Journal: Cell Death & Disease

Article Title: Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS–AMPK–mTOR axis

doi: 10.1038/s41419-019-1728-5

Figure Lengend Snippet: Semi-quantitative real-time PCR was performed to detect the gene expression of StAR and P450scc ( a ) and 3β-HSD and CYP17 ( d ) normalised by the control group. Western blot was used to evaluate the protein level of StAR and P450scc ( b ) and 3β-HSD and CYP17 ( e ). β-actin was used as internal control. Histogram displaying the densitometric analysis results of StAR and P450scc ( c ) and 3β-HSD and CYP17 ( f ) normalised by the control group. g Histogram displaying the testosterone concentration normalised by the control group. The normalised levels of gene expression are expressed as ratios of the copy number of mRNA and that of β-actin cDNA. Data were obtained from five independent experiments and expressed as mean ± SD. * P < 0.01 and # P < 0.05, compared with the corresponding control or treatment group

Article Snippet: Rabbit monoclonal antibodies against LC3-I/II (Cat. No.: 12741S), StAR (Cat. No.: 8449S), P450scc (Cat. No.: 14217S), AMPK (Cat. No.: 5831S), p-AMPK (Cat. No.: 2537S), mTOR (Cat. No.: 2983S) and p-mTOR (Cat. No.: 5536S) were from Cell Signalling Technology (Beverly, MA, USA) and rabbit monoclonal antibody against GSDMD (Cat. No.: ab219800) was from Abcam (Cambridge, England, UK).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Control, Western Blot, Concentration Assay

Journal: Particle and Fibre Toxicology

Article Title: Chronic exposure to polystyrene microplastics induced male reproductive toxicity and decreased testosterone levels via the LH-mediated LHR/cAMP/PKA/StAR pathway

doi: 10.1186/s12989-022-00453-2

Figure Lengend Snippet: PS-MPs treatment downregulated the expression of steroidogenic enzymes and StAR in testes. Mice were given drinking water comprising PS-MPs of various sizes for 180 sustained days. A , B The expression of 3β-HSD, 17β-HSD, P450scc, P450c17, and StAR in testes was measured by western blotting. The expression levels were quantified with ImageJ (n = 3). Data are expressed as means ± SD. * P < 0.05, ** P < 0.01 vs. control. # P < 0.05, ## P < 0.01 vs. 100 μg/L group. C , D The expression of StAR in mouse testicular tissues of the control group and 1000 μg/L group was tested by immunofluorescence staining. Testicular tissues were stained with StAR (red) and DAPI (blue), arrows: positive expression (scale bar = 50 µm). The expression levels of StAR were quantified with ImageJ (n = 3). Data are expressed as means ± SD. ** P < 0.01 vs. control. # P < 0.05, *** P < 0.001 vs. control

Article Snippet: Transferred blots were incubated with rabbit anti-CYP11A (P450scc) (Proteintech Group, Rosemont, IL, USA), rabbit anti-CYP17A (P450c17) (Proteintech Group, Rosemont, IL, USA), rabbit anti-StAR (Cell Signaling Technology, USA), mouse anti-HSD3β (Proteintech Group, Rosemont, IL, USA), rabbit anti-HSD17β2 (Proteintech Group, Rosemont, IL, USA), rabbit anti-LHR (Proteintech Group, Rosemont, IL, USA), and mouse anti-GAPDH (Proteintech Group, Rosemont, IL, USA) overnight at 4° C. Specific information on the antibodies was shown in Table .

