Journal: Gastro Hep Advances

Article Title: Sex Differences in Colonic Inflammation are Driven by Epithelial-Specific Expression of Estrogen Receptor Alpha

doi: 10.1016/j.gastha.2025.100624

Figure Lengend Snippet: Colonic epithelial cells express significant levels of ERα and ERβ. (A) Relative gene expression of Esr1 (ERα) and Esr2 (ERβ) in primary colonic epithelial cells obtained from healthy adult WT mice was determined by qPCR. Expression of target genes was normalized to that of Gapdh and fold change was calculated relative to male samples. (B) Ratio of Esr1:Esr2 mRNA expression was calculated for male and female samples. (C–H) Protein expression of ERα and ERβ were determined by western blot of nuclear (C–E) or cytoplasmic (F–H) protein lysates isolated from primary colonic epithelial cells of healthy adult WT mice. Expression of ERα and ERβ were normalized to levels of Lamin B1 (nuclear lysates, [D]) or β-actin (cytoplasmic lysates, [G]) using densitometry. Densitometry values were used to calculate ratios of ERα:ERβ expressed in nucleus (E) and cytoplasm (H). Subcellular localization of ERα (I) and ERβ (J) was calculated using densitometry values. (K) Expression of Cyp19A1 (aromatase) was determined by western blot of nuclear protein lysates isolated from primary colonic epithelial cells of healthy adult WT mice and (L) quantified by densitometry. For all figures, statistical analysis was performed with 2-way analysis of variance and Tukey post hoc test. Individual points represent individual animals.

Article Snippet: Antibodies used for western blots included α-Cyp19A1 (NSJ Bioreagents #RQ4643) α-ERα (Novus #NB300-560), α-ERβ (Novus #NB120-3577), α-GPER1 (Abcam #ab260033), α-Lamin B1 (Cell Signaling #12586), and α-β-Actin (Cell Signaling #12262).

Techniques: Gene Expression, Expressing, Western Blot, Isolation

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: Reagents and antibodies 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: Reagents and antibodies 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: Endocrine Connections

Article Title: 17β-Hydroxysteroid dehydrogenase type I and aromatase in ovarian cortical inclusion cysts

doi: 10.1530/ec-24-0643

Figure Lengend Snippet: Figure 2: Micrograph of sections of a CIC lined with mixed epithelium stained with hematoxylin and eosin. a) CIC with flat epithelium displays regions of ciliated cells. b) Transition from flat to mucinous epithelia is observed in a CICmix. Sections of the same cortical inclusion cyst (CIC) lined with tubal-like epithelium (CICtube) show immunoreactivity for: c) 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1), d) aromatase, and e) estrogen receptor alpha (ERα). The labeling for aromatase is lighter than that for HSD17B1 and ERα reactivity. Scale bar = 50 µm.

Article Snippet: The primary antibodies used were the HSD17B1 antibody (cat. GTX12312; GeneTex, Irvine, CA, USA), aromatase antibody H4 (cat. OASA02666; Aviva Systems Biology, San Diego, CA, USA), and Erα HC20 antibody (cat. sc-543; Santa Cruz Biotechnology, Inc).

Techniques: Staining, Labeling

Journal: Drug Design, Development and Therapy

Article Title: Semaglutide Alleviates Ovarian Oxidative Stress and Autophagy via the PI3K/AKT/mTOR Pathway in Mice with Polycystic Ovary Syndrome

doi: 10.2147/DDDT.S522730

Figure Lengend Snippet: (A ) Immunofluorescence images; ( B ) CYP17A1 expression area; ( C ) StAR expression area; ( D ) CYP19A1 (Immunohistochemistry); ( E ) CYP19A1 expression area. Data are presented as mean±SD. Vs NC, ## P <0.01; Vs PCOS, * P <0.05, ** P <0.01; n=6 per group.

