Journal: Molecular cell

Article Title: The MYCN oncoprotein is an RNA-binding accessory factor of the nuclear exosome targeting complex.

doi: 10.1016/j.molcel.2024.04.007

Figure Lengend Snippet: Figure 4. Exosome function controls the dynamics of the MYCN/MAX/MXD network (A) Immunoblot of IMR-32 cells stably expressing an shRNA targeting EXOSC10 treated for 72 h with Dox or EtOH as control. ACTB was loading control. Blot is representative of n = 3 independent replicates. (B) Read distribution of MYCN ChIP-Rx, MAX, MNT, and SIN3A cleavage under targets & release using nuclease (CUT&RUN) at the CDK5RAP3 and PHB genes. Cells treated as in (A) (n = 3 for MYCN ChIP-Rx; n = 2 for all C&Rs).

Article Snippet: 3 mg of each, MAX (Proteintech), MNT (Thermo Fisher Scientific), or SIN3A (Novus Biologicals) antibody were added per sample and incubated with shaking (800 rpm) at 4 C overnight.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control

Journal: Scientific Reports

Article Title: Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

doi: 10.1038/srep12624

Figure Lengend Snippet: ( a ) Schematic diagram of CCNY domain structure. Numbers indicate amino acid residues. Domain is predicted by ScanProsite ( http://www.expasy.ch/tools/scanprosite/ ) . ( b ) Alignment of CCNY amino acid sequences among human, rat, and mouse was performed using NCBI BLAST program. Blue color indicates amino acids showing differences among species. Orange indicates cyclin box domain in CCNY. ( c ) CCNY expression levels in the several regions of rat brain. Quantification is shown in the lower panel (n = 3; postnatal day 30 male rats). An equal amount of protein (40 μg) from each region was loaded. CTX, cortex; ST, striatum; HC, hippocampus; TH, thalamus; SN, substantia nigra; CB, cerebellum. ( d ) CCNY expression in the DG, CA3, and CA1 in the hippocampus. Postnatal day 30 male rats. ( e , f ) Hippocampal expression levels of CCNY in vivo ( e ) and in vitro ( f ) during development. P, postnatal day; DIV, days in vitro . ( g ) Distribution of CCNY in subcellular fractions of rat brains. H, homogenates; P1, nuclear pellet; P2, crude synaptosomal fraction; S3, cytosolic fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; SPM, synaptic plasma membrane fraction; T-sol, Tx-100-soluble fraction; PSD, postsynaptic density fraction. A total of 5 μg of each fraction was loaded in immunoblot experiments. GluA1, PSD-95 and synaptophysin were used as controls. ( h ) CCNY is localized adjacent to PSD-95 in spines. Scale bars, 1 μm. 3D iso-surfaced and volume rendered images with various angle views are shown in hi − hv .

Article Snippet: Primary antibodies against CCNY (Proteintech group), GFP (Roche), GluA1 (a gift from Michael Ehlers, Pfizer Neuroscience), phospho-GluA1 (S845) (Thermo scientific), PSD-95 (Thermo scientific, 7E3-1B8), Synaptophysin (Synaptic Systems), Prox1 (Proteintech group), Ctip2 (Genetex), Py (a gift from D.T.S.

Techniques: Expressing, In Vivo, In Vitro, Membrane, Clinical Proteomics, Western Blot