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining

Journal: Particle and Fibre Toxicology

Article Title: Chronic exposure to polystyrene microplastics induced male reproductive toxicity and decreased testosterone levels via the LH-mediated LHR/cAMP/PKA/StAR pathway

doi: 10.1186/s12989-022-00453-2

Figure Lengend Snippet: The expression of steroidogenic enzymes and StAR decreased in Leydig cells after PS-MPs treatment. Primary Leydig cells were exposed to 0.5 μm PS-MPs for 24 h at various concentrations as indicated. A The content of testosterone in supernatant was determined by ELISA assays (n = 3). B The mRNA expression levels of 3β-HSD, 17β-HSD, P450scc, P450c17, and StAR in Leydig cells after treatment with PS-MPs were determined by qRT-PCR (n = 3). C , D The expression of 3β-HSD, 17β-HSD, P450scc, and P450c17 in cells was measured by western blotting. The expression levels were quantified with ImageJ (n = 3). Data are expressed as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control

Article Snippet: Transferred blots were incubated with rabbit anti-CYP11A (P450scc) (Proteintech Group, Rosemont, IL, USA), rabbit anti-CYP17A (P450c17) (Proteintech Group, Rosemont, IL, USA), rabbit anti-StAR (Cell Signaling Technology, USA), mouse anti-HSD3β (Proteintech Group, Rosemont, IL, USA), rabbit anti-HSD17β2 (Proteintech Group, Rosemont, IL, USA), rabbit anti-LHR (Proteintech Group, Rosemont, IL, USA), and mouse anti-GAPDH (Proteintech Group, Rosemont, IL, USA) overnight at 4° C. Specific information on the antibodies was shown in Table .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Particle and Fibre Toxicology

Article Title: Chronic exposure to polystyrene microplastics induced male reproductive toxicity and decreased testosterone levels via the LH-mediated LHR/cAMP/PKA/StAR pathway

doi: 10.1186/s12989-022-00453-2

Figure Lengend Snippet: Overexpression of LHR alleviated the reduction in StAR, steroidogenic enzymes and testosterone levels induced by PS-MPs treatment in primary Leydig cells. A Primary cells were infected with LHR or empty vector with lentivirus for 72 h. The mRNA levels of LHR were tested by qRT-PCR (n = 3; *** P < 0.001 vs. LV-NC group). B Primary cells were infected with LHR or empty vector with lentivirus for 72 h. Then, the cells were exposed to 0.5 μm PS-MPs at a concentration of 0.2 mg/mL for 24 h. The testosterone content in the supernatant was examined by ELISA (n = 3; *** P < 0.001 vs. LV-NC group; ## P < 0.01 vs. LV-NC + PS-MPs group). C , D Primary cells were infected with LHR or empty vector with lentivirus for 72 h. Then, the cells were treated with 0.5 μm PS-MPs with a concentration at 0.2 mg/mL for 24 h. The expression of 3β-HSD, 17β-HSD, P450scc, P450c17, StAR, and LHR in cells was measured by western blotting. The expression levels were quantified with ImageJ (n = 3; * P < 0.05, ** P < 0.01 vs. LV-NC group; # P < 0.05, ## P < 0.01, ### P < 0.01 vs. LV-NC + PS-MPs group)

Article Snippet: Transferred blots were incubated with rabbit anti-CYP11A (P450scc) (Proteintech Group, Rosemont, IL, USA), rabbit anti-CYP17A (P450c17) (Proteintech Group, Rosemont, IL, USA), rabbit anti-StAR (Cell Signaling Technology, USA), mouse anti-HSD3β (Proteintech Group, Rosemont, IL, USA), rabbit anti-HSD17β2 (Proteintech Group, Rosemont, IL, USA), rabbit anti-LHR (Proteintech Group, Rosemont, IL, USA), and mouse anti-GAPDH (Proteintech Group, Rosemont, IL, USA) overnight at 4° C. Specific information on the antibodies was shown in Table .

Techniques: Over Expression, Infection, Plasmid Preparation, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Particle and Fibre Toxicology

Article Title: Chronic exposure to polystyrene microplastics induced male reproductive toxicity and decreased testosterone levels via the LH-mediated LHR/cAMP/PKA/StAR pathway

doi: 10.1186/s12989-022-00453-2

Figure Lengend Snippet: Specifications of primary antibodies

Article Snippet: Transferred blots were incubated with rabbit anti-CYP11A (P450scc) (Proteintech Group, Rosemont, IL, USA), rabbit anti-CYP17A (P450c17) (Proteintech Group, Rosemont, IL, USA), rabbit anti-StAR (Cell Signaling Technology, USA), mouse anti-HSD3β (Proteintech Group, Rosemont, IL, USA), rabbit anti-HSD17β2 (Proteintech Group, Rosemont, IL, USA), rabbit anti-LHR (Proteintech Group, Rosemont, IL, USA), and mouse anti-GAPDH (Proteintech Group, Rosemont, IL, USA) overnight at 4° C. Specific information on the antibodies was shown in Table .

