Journal: Endocrines

Article Title: The Urokinase-Type Plasminogen Activator Contributes to cAMP-Induced Steroidogenesis in MA-10 Leydig Cells

doi: 10.3390/endocrines3030037

Figure Lengend Snippet: Figure 1. cAMP stimulation of Plau expression is blunted by AMPK. MA-10 Leydig cells were treated with vehicle (DMSO, open bars), AICAR (1 mM, AMPK agonist, gray bars), forskolin (Fsk, 1 µM, adenylate cyclase agonist, hatched bars), or Fsk+AICAR (black bars). RNA was isolated, reverse transcribed, and quantitative real-time PCRs were performed with primers specific for Plau (A) or Star (B). Results were corrected with the Rpl19 cDNA. Results are the mean of three individual experiments each performed in duplicate (±SEM). For a given reporter, different letters indicate a statistically significant difference (p < 0.05). The number of replicates is indicated.

Article Snippet: The AMPK activator AICAR was obtained from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Expressing, Isolation, Reverse Transcription

Journal: Endocrines

Article Title: The Urokinase-Type Plasminogen Activator Contributes to cAMP-Induced Steroidogenesis in MA-10 Leydig Cells

doi: 10.3390/endocrines3030037

Figure Lengend Snippet: Figure 2. Depletion of Urokinase (uPA) in Leydig cells reduces cAMP-induced STAR protein levels and steroidogenesis. MA-10 Leydig cells were transfected with control siRNA (open bars) or siRNA targeting uPA (black bars). After 48 h, cells were treated with vehicle, AICAR (1 mM), 8Br-cAMP (0.5 mM), or 8Br-cAMP+AICAR for 4 h in serum-free medium. (A) The efficiency of uPA depletion was determined by Western blot and normalized to Tubulin levels used as a loading control. (B) STAR protein levels were quantified by Western blot and normalized to Tubulin levels used as a loading control. (C) Progesterone secreted by MA-10 Leydig cells was quantified by ELISA. Data are presented as mean ± SEM. Different letters indicate a statistically significant difference (p < 0.05). The number of replicates is indicated.

Article Snippet: The AMPK activator AICAR was obtained from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Endocrines

Article Title: The Urokinase-Type Plasminogen Activator Contributes to cAMP-Induced Steroidogenesis in MA-10 Leydig Cells

doi: 10.3390/endocrines3030037

Figure Lengend Snippet: Figure 3. The proximal region of the Plau promoter responds to cAMP stimulation and this stimulation is blunted by AMPK. MA-10 Leydig cells were transiently transfected with a series of 5′ deletion constructs (−2082, −1501, −1005, −503, −406, −313, −203, −81, and −37 to +44 bp) of the mouse Plau promoter and treated with vehicle (DMSO, open bars), AICAR (1 mM, gray bars), 8Br-cAMP (0.5 mM, hatched bars), or 8Br-cAMP+AICAR (black bars) for 4 h. Results are shown as Fold Activation over vehicle (mean ± SEM). For a given reporter, different letters indicate a statistically significant difference (p < 0.05). The number of replicates is indicated.

Article Snippet: The AMPK activator AICAR was obtained from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Transfection, Construct, Activation Assay

Journal: Endocrines

Article Title: The Urokinase-Type Plasminogen Activator Contributes to cAMP-Induced Steroidogenesis in MA-10 Leydig Cells

doi: 10.3390/endocrines3030037

Figure Lengend Snippet: Figure 6. The GC box in the proximal Plau promoter is required for maximal KLF6-dependent activation and cAMP responsiveness. (A) MA-10 Leydig cells were transiently transfected with 450 ng of different Plau promoter constructs (−2082/+44 bp wild-type, −2082/+44 bp harboring a mutation in the GC box, −37/+44 bp), as indicated along with 50 ng of either an empty vector (pcDNA3, open bars) or an expression vector for KLF6 (black bars). Results are shown as Fold Activation over control (empty expression vector, value set at 1) ± SEM. For a given reporter, different letters indicate a statistically significant difference (p < 0.05). The number of replicates is indicated. (B) MA-10 Leydig cells were transfected with the same reporters described in A and treated with vehicle (DMSO, open bars), AICAR (1 mM, gray bars), 8Br-cAMP (0.5 mM, hatched bars), or 8Br- cAMP+AICAR (black bars) for 4 h. Results are shown as Fold Activation over control (vehicle, value set at 1) ± SEM. For a given reporter, different letters indicate a statistically significant difference (p < 0.05).

Article Snippet: The AMPK activator AICAR was obtained from Tocris Bioscience (Minneapolis, MN, USA).

Techniques: Activation Assay, Transfection, Construct, Mutagenesis, Plasmid Preparation, Expressing, Control