Journal: Journal of cell science

Article Title: Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

doi: 10.1242/jcs.091736

Figure Lengend Snippet: Fig. 3. P2Y1 silencing impairs ADP-dependent axon elongation. (A) Hippocampal neurons were nucleofected with scrambled shRNA, P2Y1 shRNA or P2Y13 shRNA. Neurons were fixed at 3 DIV and stained with an anti-a-tubulin antibody. Nucleofected neurons were identified by their GFP fluorescence. (B) HEK- 293T cells were co-transfected with GFP, P2Y1–GFP or P2Y13 plasmids, in combination with different P2Y1 or P2Y13 shRNAs. Data are means ± s.e.m. of three independent experiments. P2Y1–GFP and P2Y13 protein expression was normalized to a-tubulin expression levels; ***P,0.001. (C) Axon length of hippocampal neurons expressing scrambled shRNA, two different P2Y1 shRNAs or two different P2Y13 shRNAs was quantified after staining with antibodies against MAP2 and Tau-1. Data are mean axon lengths ± s.e.m. from three independent experiments, analyzing 100 neurons for each condition in each experiment; ***P,0.001. The dotted grey line indicates the mean axon length of scrambled-shRNA-nucleofected neurons. (D,E) Hippocampal neurons nucleofected with scrambled shRNA or P2Y1 shRNA and treated with ADP (5 mM) from day 1 to day 3 in vitro. The graph in D shows the axon length in nucleofected neurons (GFP-positive) incubated in the presence or absence of ADP. (F–H) Hippocampal neurons nucleofected with scrambled shRNA, P2Y1 shRNA or P2Y13 shRNA and treated with the P2Y1 antagonist (MRS-2179) or the P2Y13 antagonist (MRS-2211) from day 1 to day 3 in vitro. Scale bars: 50 mm. Note that in all cases P2Y1 expression and function is necessary for axon elongation. Data in G are the mean axon lengths ± s.e.m. from three independent experiments, analyzing 100 neurons for each condition in each experiment; ***P,0.001. H shows the distribution of the axon length for all neurons from three independent experiments for each condition (n5300). (I) Hippocampal neurons that had been nucleofected with plasmids expressing GFP, P2Y1–GFP and P2Y13. After 3 DIV neurons were stained for MAP2 and Tau-1 to identify the axon. (J) P2Y1 or P2Y13 mean fluorescence intensity along the axon in control, scrambled shRNA, P2Y1 shRNA or P2Y13 shRNA nucleofected neurons. (K) Graph of the mean axon lengths ± s.e.m. of neurons nucleofected with GFP, P2Y1–GFP or P2Y13 and GFP. Neurons were quantified in three independent experiments, analyzing 100 neurons for each condition in each experiment; ***P,0.001. Scale bars: 100 mm. Box-plot shows the distribution of axon lengths for all the neurons quantified in K.

Article Snippet: The adenylate cyclase 5 interference shRNAs (79 and 84) and control scrambled shRNA were purchased from Origene (TG506651).

Techniques: shRNA, Staining, Fluorescence, Transfection, Expressing, In Vitro, Incubation, Control

Journal: Journal of cell science

Article Title: Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

doi: 10.1242/jcs.091736

Figure Lengend Snippet: Fig. 5. Adenylate cyclase activity is necessary for ADP–P2Y1-dependent axon elongation. (A) Hippocampal neurons treated with the indicated compounds from day 1 to day 3 in vitro and stained for MAP2 and Tau-1. Scale bar: 100 mm. (B) Axon length in neurons treated with vehicle (black bars) or the indicated adenylate cyclase or cAMP regulators (white bars), in combination with agonists or antagonists of P2Y1 or P2Y13. Graphs represent the mean axon length ± s.e.m. from three independent experiments, analyzing 100 neurons for each condition in each experiment; ***P,0.001, **P,0.01; n.s., not significant. (C,E) Hippocampal neurons nucleofected with P2Y1 shRNA and stained at 3 DIV for Tau-1 or a-tubulin (red). Nucleofected neurons were identified by GFP fluorescence. Neurons were treated with the adenylate cyclase activator forskolin (5 mM) or a PDE4 inhibitor (20 nM). Note that both treatments reversed the negative effects of P2Y1 silencing or P2Y13 expression on axon elongation. The graphs in E show the axonal lengths ± s.e.m. from three independent experiments, analyzing 100 GFP positive neurons for each condition in each experiment; ***P,0.001. (D,F) Neurons nucleofected with P2X7 shRNA or P2X7– GFP expression plasmids and treated from day 1 to day 3 in vitro with the adenylate cyclase inhibitor (SQ-22536) or the adenylate cyclase activator forskolin, respectively. Note that adenylate cyclase activation or increased cAMP levels reversed the negative effect of P2X7–GFP expression on axon elongation. The graphs in F show the axon length ± s.e.m. from three independent experiments analyzing 100 neurons for each condition in each experiment; ***P,0.001. Scale bars: 100 mm.

Article Snippet: The adenylate cyclase 5 interference shRNAs (79 and 84) and control scrambled shRNA were purchased from Origene (TG506651).