Techniques:

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Identification of a novel androgen receptor mutation in a family with multiple components compatible with the testicular dysgenesis syndrome.

doi: 10.1210/jc.2013-1278

Figure Lengend Snippet: Figure 3. Histology and immunohistochemical expression of selected proteins in the testis tissue from the proband (III:2) and his cousin (III:5). Section of a biopsy from the contralateral testis from the proband stained with hematoxylin and eosin shows areas of testicular dysgenesis including Sertoli cell-only tubules (solid arrow), hyalinized tubules (barbed arrows), decreased spermatogenesis (black solid arrowheads), Leydig cell hyperplasia (asterisk), and normal spermatogenesis (white solid arrowheads) (A). Microlithiasis (B) and decreased spermatogenesis (C) were observed in the contralateral testis from patient III:5. The remaining images show immunohistochemical analysis of the orchidectomy sample from the proband (II:2). M2A/D2-40 was expressed in seminoma cells of the tumor (D) and in carcinoma in situ cells in the surrounding tissue (E). The AR was expressed in Sertoli, Leydig, and peritubular cells (F), although at a lower level than in control testis samples from patients without an AR defect (F inset). GATA-4 was expressed in Sertoli cells (G) and CYP11A1 in Leydig cells (H). Extensive lymphocytic infiltration was observed (E and H). The bars in each picture correspond to 100 m.

Article Snippet: Tumor tissue and surrounding testis parenchyma from the orchidectomy specimen of the proband were analyzed by immunohistochemistry, using a standard indirect peroxidase method (11, 12) and antibodies for the AR (MS-443-P; Lab Vision Corp., Fremont, California), cholesterol side chain cleavage enzyme (CYP11A1) (HPA016436; Atlas Antibodies AB, Stockholm, Sweden), anti-Müllerian hormone (AMH) (gift from Dr R. Cate, Biogen, Cambridge, Massachusetts), GATA-binding factor 4 (GATA-4) (sc-1237; Santa Cruz Biotechnology, Dallas, Texas), and M2A/D2-40 (M3619; Dako, Glostrup, Denmark).

Techniques: Immunohistochemical staining, Expressing, Staining, In Situ, Control

Journal: PLoS ONE

Article Title: Expression and activation of the steroidogenic enzyme CYP11A1 is associated with IL-13 production in T cells from peanut allergic children

doi: 10.1371/journal.pone.0233563

Figure Lengend Snippet: PBMCs from PA children and healthy controls were activated with anti-human CD3/CD28 for 48 hours. (A) CYP11A1 mRNA expression detected by qPCR in PBMCs from PA children (n = 33) and controls (n = 11). (B) Representative flow- cytometric analysis of CYP11A1 expression in CD4 + T cells. (C) Percentages of CYP11A1 + CD4 + T cells (PA children (n = 18) and controls (n = 11). (D) Representative immunocytochemistry staining for CYP11A1. CYP11A1 (green), CD4 + T cells (red), cell nuclei (DAPI, blue), and coincidence of CYP11A1 + and CD4 + T cell staining (yellow). (magnification x200). (E) Percentages of CYP11A1 + CD4 + T cells detected by immunocytochemistry (PA children (n = 18) and controls (n = 11). Data are expressed as means±SEM. #P<0.001, n.s.: not significant.

Article Snippet: Cells were transfected with a human CYP11A1 CRISPR/Cas9 knockout plasmid with a GFP insert (Santa Cruz Biotechnology, Santa Cruz, CA) or a scrambled control CRISPR/Cas9 plasmid using Xfect transfection reagent (Clontech, Mountain View, CA) according to the manufacturer's instructions.

