Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01. ( B ) Effect of RARB overexpression in absence or presence of ATRA was studied with respect to endogenous StAR and HSD3B2 expression. H295R cells were transiently transfected with RARB or the empty vector for control. Transfected cells were optionally treated with ATRA for 24 h in starvation medium. Finally, the effect of RARB on StAR and HSD3B2 was assessed by performing specific Western blot analyses. A representative blot is shown on the left and quantitative analysis on the right. Data are the mean ± SD of three independent experiments. ( C ) Effect of RARB overexpression and ATRA treatment on H295R steroid production. H295R cells were transiently transfected with RARB or the empty vector for control. Transfected cells were optionally treated with 1 μM ATRA for 24 h in starvation medium. Steroid production was labeled with [ 3 H] pregnenolone for 90 min and extracted steroids were resolved by TLC. A representative TLC is shown on the left and the quantitative analysis of steroids shown on the right. Data are the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01. Prog, progesterone; Δ4A, androstenedione; Preg, pregnenolone; 17OH Preg, 17α-hydroxypregnenolone; 17OH Prog, 17OHP; 11DOC, 11-deoxycortisol.

Article Snippet: Rabbit polyclonal antibody against human CYP17A1 was custom made by Genscript (Piscataway, NJ, USA) .

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Activation Assay, Luciferase, Control, Over Expression, Western Blot, Labeling

Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: ANGPTL1 suppresses CYP17A1 expression through inducing ERK1/2 phosphorylation in starved H295R cells. ( A ) Effect of rANGPTL1 protein on ERK and AKT kinase phosphorylation: Western blot analysis was performed on starved H295R cells treated with 50 ng/ml rANGPTL1 for 0-4 h to study the changes in phosphorylation of ERK1/2 and AKT kinases. Western blot was performed on total protein preparations from treated starved H295R cells. Levels of total ERK and AKT were also analyzed. ( B ) Effect of rANGPTL1 treatment on steroidogenic CYP17A1 expression. Western blot analysis showed CYP17A1 expression was suppressed under treatment with rANGPTL1 in 30 min. We used β-actin as loading control. ( C ) The effect of inhibiting MEK/ERK1/2 phosphorylation on CYP17A1 expression in ANGPTL1 treated starved H295R cells. Cells were starved for 48 hrs and treated or not with rANGPTL1. When indicated, cells were pretreated with 10 μM U0126 for 30 min before rANGPTL1 treatment. Cells were lysed and levels of CYP17A1 and pERK1/2 were analyzed by Western blotting. Levels of total ERK were taken as a loading control. ( D ) Effect of rANGPTL1 on DUSP6 expression. Levels of DUSP6 were assessed by Western blotting. Representative blots are shown on the left and the quantitative analysis of blots is shown on the right. Data are the mean ± SEM of two or three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit polyclonal antibody against human CYP17A1 was custom made by Genscript (Piscataway, NJ, USA) .

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: Scheme of suggested regulation of androgen production by ANGPTL1 in H295R cells based on presented findings (arrows) and information from literature . ANGPTL1 enhances ERK1/2 phosphorylation and thereby modulates CYP17A1 expression. ANGPTL1 also stimulates DUSP6 expression. DUSP6 and ERK1/2 regulate each other; while DUSP6 may dephosphorylate ERK1/2, phospho-ERKs phosphorylate DUSP6 prompting its degradation.

Article Snippet: Rabbit polyclonal antibody against human CYP17A1 was custom made by Genscript (Piscataway, NJ, USA) .

Techniques: Phospho-proteomics, Expressing