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

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: siRNA silencing of PDE3A (siPDE3A) blunted NO‐induced AMPK activation. Human PASMC were transfected with siPDE3A, siPDE3B, or scramble siRNA. After 48 hr, cells were treated with DETA NONOate (250 μM) for 24 hr. Protein was harvested, analyzed by Western blot, and expression quantified by densitometry. (a) Representative Western blots for each PDE3A, PDE3B, β‐actin, and p‐ and total AMPK. Data are shown as fold changes ± SEM relative to scramble siRNA control for (b) PDE3A/β‐actin after siPDE3A or siPDE3B transfection; n = 5–6 for each group, * p < .025 different from scramble siRNA (two‐way ANOVA). (c) PDE3B/β‐actin after siPDE3A or siPDE3B transfection; n = 6 for each group, * p < .005 different from scramble siRNA (two‐way ANOVA), and (d) p/T‐AMPK after siPDE3A or siPDE3B transfection; n = 5–6 for each group, * p < .02 different from scramble siRNA, control; # p < .001 different from scramble siRNA, DETA NONOate; & p < .003 different from siPDE3A, DETA NONOate (two‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Control

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: sGC stimulator increased PDE3A protein expression and AMPK phosphorylation, whereas sGC inhibitor prevented the NO‐induced increase in PDE3A protein expression and AMPK phosphorylation in hPASMC. Human PASMC were treated with the NO donor DETA NONOate (250 μM) or sGC stimulator, BAY 41‐2272 (10 μM). Protein was harvested, analyzed by Western blot, and expression quantified by densitometry. Representative Western blots with data shown below as fold changes ± SEM relative to vehicle control for (a) PDE3A and β‐actin; n = 6–11 for each group, * p < .04 different from control (one‐way ANOVA) and (b) p‐AMPK and total AMPK; n = 6–9 for each group, * p < .03 different from control (one‐way ANOVA)l. Alternatively, hPASMC were pretreated for 4 hr with the sGC inhibitor, ODQ (10 μM), then incubated with DETA NONOate (250 μM) for 48 hr. Representative Western blots with data below shown as fold changes ± SEM relative to vehicle control for (c) PDE3A and β‐actin; n = 8–11 for each group, * p < .001 different from control, # p < .001 different from DETA NONOate (one‐way ANOVA) and (d) p‐AMPK and total AMPK; n = 8–9 for each group, * p < .001 different from control, # p < .001 different from DETA NONOate (one‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

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

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: Treatment with NO, AMPK agonist, and PDE3 inhibitor decreased hPASMC proliferation. Human PASMC were treated with DETA NONOate (250 μM), the AMPK agonist, AICAR (5 μM), the PDE3 inhibitor, milrinone (10 μM), the combination of DETA NONOate + milrinone, or vehicle and incubated for 48 hr. MTT proliferation assay was performed. Data shown are means ± SEM . n = 6–10, * p < .001 different from control, # p < .003 different from AICAR (one‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Proliferation Assay, Control