Techniques: Expressing, Immunocytochemistry, Staining

Journal: PLoS ONE

Article Title: Expression and activation of the steroidogenic enzyme CYP11A1 is associated with IL-13 production in T cells from peanut allergic children

doi: 10.1371/journal.pone.0233563

Figure Lengend Snippet: lower delta Ct values correspond to higher mRNA levels. (A) Significant correlation between CYP11A1 mRNA expression and IL-13 levels (P<0.001, lower Ct values mean higher mRNA levels). (B) Significant correlation between CYP11A1 mRNA expression and IL13 mRNA expression (P<0.001). (C) No significant correlation between CYP11A1 mRNA expression and IFNγ levels (P>0.05). (D) No significant correlation between CYP11A1 mRNA expression and IFNγ mRNA expression (P>0.05). (E) Significant correlation between CYP11A1 mRNA and serum levels of sIgE to Ara h 2 (P<0.05). (F) Significant correlation between CYP11A1 mRNA and outcomes of oral food challenge (1 = positive, 0 = negative (no reaction) (P<0.05). Analyses were performed using Pearson correlation. The data were expressed as log-transformed data.

Article Snippet: Cells were transfected with a human CYP11A1 CRISPR/Cas9 knockout plasmid with a GFP insert (Santa Cruz Biotechnology, Santa Cruz, CA) or a scrambled control CRISPR/Cas9 plasmid using Xfect transfection reagent (Clontech, Mountain View, CA) according to the manufacturer's instructions.

Techniques: Expressing, Transformation Assay

Journal: PLoS ONE

Article Title: Expression and activation of the steroidogenic enzyme CYP11A1 is associated with IL-13 production in T cells from peanut allergic children

doi: 10.1371/journal.pone.0233563

Figure Lengend Snippet: ( A ) Representative flow-cytometric analysis of CYP11A1 expression after transfection with CYP11A1 CRISPR/Cas9 KO plasmid or scrambled control CRISPR/Cas9 plasmid. ( B ) Quantitative analysis of double CYP11A-positive and CD4-positive cells. ( C ) Representative flow cytometric analysis of cytokine expression in CD4 + T cells after transfection. ( D ) Percentages of CD4 + IL-13 + cells and CD4 + IFNγ + cells. Data are from two independent experiments. ** P < 0.01, n.s.: not significant, compared with scramble CRISPR/Cas9 plasmid group.

Article Snippet: Cells were transfected with a human CYP11A1 CRISPR/Cas9 knockout plasmid with a GFP insert (Santa Cruz Biotechnology, Santa Cruz, CA) or a scrambled control CRISPR/Cas9 plasmid using Xfect transfection reagent (Clontech, Mountain View, CA) according to the manufacturer's instructions.

Techniques: Expressing, Transfection, CRISPR, Plasmid Preparation, Control

Journal: Frontiers in Pharmacology

Article Title: Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension

doi: 10.3389/fphar.2024.1387756

Figure Lengend Snippet: CYP11A1 is identified as the direct target of Tet. (A) Chemical structure of Tet and the Tet probe. (B) Target identification, including SDS‒PAGE (B, upper panel) and Western blotting (below panel), as well as (C) target protein profiling. Lane 1, total protein of the adrenal glands; Lane 2, negative magnetic microspheres; Lane 3, target fishing by the Tet probe. (D) Colocalization (pseudo-yellow) of the Tet probe (pseudo-green) and CYP11A1 protein (pseudo-red) in the adrenal gland tissue slices. Tet was used as the control (n = 3). (E) Colocalization (pseudo-yellow) of the Tet probe (pseudo-green) and CYP11A1 protein (pseudo-red) in the NCI-H295R cells. The Con group was not treated with drugs; the Tet probe group was administered 1 μmol/L Tet probe, and the Com group was administered 1 μmol/L Tet probe +10 μmol/L Tet (n = 3).

Article Snippet: The CYP11A1 antibody (bs-10099R) and CYP11A1 siRNA (h) were obtained from Bioss Co., Ltd. (Beijing, China) and Santa Cruz Biotechnology, Inc. (Santa Cruz, United States), respectively.