Article Snippet: The antibodies used in the study were CYP19A1 antibodies (No. ba3704, BOSTER, China; dilution 1:50;), Beclin-1 antibodies (dilution 1:100; No.#3495, CST, USA), p62 antibodies (dilution 1:500; No. #23214, CST, USA), LC3B antibodies (dilution 1:200; No.#83506, CST, USA) was increased for half an hour and then observed.

Techniques: Immunofluorescence, Expressing, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: Astilbin Activates the Reactive Oxidative Species/PPARγ Pathway to Suppress Effector CD4 + T Cell Activities via Direct Binding With Cytochrome P450 1B1

doi: 10.3389/fphar.2022.848957

Figure Lengend Snippet: CYP1B1 provides a dock site for astilbin. (A) Variations of CYP1B1 and CYP19A1 in astilbin-treated CD4 + T cells. (B,C) Astilbin promotes cellular ROS production similar to ABT (a non-specific inhibitor of CYP). Mean ± SD; n = 3. (D) Effects of astilbin on IFN-γ, TNF-α, CD36, and CCR9 of CD4 + T cells similar to ABT. Mean ± SD; n = 3. (E) Three-dimensional model of the CYP1B1 active site with bound astilbin. (F) Binding of CYP1B1 with astilbin and the inset showing the principal interactive residues of astilbin at the CYP1B1 binding pocket. Green dashed lines represent hydrogen bonds. The hydrophobic interaction is depicted by pink dashed lines. (G) Real binding of CYP1B1 with astilbin confirmed by SPR assay. Production of cROS and mROS (H) and TNF-α (I) in CYP1B1 shRNA-transfected CD4 + T cells. Mean ± SEM; n = 3. All experiments were performed three times. The inhibitor of CYP, ABT, is abbreviated as CYPi. p values (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, no significant difference) determined by one-way ANOVA (C,D) and two-way ANOVA (H,I) .

Article Snippet: The following antibodies were used for western blot: ACC (Cat# 3676, RRID:AB_2219397), p-ACC (Cat# 11818, RRID:AB_2687505), HKII (Cat# 2867, RRID:AB_2232946), mTOR (Cat# 2983, RRID:AB_2105622), p-mTOR (Cat# 5536, RRID:AB_10691552), Akt (Cat# 4691, RRID:AB_915783), p-Akt (Cat# 4060, RRID:AB_2315049), AMPK (Cat# 5831, RRID:AB_10622186), p-AMPK (Cat# 50081, RRID:AB_2799368), LKB1 (Cat# 3047, RRID:AB_2198327), p-LKB1 (Cat# 3482, RRID:AB_2198321), PTEN (Cat# 9188, RRID:AB_2253290), PPAR-γ (Cat# 2443, RRID:AB_823598), PI3K p110α (Cat# 4249, RRID:AB_2165248), β-actin (Cat# 4970, RRID:AB_2223172), NF-κB p65 (Cat# 8242, RRID:AB_10859369), NF-κB p-p65 (Cat# 3033, RRID:AB_331284), p38 (Cat# 8690, RRID:AB_10999090), p-p38 (Cat# 4511, RRID:AB_2139682), JNK (Cat# 9252, RRID:AB_2250373), p-JNK (Cat# 4668, RRID:AB_823588), Erk (Cat# 4695, RRID:AB_390779), p-Erk (Cat# 4370, RRID:AB_2315112), Stat3 (Cat# 4904, RRID:AB_331269), p-Stat3 (Cat# 9145, RRID:AB_2491009), SOCS3 (Cat# 52113, RRID:AB_2799408) (Cell Signaling Technology, MA, United States), Glut1 (Cat# ab115730, RRID:AB_10903230), CPT1α (Cat# ab234111, RRID:AB_2864319), LPL (Cat# ab21356, RRID:AB_446221) (Abcam, Cambridge, Britain), CYP1B1 (Cat# 18505-1-A, RRID:AB_2878548) (Proteintech, IL, United States), CYP19A1 (Cat# YT1190, RRID:AB_2864736) (Immunoway, DE, United States) and P-PPARγ (abs130911, absin, Shanghai China).

Techniques: Binding Assay, SPR Assay, shRNA, Transfection