 30 min later received AEA treatment. ** p < 0.01, * p < 0.05 vs. the control group.

Article Snippet: Aromatase (Cyp19) anti-rabbit polyclonal IgG (E-AB-64300, Elabscience: Houston, TX, USA) , 1:2000.

Techniques: Expressing, Western Blot, Standard Deviation, Control, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of the intratesticular markers of steroidogenesis and GnRH . Representative Western blots for 3βHsd and Cyp19 (A) and normalization of 3βHsd (B) and Cyp19 (C) signals carried out against α-tubulin. Quantitative expression of 3βHsd , Cyp19 , and GnRH mRNA carried out by qPCR (D) . Data are representative of n = 6 different animals and are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin and the control group used as a reference sample. C, control group (placebo-injected animals); KP, animals injected with Kp10; * p < 0.05.

Article Snippet: Aromatase (Cyp19) anti-rabbit polyclonal IgG (E-AB-64300, Elabscience: Houston, TX, USA) , 1:2000.

Techniques: Western Blot, Expressing, Standard Deviation, Control, Injection

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: A novel variant of ER-alpha, ER-alpha36 mediates testosterone-stimulated ERK and Akt activation in endometrial cancer Hec1A cells

doi: 10.1186/1477-7827-7-102

Figure Lengend Snippet: Letrozole inhibits ER-α36-mediated ERK and Akt phosphorylation . Hec1A cells were pre-treated with 10 nM aromatase inhibitor letrozole (Lanes 3 and 4) for 1 h and then the cells were treated with vehicle (DMSO) (Lanes 1) and 10 nM testosterone (Lanes 2 and 4) for 5 min. Phospho-ERK1/2 (A) or phospho-Akt (B) were examined with specific antibodies. The same blot was stripped and probed with anti-total ERK or Akt antibodies. Hec1A cells were treated with 10 nM testosterone (Lanes 2) or together with 10 nM aromatase inhibitor letrozole (Lanes 3) overnight, and then aromatase expression was detected by Western blot using specific antibody against aromatase (C).

Article Snippet: Anti-aromatase antibody was purchased from Novus Biologicals (Novus Biologicals, Littleton, CO).

Techniques: Phospho-proteomics, Expressing, 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: Frontiers in Endocrinology

Article Title: SCM-198 Prevents Endometriosis by Reversing Low Autophagy of Endometrial Stromal Cell via Balancing ERα and PR Signals

doi: 10.3389/fendo.2022.858176

Figure Lengend Snippet: TNF-α promotes an imbalance of estrogen and progesterone signaling in eESCs. (A) The mRNA expression (n = 5) and concentration (n = 6) of TNF-α in eESCs and nESCs were detected via RT-PCR and ELISA. (B) The aromatase expression of eESCs and nESCs was detected via Western blotting (n = 3). (C, D) After treatment with TNF-α (10 ng/ml), R-7050 (5 µM), or TNF-α+R-7050 (10 ng/ml, 5 µM), the expressions of aromatase, ERα, Erβ, and PRB in eESCs were analyzed via Western blotting (C) , and the concentration of estrogen was detected via ELISA (n = 4) (D) . (E–H) Aromatase was silenced or overexpressed in eESCs for 48 h. Then, the expressions of aromatase and ERα were detected via Western blotting (E, G) , and the level of estrogen was assayed via ELISA (n = 4) (F, H) . (I, J) The protein expressions of aromatase and ERα were analyzed via Western blotting (I) , and the concentration of estrogen was detected via ELISA (n = 4) (J) . Continuous data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant).

Article Snippet: Nonspecific binding sites were blocked by incubating the membranes with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Next, the membranes were incubated overnight at 4°C with primary antibodies (1:1,000) against aromatase (#14528, CST, Boston, USA), ERα (#ab32063, Abcam, UK), PRB (#ab32085, Abcam, UK), LC3B (#3868, CST, USA), BECN1 (#ab207612, Abcam, UK), Bcl-2 (#2870, CST, USA), Bax (#12105, CST, USA), FN1 (#ab2413, Abcam, UK), vimentin (#5741, CST, USA), α-tubulin (#ab7291, Abcam, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#10112, Arigo, Taiwan, China).

Techniques: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Endocrinology

Article Title: SCM-198 Prevents Endometriosis by Reversing Low Autophagy of Endometrial Stromal Cell via Balancing ERα and PR Signals

doi: 10.3389/fendo.2022.858176

Figure Lengend Snippet: The proapoptotic effects of SCM-198 are brought about by downregulating the aromatase-estrogen pathway via the inhibition of TNF-α. (A) ELISA was utilized to detect the TNF-α concentration of eESCs treated with different doses of SCM-198 (n = 4). (B) The protein expressions of aromatase and ERα in eESCs treated with different doses of SCM-198 were measured via Western blotting (n = 3). (C, D) After treatment with TNF-α (10 ng/ml), SCM-198 (200 µM), or TNF-α+SCM-198 (10 ng/ml, 200 µM), the expressions of aromatase, ERα, PRB, LC3B-II/I, BECN1, Bcl-2, and Bax were analyzed via Western blotting (n = 3) (C) , and the concentration of estrogen was detected via ELISA (n = 4) (D) . Continuous data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant).

Article Snippet: Nonspecific binding sites were blocked by incubating the membranes with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Next, the membranes were incubated overnight at 4°C with primary antibodies (1:1,000) against aromatase (#14528, CST, Boston, USA), ERα (#ab32063, Abcam, UK), PRB (#ab32085, Abcam, UK), LC3B (#3868, CST, USA), BECN1 (#ab207612, Abcam, UK), Bcl-2 (#2870, CST, USA), Bax (#12105, CST, USA), FN1 (#ab2413, Abcam, UK), vimentin (#5741, CST, USA), α-tubulin (#ab7291, Abcam, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#10112, Arigo, Taiwan, China).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot

Journal: Frontiers in Endocrinology

Article Title: SCM-198 Prevents Endometriosis by Reversing Low Autophagy of Endometrial Stromal Cell via Balancing ERα and PR Signals

doi: 10.3389/fendo.2022.858176

Figure Lengend Snippet: Schematic showing the therapeutic mechanism of SCM-198 on EMS. The production of TNF-α was higher in eESCs than that in nESCs. Elevated TNF-α levels augmented the activation of aromatase-estrogen-ERα signaling and aggravated PRB reduction. The upregulated estrogen signaling and downregulated progesterone signaling synergistically suppressed the autophagy level, which further led to the growth of eESCs. SCM-198 inhibited aromatase-estrogen-ERα signaling and increased PRB expression by downregulating TNF-α. Consequently, SCM-198 promoted the autophagy-mediated apoptosis of eESCs by reconstructing the balance of estrogen and progesterone signals.

Article Snippet: Nonspecific binding sites were blocked by incubating the membranes with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h. Next, the membranes were incubated overnight at 4°C with primary antibodies (1:1,000) against aromatase (#14528, CST, Boston, USA), ERα (#ab32063, Abcam, UK), PRB (#ab32085, Abcam, UK), LC3B (#3868, CST, USA), BECN1 (#ab207612, Abcam, UK), Bcl-2 (#2870, CST, USA), Bax (#12105, CST, USA), FN1 (#ab2413, Abcam, UK), vimentin (#5741, CST, USA), α-tubulin (#ab7291, Abcam, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#10112, Arigo, Taiwan, China).

Techniques: Activation Assay, Expressing