Techniques: Drug discovery, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension

doi: 10.3389/fphar.2024.1387756

Figure Lengend Snippet: Tet inhibits ALD synthesis by targeting CYP11A1. (A) CYP11A1 participates in the ALD and Cort synthesis processes as a key rate-limiting enzyme. (B) Western blotting analysis was performed after transfection of the 293T cells with CYP11A1 DNA for 48 h (n = 3) and CYP11A1 enzyme activity assay of the CYP11A1-overexpressing 293T cells (n = 6). (C) ALD and (D) Cort level assays in the NCI-H295R cells with CYP11A1 interference and normal cells (n = 6). The data are expressed as mean ± SD; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs Con group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Mod group; N.S., no significant difference.

Article Snippet: The CYP11A1 antibody (bs-10099R) and CYP11A1 siRNA (h) were obtained from Bioss Co., Ltd. (Beijing, China) and Santa Cruz Biotechnology, Inc. (Santa Cruz, United States), respectively.

Techniques: Western Blot, Transfection, Enzyme Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension

doi: 10.3389/fphar.2024.1387756

Figure Lengend Snippet: CYP11A1 C423 is identified as the key site for Tet binding. (A) Irreversible binding of the Tet probe to CYP11A1. In the preincubation group, the recombinant CYP11A1 protein was preincubated with the Tet probe for 12 h and then incubated with Tet for another 12 h for competitive binding. In the coincubation group, the recombinant CYP11A1 protein was incubated for 12 h with the Tet probe and Tet for competitive binding. (B) CO difference spectra analyses of Tet and CYP11A1; the recombinant CYP11A1 protein (0.1 mg/mL) was incubated with Tet (0–100 μmol/L), and the products were reduced with sodium dithionite and complexed with CO. The reduced CO complex was scanned between 400 and 700 nm. (C) Schematic of the CYP11A1 catalytic mechanism. (D) CO difference spectra between CYP11A1 and C423G. (E) Fluorescence in-gel imaging assay between the Tet probe and CYP11A1 or CYP11A1 mutant C423G; (F) MST assay of Tet with CYP11A1 or CYP11A1 mutant C423G.

Article Snippet: The CYP11A1 antibody (bs-10099R) and CYP11A1 siRNA (h) were obtained from Bioss Co., Ltd. (Beijing, China) and Santa Cruz Biotechnology, Inc. (Santa Cruz, United States), respectively.

Techniques: Binding Assay, Recombinant, Incubation, Fluorescence, Imaging, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension

doi: 10.3389/fphar.2024.1387756

Figure Lengend Snippet: Tet targets CYP11A1 for its antihypertensive effect. (A–C) Heart rate as well as systolic and diastolic blood pressures of the SHRs. (D–I) Na + , K + , and Cl − levels in the urine and serum of the SHRs analyzed using ion detection kits. (J–L) . The levels of Preg, Cort, and ALD in the serum of the SHRs were analyzed using ELISA kits. The data are expressed as mean ± SD, n = 6; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. Con group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Mod group.

Article Snippet: The CYP11A1 antibody (bs-10099R) and CYP11A1 siRNA (h) were obtained from Bioss Co., Ltd. (Beijing, China) and Santa Cruz Biotechnology, Inc. (Santa Cruz, United States), respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Tetrandrine inhibits aldosterone synthesis by covalently targeting CYP11A1 to attenuate hypertension

doi: 10.3389/fphar.2024.1387756

Figure Lengend Snippet: Schematic of the antihypertensive mechanism of Tet targeting CYP11A1. Tet exerts diuretic and antihypertensive effects on the SHRs by covalently binding to the Cys423 site of CYP11A1 to inhibit the biosynthesis of ALD.

Article Snippet: The CYP11A1 antibody (bs-10099R) and CYP11A1 siRNA (h) were obtained from Bioss Co., Ltd. (Beijing, China) and Santa Cruz Biotechnology, Inc. (Santa Cruz, United States), respectively.

Techniques: Binding Assay