pde10a sirna  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

doi: 10.1371/journal.pone.0018136

Figure Lengend Snippet: Primer pairs of rat PDEs for quantitative realtime-PCR.

Article Snippet: siRNA oligonucleotides specific for PDE10A (sense: 5′-GGACAGCUUGGAUUCUACA-3′ ; anti-sense: 5′-UGUAGAAUCCAAGCUGUCC-3′ ) and scramble siRNA (dTdT 3′ overhang) were purchased from Eurogentec and transient transfection of siRNA was performed with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's protocols.

Techniques:

Journal: PLoS ONE

Article Title: Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

doi: 10.1371/journal.pone.0018136

Figure Lengend Snippet: A ) Immunohistochemistry staining of PDE10A in lung sections from control (a, b) and MCT (c, d) rats. Scale bar: 20 µm. B ) PDE10A mRNA expression in the pulmonary artery (P.A.), aortic artery (A.A.) and femoral artery (F.A.) from control rats (gray bars) and 4-week MCT-PH rats (black bars) are shown as a percentage of control by qRT-PCR after normalization to PBGD. **P<0.01 vs control PASMCs. n = 4 in each group. Values are expressed as the mean ± SEM.

Article Snippet: siRNA oligonucleotides specific for PDE10A (sense: 5′-GGACAGCUUGGAUUCUACA-3′ ; anti-sense: 5′-UGUAGAAUCCAAGCUGUCC-3′ ) and scramble siRNA (dTdT 3′ overhang) were purchased from Eurogentec and transient transfection of siRNA was performed with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's protocols.

Techniques: Immunohistochemistry, Staining, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

doi: 10.1371/journal.pone.0018136

Figure Lengend Snippet: A ) A representative blot of PDE10A protein expression in MCT and control PASMCs, which was determined by immunoblots using GAPDH as the loading control. B ) Densitometric quantification of PDE10A expression in PASMCs is shown as the ratio to GAPDH in the bar graph. *P<0.05 vs control PASMCs, n = 3 in each group. Values are expressed as the mean ± SEM. C ) Cellular localization of PDE10A in both control and MCT PASMCs was determined by representative immunofluorescence: green (PDE10A, FITC-conjugated), Blue (DAPI). Staining is shown at 400× magnification.

Article Snippet: siRNA oligonucleotides specific for PDE10A (sense: 5′-GGACAGCUUGGAUUCUACA-3′ ; anti-sense: 5′-UGUAGAAUCCAAGCUGUCC-3′ ) and scramble siRNA (dTdT 3′ overhang) were purchased from Eurogentec and transient transfection of siRNA was performed with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's protocols.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

doi: 10.1371/journal.pone.0018136

Figure Lengend Snippet: A ) Total cAMP activity in control and MCT PASMCs. †P<0.05 vs control PASMCs, ***P<0.001 vs total activity, n = 3 in each group. cAMP activity of B ) control PASMCs and C ) MCT PASMCs mediated by PDE10A or other PDEs (PDE1, PDE2, PDE3 and PDE4). Total cAMP PDE activity was suppressed in varying degrees by a PDE10A inhibitor (papaverine, 10 µM) or by a combination of inhibitors against PDE1 (8 mm-IBMX, 30 µM), PDE2 (EHNA, 30 µM), PDE3 (milrinone, 5 µM) and PDE4 (rolipram, 10 µM). ***P<0.001 vs total activity, n = 3 in each group. Values are expressed as the mean ± SEM.

Article Snippet: siRNA oligonucleotides specific for PDE10A (sense: 5′-GGACAGCUUGGAUUCUACA-3′ ; anti-sense: 5′-UGUAGAAUCCAAGCUGUCC-3′ ) and scramble siRNA (dTdT 3′ overhang) were purchased from Eurogentec and transient transfection of siRNA was performed with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's protocols.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

doi: 10.1371/journal.pone.0018136

Figure Lengend Snippet: Representative blots showing PDE10A protein expression in A ) control PASMCs and B ) MCT PASMCs transiently transfected with 100 nM scramble siRNA or PDE10A siRNA for 48 h. PASMCs treated with transfection reagent alone served as a negative control (NTC), and GAPDH was used as a loading control. C ) PASMC proliferation stimulated by 10% FBS with siRNA transfection was determined by the [ 3 H]thymidine incorporation assay. †††P<0.001 vs 0.1% FBS, *P<0.05 vs 10% FBS, n = 4 in each group. Values are expressed as the mean ± SEM.