Techniques: Activity Assay, In Vitro, Staining, shRNA, Fluorescence, Expressing, Activation Assay

Journal: Journal of cell science

Article Title: Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

doi: 10.1242/jcs.091736

Figure Lengend Snippet: Fig. 6. Adenylate cyclase 5 activity is required for proper axonal elongation in response to ADP or P2X7 inhibition. (A,D) Hippocampal neurons cultured from day 1 to day 3 in vitro in the presence or absence of the PKCf inhibitor, PKCf pseudosubstrate (10 mM) in combination with ADP 5 mM, BBG 100 nM, MRS-2211 5 mM or forskolin 5 mM. Neurons were stained with anti-MAP2 and anti-Tau-1 antibodies. Graph in D shows the mean axonal lengths ± s.e.m. from three independent experiments. (B) Distribution of adenylate cyclase 5 in hippocampal neurons at 3 DIV. Arrow indicates the AC5 in the distal region of the axon. Right panels show the distal region of the axon stained for AC5 and F-actin. Scale bar: 100 mm. (C) Hippocampal neurons treated from day 1 to day 3 in vitro with ADP (5 mM) in the presence or absence of the adenylate cyclase 5 inhibitor NY80 (10 mM). Scale bar: 100 mm. (E) Mean axon lengths ± s.e.m. of 3 DIV neurons treated with vehicle (black bars) or NKY80 (white bars), in combination with ADP (5 mM), the P2Y13 antagonist MRS-2211, dbcAMP (2 mM), rPMT or PTX. Note that addition of dbcAMP impaired the inhibitory effect of NKY80 on axon growth; ***P,0.001. Data are from three independent experiments analyzing 100 neurons for each condition in each experiment. (F,H) Neurons nucleofected with GFP, P2Y1–GFP, scrambled shRNA, P2X7 shRNA or P2Y13 shRNA were cultured from day 1 to day 3 in vitro with vehicle or the adenylate cyclase 5 inhibitor, NKY80. (F) Representative images of these neurons. (H) Mean axonal lengths ± s.e.m. from three independent experiments analyzing 100 neurons for each condition in each experiment; ***P,0.001. (G,I) Neurons were nucleofected with scrambled shRNA or AC5 shRNA. (G) Representative images of 3 DIV neurons. (I) Mean axon length ± s.e.m. of neurons shown in G cultured in the presence of the indicated compounds at the concentrations shown previously. Scale bars: 100 mm.

Article Snippet: The adenylate cyclase 5 interference shRNAs (79 and 84) and control scrambled shRNA were purchased from Origene (TG506651).

Techniques: Activity Assay, Inhibition, Cell Culture, In Vitro, Staining, shRNA

Journal: Experimental neurology

Article Title: Role of PDGF-D and PDGFR-β in neuroinflammation in experimental ICH mice model.

doi: 10.1016/j.expneurol.2016.06.010

Figure Lengend Snippet: Fig. 5. Effects of plasmin on PDGF-D induced inflammation. The plasmin antagonist (EACA) decreased ICH induced production of PDGF-D and p-PDGFRβ increases 24 h after ICH (A, #p b 0.05 vs sham. *p b 0.05 vs ICH. n = 6 mice per group), resulting in decreased microglia activation (B). Injection of the recombinant plasmin induced significant production of the PDGF-D in ipsilateral hemisphere (C, #p b 0.05 vs sham, @p b 0.05 vs contralateral. n = 6 mice per group). Increased MPO levels (D) were also observed after plasmin injection. While scrambled RNA has no effect, PDGF-D siRNA attenuated plasmin induced increase of MPO production (D, #p b 0.05 vs sham. αp b 0.05 vs Plasmin. n = 6 mice per group). Error bars represent mean ± standard error of the mean. Scale bar = 50um.

Article Snippet: Experiment 5: The PDGF-D siRNAs mixture or scramble siRNA (100 pmol in 2 μl, OriGene) was administered intraventricularly 24 h before plasmin was injected into the right basal ganglia in naïve mice.

Techniques: Activation Assay, Injection, Recombinant

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH inhibits the proliferation and induces apoptosis of NP cells. ( A ) The expression levels of circITCH were measured by qPCR in the NP tissues of IDD patients (n=90) and normal cases (n=90). ( B – F ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( B ) The expression levels of circITCH were examined by qPCR in the cells. ( C , D ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( E ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( F ) The expression of Bax, caspase3, cleaved caspase3 (c-caspase3), caspase9, and cleaved caspase9 (c-caspase9) was measured by Western blot analysis. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH promotes ECM degradation of degenerative NP cells. ( A – E ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( A – D ) The mRNA expression of collagen II ( A ), aggrecan ( B ), MMP13 ( C ), and ADAMTS4 ( D ) was measured by qPCR in the cells. ( E ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was tested by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH serves as a miR-17-5p sponge in NP cells. ( A ) The potential interaction between circITCH and miR-17-5p was identified by the bioinformatic analysis using ENCORI ( http://starbase.sysu.edu.cn/index.php ). ( B , C ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type circITCH (circITCH WT) and circITCH with the miR-17-5p-binding site mutant (circITCH MUT) were determined by luciferase reporter gene assays in the cells. ( D ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. The expression of miR-17-5p was analyzed by qPCR in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Control, Expressing, Luciferase, Binding Assay, Mutagenesis, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH activates Wnt/β-catenin signaling by targeting miR-17-5p/SOX4 axis. ( A ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and miR-17-5p inhibitor. The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( B , C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, lentiviral plasmids carrying circITCH shRNA, pcDNA3.1-SOX4 overexpression vector, and miR-17-5p mimic, or lentiviral plasmids carrying circITCH shRNA and LiCl. The expression of Wnt1, β-catenin, c-Myc, Cyclin D1, and β-actin was analyzed by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Expressing, Western Blot, Over Expression, Plasmid Preparation, Software

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH contributes to ECM degradation of degenerative NP cells by modulating miR-17-5p/SOX4/Wnt/β-catenin signaling. ( A – C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, miR-17-5p inhibitor, or LiCl. ( A ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( B ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( C ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot