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: Scientific Reports

Article Title: Impact of the secretome of activated pancreatic stellate cells on growth and differentiation of pancreatic tumour cells

doi: 10.1038/s41598-019-41740-x

Figure Lengend Snippet: The effect of knocking down eIF4E protein expression by small interfering RNA. Tumour cells PT45P1 and Panc-1 were transfected with 100 nM of an eIF4E-specific siRNA or a control siRNA of scrambled sequence for 48 h, followed by serum starvation overnight. Subsequently, they were grown in presence or absence of activated PSC secretome for 24 h. The effect of the various conditions on eIF4E is shown, determined by Western blotting. Also, the results of caspase-3/7 assays are shown for each column (c1 to c6). Note: the two lanes c1 and c2 are exchanged in the Western blots generated from Panc-1 cells.

Article Snippet: We used the siRNA gene silencer system (siRNA #6554) as well as a control siRNA (#6568) of Cell Signaling Technology (Danvers, USA) to perform the EIF4E gene silencing in the pancreatic cancer cell lines PT45P1, Panc-1 and Capan-1 according to the manufacturer’s protocol.

Techniques: Expressing, Small Interfering RNA, Transfection, Control, Sequencing, Western Blot, Generated

Journal: Scientific Reports

Article Title: Impact of the secretome of activated pancreatic stellate cells on growth and differentiation of pancreatic tumour cells

doi: 10.1038/s41598-019-41740-x

Figure Lengend Snippet: Transient silencing eIF4E in Capan-1 pancreatic cancer cells inhibits cell migration. Capan-1 cells were transfected with 100 nM of either eIF4E-specific siRNA or a control siRNA of scrambled sequence for 48 h. Cells were serum starved overnight and either left untreated or treated with activated PSC secretome. A gap was generated by physically scraping off cells. The gap was inspected at different time intervals of up to 48 h at the growth conditions indicated in the figure.

Article Snippet: We used the siRNA gene silencer system (siRNA #6554) as well as a control siRNA (#6568) of Cell Signaling Technology (Danvers, USA) to perform the EIF4E gene silencing in the pancreatic cancer cell lines PT45P1, Panc-1 and Capan-1 according to the manufacturer’s protocol.

Techniques: Migration, Transfection, Control, Sequencing, Generated

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 5. AMPK activator A769662 mimics the effect of metformin in MGO-stimulated cells (A) Cells were pretreated with metformin (6 mM) or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) treatment for indicated time points. AMPK phosphorylation was measured by immunoblotting. (B) Cells were 30 min pretreated with A769662 (25 μM) prior to MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. (C, D, E, F) Cells were pretreated with A769662 (25 μM) for 30 min followed by MGO (300 μg/ml) treatment for 4 h. DCFDA (C), DHE (D), mitoSOX (E), and mitoPY1 (F) were used to measure cellular ROS. (G) Cells were 30 min pretreated with compound C (10 μM) followed by MGO (300 μg/ml) stimulation for 6 h. In some experiments, ARPE-19 cells were treated with siRNA followed by stimulation with MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. AMPK expression after siRNA treatment was determined by immunoblotting. (H) Immediately after treatment with A769662 (25 μM), cells were subjected into SFe24 analyzer for OCR measurement, as described in Fig. 3D. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO; #p < 0.05, indicating the significant effects of A769662, compound C and AMPK silencing on MGO- induced responses.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 8. Metformin and A769662 reverse MGO-induced GLO1 downregulation. (A) Cells were pretreated with BBGC (10 μM), metformin (6 mM) and/or A769662 (25 μM) 30 min before MGO (100 μg/ml) stimulation. After 4 h, cell viability was determined by Annexin V-FITC/PI staining using FACS. (B) Cells were treated with siRNA to silence GLO1, then treated with MGO (100 or 300 μg/ml) for 6 h. Cell viability was determined by Annexin V-FITC/PI staining using FACS. (C–E) Cells were pretreated with metformin (6 mM) and/or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) stimulation. (C) After incubation for 1, 3, or 6 h, cell lysates were prepared for immunoblotting. (D) After incubation for 2 or 4 h, GLO1 gene expression was measured using PCR analysis. (E) After incubation for 3 or 6 h, GLO1 and GLO2 activities were determined by commercial kits according to the manufacturer’s instructions. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO. #p < 0.05, indicating the blockade effects of metformin and A769662.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Staining, Incubation, Western Blot, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Hyaluronic Acid Receptor CD44 Deficiency Is Associated with Decreased Cryptococcus neoformans Brain Infection

doi: 10.1074/jbc.m112.353375

Figure Lengend Snippet: FIGURE6.Effectofanti-CD44andanti-RHAMMsiRNAtreatmentsonasso- ciation of C. neoformans cells with mouse BMEC. A, mouse BMEC was treated with siRNA (20 pmol) individually or in combination for 5 h before the in vitro C. neoformans adhesion assay. C. neoformans CPS1 wild-type strain (B-4500FO2) and its isogenic cps1 deletion strain C559 were used in parallel. ,anti-CD44siRNAand/oranti-RHAMMsiRNA-treatedsampleasindicatedat the bottom; , control oligonucleotide (n 4). In all treatments, the MBMEC without any siRNA treatment was taken as a control for comparison, and the statistical package GraphPad Prism 5 was used to quantify the readings. Sig- nificant differences with regard to the controls: *, p 0.05; **, p 0.01). B, as a control, a Western blot was used to show the CD44 and RHAMM protein levels after siRNA treatment.

Article Snippet: All samples were examined under a fluorescence microscope at the Congressman Dixon Cellular Imaging Core Facility, Children’s Hospital Los Angeles. siRNA Treatment—Anti-CD44 siRNA (sc-35534) and antiRHAMM siRNA (sc-40182) were purchased from Santa Cruz Biotechnology, and a control oligonucleotide (sc-36869) was used in parallel.

Techniques: In Vitro, Cell Adhesion Assay, Control, Comparison, Western Blot