Article Snippet: siRNA oligonucleotides specific for PDE10A (sense: 5′-GGACAGCUUGGAUUCUACA-3′ ; anti-sense: 5′-UGUAGAAUCCAAGCUGUCC-3′ ) and scramble siRNA (dTdT 3′ overhang) were purchased from Eurogentec and transient transfection of siRNA was performed with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's protocols.

Techniques: Expressing, Transfection, Negative Control, Thymidine Incorporation Assay

Journal: PLoS ONE

Article Title: Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling

doi: 10.1371/journal.pone.0018136

Figure Lengend Snippet: Representative PDE10A immunostaining microphotographs of the human lung sections from donors (A–D) and IPAH patients (E–H). Scale bar: 20 µm.

Article Snippet: siRNA oligonucleotides specific for PDE10A (sense: 5′-GGACAGCUUGGAUUCUACA-3′ ; anti-sense: 5′-UGUAGAAUCCAAGCUGUCC-3′ ) and scramble siRNA (dTdT 3′ overhang) were purchased from Eurogentec and transient transfection of siRNA was performed with X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's protocols.

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

doi: 10.1371/journal.pone.0063333

Figure Lengend Snippet: ( A&B ) Immuno-blot and quantification analysis for various PDEs that can affect cAMP/PKA signaling. Data show that the expression of PDE1C, PDE4B, and PDE7B are similar between control (parent) and HPRT-deficient (mutant) MN9D cell lines. Expression of PDE10A protein is significantly increased in HPRT-deficient cells. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents the statistical significant between the control (open bars) and HPRT-deficient (closed bars) MND9 cells (p<0.05 t-test).

Article Snippet: 2×10 5 HPRT-deficient MN9D cells in 6 well-plate were exposed to 80 pmole of control (scramble siRNA) or PDE10A siRNA (directed to mouse PDE10A) from Santa cruz biotechnology and transfected according to the established transfection protocol ( http://datasheets.scbt.com/siRNA_protocol.pdf ).

Techniques: Expressing, Control, Mutagenesis

Journal: PLoS ONE

Article Title: HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

doi: 10.1371/journal.pone.0063333

Figure Lengend Snippet: ( A & B ), immuno-blot and quantification analysis of p-Syn (Ser9), data show that the lower expression of phospho-PKA substrate and p-Syn (Ser9) in response to forskolin treatment in HPRT-deficient MN9D cells is restored in the presence of Papaverine (200 µM). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisks (*) represent statistical significance between forskolin treated cells without papaverine treatment (p<0.05, t-test). ( C & D ), immuno-blot and quantification analysis of PDE10A expression after transfection of HPRT-deficient MN9D cells with siRNA directed to the mouse PDE10 gene. The data show lower expression PDE10A protein in cells transfected with siRNA-PDE10A relative to cells transfected with scramble control siRNA (siRNA-CTL). Error bars represent mean ± SEM of duplicate measurements carried out independently twice (n = 4). The asterisks (*) represent the statistical significant (p<0.05, t-test) between the control (open bar, siRNA-CTL) and siRNA-PDE10A transfected cells (closed bar). This significant reduction of PDE10A protein contributes to the enhanced expression of phospho-PKA-substrates including p-Syn observed in ( E & F ). Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisk (*) represents the statistical significant (p<0.05, t-test) between the siRNA-CTL and siRNA-PDE10A HPRT-deficient transfected MND9 cells treated with forskolin.

Article Snippet: 2×10 5 HPRT-deficient MN9D cells in 6 well-plate were exposed to 80 pmole of control (scramble siRNA) or PDE10A siRNA (directed to mouse PDE10A) from Santa cruz biotechnology and transfected according to the established transfection protocol ( http://datasheets.scbt.com/siRNA_protocol.pdf ).

Techniques: Expressing, Transfection, Control