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: Cell death and differentiation

Article Title: TFEB controls syncytiotrophoblast formation and hormone production in placenta.

doi: 10.1038/s41418-024-01337-y

Figure Lengend Snippet: Fig. 4 TFEB drives STB endocrine functions. A Analysis of secreted proteins through LC–MS/MS in wild-type (WT) and TFEB knock-out (KO) cells. (Left) Pie chart showing the percentage of detected proteins classified into “not secreted” (92.83%) and “secreted” (7.17%) according to Human Protein Atlas. (Right) Bar plot showing the number of secreted proteins (x-axis) belonging to the indicated Biological Process categories (y-axis). B (Left) Volcano plot showing secreted proteins displayed as Log2 Fold Change in wild-type (WT) cells upon Forskolin (FRSK) treatment compared to DMSO conditions - FRSK vs DMSO (WT) on the x-axis - and in TFEB knock-out (KO) cells treated with FRSK with respect to WT in the same conditions - KO vs WT (FRSK) on the y-axis. Proteins upregulated upon FRSK treatment in WT cells and downregulated in KO versus WT cells in FRSK conditions are represented as light blue dots. (Right) Table displaying selected proteins and their corresponding Log2 Fold Change in indicated conditions. C Schematic representation of the estrogen biosynthetic pathways from cholesterol to 17β-Estradiol (E2). Major enzymes synthesizing indicated steroid intermediates are displayed in red (directly bound by TFEB from our ChiPseq analysis) and in blue (not directly bound by TFEB but affected by its downregulation). D Representative image of immunoblot analysis of CYP19A1 levels in cells wild-type (WT) and TFEB knock-out cells (KO#1 and #2) in DMSO e Forskolin (FRSK) conditions. GAPDH was used as a loading control. E Bar chart graph representations of the quantitative determination of 17β-Estradiol (E2) and Estrone (E1) in wild-type (WT) and TFEB knock-out (KO) cells in DMSO and Forskolin (FRSK) conditions detected by UHPLC–MS/MS-based targeted steroidomics. Steroid concentration values are expressed as ng/mg of protein. Statistical analysis was performed by One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (*p ≤0.05; **p ≤0.01; ***p ≤0.001).

Article Snippet: Primary antibodies (in TBST 0.1% BSA 3%) were incubated overnight at 4 °C (TFEB, Cell Signaling, # 4240 s; E-Cadherin, Cell Signaling, # 24E10; GAPDH, Santacruz, # sc32233; CYP19A1, Cell Signaling, # 14528; OVOL1, Aviva Systems Biology, #ARP38500T100).

Techniques: Liquid Chromatography with Mass Spectroscopy, Knock-Out, Western Blot, Control, Tandem Mass Spectroscopy, Concentration Assay

Journal: Cell death and differentiation

Article Title: TFEB controls syncytiotrophoblast formation and hormone production in placenta.

doi: 10.1038/s41418-024-01337-y

Figure Lengend Snippet: Fig. 5 TFEB overexpression restores the STB phenotype in TFEB KO cells. A Gene-set Enrichment Analysis was performed on genes ranked by their fold-change and significance (see “Methods”) in TFEB knock-out cells transduced with a TFEB overexpressing vector (KO + TFEB) against an empty control vector (KO + EMPTY), upon treatment with Forskolin (FRSK - left) or Torin-1 (TORIN - right) in RNAseq (upper panels) and LC–MS/MS (lower panels). Upregulated genes from panel S1A (STB signature - BeWo) were used as geneset. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are reported. B Representative image of immunoblot analysis of CYP19A1 and TFEB levels in TFEB knock- out cells transduced with an empty control vector (KO + EMPTY) or TFEB overexpressing vector (KO + TFEB) upon treatment with DMSO, Forskolin (FRSK) and Torin-1 (TORIN). GAPDH was used as a loading control. C Bar chart graph representations of the quantitative determination of 17β-Estradiol (E2) and Estrone (E1) in TFEB knock-out cells transduced with an empty control vector (KO + EMPTY) or TFEB overexpressing vector (KO + TFEB) upon treatment with DMSO, Forskolin (FRSK) and Torin-1 detected by UHPLC–MS/MS-based targeted steroidomics. Steroid concentration values are expressed as ng/mg of protein. Statistical analysis was performed by One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (*p ≤0.05; **p ≤0.01; ***p ≤0.001).

Article Snippet: Primary antibodies (in TBST 0.1% BSA 3%) were incubated overnight at 4 °C (TFEB, Cell Signaling, # 4240 s; E-Cadherin, Cell Signaling, # 24E10; GAPDH, Santacruz, # sc32233; CYP19A1, Cell Signaling, # 14528; OVOL1, Aviva Systems Biology, #ARP38500T100).

Techniques: Over Expression, Knock-Out, Transduction, Plasmid Preparation, Control, Liquid Chromatography with Mass Spectroscopy, Western Blot, Tandem Mass Spectroscopy, Concentration Assay

Journal: Journal of Virology

Article Title: Testicular Degeneration and Infertility following Arbovirus Infection

doi: 10.1128/JVI.01131-18

Figure Lengend Snippet: 3β-HSD and P450 expression in BTV-1 experimentally infected rams and mock-infected controls. (A and B) Immunohistochemistry showing expression of 3β-HSD (A) and P450 (B) (in brown) by the Leydig cells in sections of the testes of mock-infected healthy control rams. Arrows point to Leydig cells. (C to F) Immunohistochemistry showing lack of expression of 3β-HSD (C and E) and P450 (D and F) by Leydig cells in the testes of rams infected with BTV-1 IT2006 (C and D) or BTV-1 IT2013 (E and F). The peritubular areas where the Leydig cells are located are highlighted with a broken line. Scale bars, 100 μm. (G) Graph representing the relative levels of testosterone, assessed by ELISA, in the blood of rams infected with either BTV-1 IT2006 or BTV-1 IT2013 . Values are shown as percentages of the values of testosterone taken at day 0 before virus infection. Note the significant reduction in the levels of testosterone in rams infected with BTV-1 IT2006 between 0 and 8 to 11 dpi ( P < 0.05; one-way ANOVA) and in rams infected with BTV-1 IT2013 between 0 and either 5 or 15 dpi ( P < 0.05; one-way ANOVA).

Article Snippet: Sections were incubated overnight at 4°C using primary antibodies specific against the following markers: BTV NS2 ( , ), vimentin (Dako Agilent), von Willebrand factor (Dako Agilent), CD3 (Dako Agilent), MX-1 ( , ), 3β-HSD (Santa Cruz Biotechnology), P450 Aromatase (AcrisAntibodies GmbH), inhibin α (Ventana Medical Systems), smooth muscle actin (Dako Agilent), and KI-67 (Dako Agilent).

Techniques: Expressing, Infection, Immunohistochemistry, Control, Enzyme-linked Immunosorbent Assay, Virus

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: 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