Journal: Scientific reports

Article Title: TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/ERK/NF-kB signaling pathways.

doi: 10.1038/s41598-023-41514-6

Figure Lengend Snippet: Figure 3. ACSL1 siRNA transfection reduced TNFα-mediated MMP-9 production. We transfected THP-1 monocytic cells with siRNA targeting human ACSL1 gene expression or scrambled siRNA (a control siRNA). (A,B) After 36 h, we performed real-time PCR to measure ACSL1 gene expression or western blotting for protein to test the knocking down efficiency. (C) We then incubated ACSL1-deficient cells with TNFα for 24 h. We determined mRNA expression of MMP-9 by real-time PCR. (D) We determined MMP-9 protein in culture media using ELISA. (E) The effect of siRNA transfection in combination with TNFα on cell viability was evaluated by measuring cell metabolic activity (MTT assay). The cell viability is expressed as the percentage of cells compared to the condition of vehicle control. Three independent experiments were performed with similar results. All data are expressed as mean ± SEM (n ≥ 3). t test or onene way ANOVA (Dunnett’s Test) for comparing treatments vs control) were used). **p < 0.01, ***p < 0.001.

Article Snippet: We transfected the cells separately with siRNA against ACSL1 (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), scramble siRNA (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), and pmaxGFP (0.5 ug; Amaxa Nucleofector Kit V for THP-1cells, Lonza, Cologne, Germany).

Techniques: Transfection, Gene Expression, Control, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, MTT 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. 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: Oncogene

Article Title: Oncogenic suppression of PHLPP1 in human melanoma.

doi: 10.1038/onc.2013.420

Figure Lengend Snippet: Figure 4. Knockdown of PHLPP1 results in anchorage-independent growth of melanocytes, whereas overexpression of PHLPP1 inhibits melanoma growth in a xenograft mouse model. (a) HEMa-LP melanocytes were transduced with the control shRNA, PHLPP1 shRNA1 or PHLPP1 shRNA2. After 24 h, whole-cell lysates were subjected to western blot analysis. Data shown are representative of three individual western blot analyses. (b) Left panel: anchorage-independent growth assay of HEMa-LP melanocytes transduced with the control, PHLPP1 shRNA1, or PHLPP1 shRNA2. Scale bar, 0.5 mm. Right panel: quantitation of colony formation as shown in left panel. Data shown are the numbers of colonies in 10 random phase-contrast microscopic fields (n ¼ 3, mean±s.e.m; Student’s t-test, *Po0.05). (c) HEMa-LP melanocytes transduced with the control shRNA, PHLPP1 shRNA1, PHLPP1 shRNA2 were counted on days 3, 6 and 9 using an automated cell counter (n ¼ 3, mean±s.e.m; Student’s t-test, *Po0.05). (d) A total of 5 104 HEMa-LP melanocytes transduced with the control shRNA, PHLPP1 shRNA1 or PHLPP1 shRNA2 with or without exposure to the vehicle control (DMSO) or LY294002 (20 mM) were counted on day 9 using an automated cell counter (n ¼ 3, mean±s.e.m; Student’s t-test, *Po0.001). (e) MM200.PHLPP1 and Mel-RM.PHLPP1 cells and their corresponding control counterparts carrying vector alone were xenografted into flanks of nu/nu mice via subcutaneous (s.c.) injection. The data shown are photographs of representative melanomas harvested at day 36 after intraperitoneal (i.p.) injection. Scale bars, 5 mm. (f) MM200.PHLPP1 and Mel-RM.PHLPP1 cells and their corresponding control counterparts carrying vector alone were xenografted into flanks of nu/nu mice via s.c. injection. The data shown are growth curves of melanoma tumors represented by the volume calculated with the modified ellipsoidal formula (tumor volume ¼ 1/2(length width2)), which are mean±s.e.m. of all tumors in each experimental group (*Po 0.001, Student’s t-test). (g) MM200.PHLPP1 and Mel-RM.PHLPP1 cells and their corresponding control counterparts carrying vector alone were xenografted into flanks of nu/nu mice via s.c. injection. The data shown tumor weight (mean±s.e.m.) generated by PHLPP1-overexpressing melanoma sub-lines (MM200.PHLPP1 and Mel-RM.PHLPP1) in comparison with that formed corresponding control lines carrying vector alone. (h) Whole-cell lysates of crude tumor tissues from randomly sampled tumors generated by PHLPP1-overexpressing melanoma sub-lines (MM200.PHLPP1 and Mel- RM.PHLPP1) and those formed by corresponding control lines carrying vector alone were subjected to western blot analysis. Data shown are representative of three individual western blot analyses. (i) MM200.PHLPP1 cells with or without co-transduced with Akt/S473D were xenografted into flanks of nu/nu mice via s.c. injection. The data shown are photographs of representative melanomas harvested at day 36 after i.p. injection. Scale bars, 5 mm. (j) MM200.PHLPP1 cells with or without co-transduced with Akt/S473D were xenografted into flanks of nu/nu mice via s.c. injection. The data shown are tumor volume at 36 days after i.p. injection (mean±s.e.m; Student’s t-test, *Po0.05).

Article Snippet: Human PHLPP1 shRNA kit (TG302501) and scramble shRNA kit (TG300130) were purchased from Origene (Karrinyup, WA, Australia).

Techniques: Knockdown, Over Expression, Transduction, Control, shRNA, Western Blot, Growth Assay, Quantitation Assay, Plasmid Preparation, Injection, Generated, Comparison