Journal: Scientific Reports

Article Title: Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

doi: 10.1038/srep12624

Figure Lengend Snippet: ( a ) Knockdown of CCNY increases basal EPSC AMPA amplitudes with no change in EPSC NMDA amplitudes. Pairwise analysis on the effect of the CCNY shRNA on basal EPSC AMPA amplitude (•, recorded at a holding potential of −70 mV, n = 16) and EPSC NMDA amplitude (●, recorded at a holding potential of +40 mV, n = 16) in the same slice using the same stimulus position and intensity. Red symbols and error bars indicate mean ± SEM. ( b ) The CCNY shRNA-mediated enhancement of basal EPSC AMPA amplitudes was rescued back to the level of untransfected neurons by co-transfecting with shRNA-resistant CCNY-WT construct (●, n = 16) with no effect on EPSC NMDA amplitude (•, n = 16). Red symbols and error bars indicate mean ± SEM. ( c ) Confocal immunostaining of endogenous surface GluA1 in CCNY shRNA transfected or CCNY shRNA-resistant CCNY-WT (Rescue) co-transfected neurons. Scale bar, 5 μm. ( d , e ) Cumulative distribution of surface GluA1 ( d ) and GluN1 ( e ) in dendritic protrusions. Insets display means ± SEM of surface GluA1 ( d ) and GluN1 ( e ) intensity. n = 847, 829, 515 protrusions from n = 24, 27, 17 neurons, respectively in ( d ). n = 227, 361, 287 protrusions from n = 7, 11, 11 neurons, respectively in ( e ). ** p < 0.005 relative to control. ## p < 0.005 relative to shCCNY. ( f – h ) Knockdown of CCNY does not change the total expression level of endogenous GluA1. ( f , g ) Confocal images of endogenous total GluA1 in CCNY shRNA or scrambled shRNA transfected neurons. Neurons were transfected at DIV13−14 and immunostained at DIV16−18. NS, not significant, Scale bar, 20 μm. ( h ) Cultured neurons infected with lentivirus overexpressing CCNY shRNA were applied to immunoblot analysis.

Article Snippet: Primary antibodies against CCNY (Proteintech group), GFP (Roche), GluA1 (a gift from Michael Ehlers, Pfizer Neuroscience), phospho-GluA1 (S845) (Thermo scientific), PSD-95 (Thermo scientific, 7E3-1B8), Synaptophysin (Synaptic Systems), Prox1 (Proteintech group), Ctip2 (Genetex), Py (a gift from D.T.S.

Techniques: Knockdown, shRNA, Construct, Immunostaining, Transfection, Control, Expressing, Cell Culture, Infection, Western Blot

Journal: Scientific Reports

Article Title: Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

doi: 10.1038/srep12624

Figure Lengend Snippet: ( a , b ) Overexpression of CCNY reduces basal EPSC AMPA amplitudes with no change in EPSC NMDA amplitudes. Pairwise analysis of the effect of CCNY-WT (21 pairs of transfected and untransfected neighboring cells) on basal EPSC AMPA amplitude ( a ) and EPSC NMDA amplitude ( b ). Pairs of transfected and untranfected neighboring cells in the same slice using the same stimulus position and intensity are individually plotted. Red symbol and error bars indicate mean ± SEM. ( c ) Overexpression of CCNY-WT decreases surface level of endogenous GluA1. Confocal immunostaining of endogenous surface GluA1 in CCNY-WT transfected neurons. Neurons were transfected at DIV14−15 and immunostained at DIV15−17. Scale bar, 5 μm. ( d , e ) Cumulative distribution of surface GluA1 ( d ) and GluN1 ( e ) in dendritic protrusions. Insets display means ± SEM of surface GluA1 ( d ) and GluN1 ( e ) intensity. n = 1827, 1699 protrusions from n = 31, 27 neurons, respectively in ( d ). n = 652, 509 protrusions from n = 10, 10 neurons, respectively in ( e ). ** p < 0.0001 relative to control. ( f – h ) Overexpression of CCNY does not change the total level of endogenous GluA1. ( f , g ) Confocal images of endogenous total GluA1 in CCNY-WT transfected neurons. Neurons were transfected at DIV14−15 and immunostained at DIV15−17. NS, not significant, Scale bar, 20 μm. ( h ) Cultured neurons infected with lentivirus overexpressing CCNY-WT were applied to immunoblot analysis.

Article Snippet: Primary antibodies against CCNY (Proteintech group), GFP (Roche), GluA1 (a gift from Michael Ehlers, Pfizer Neuroscience), phospho-GluA1 (S845) (Thermo scientific), PSD-95 (Thermo scientific, 7E3-1B8), Synaptophysin (Synaptic Systems), Prox1 (Proteintech group), Ctip2 (Genetex), Py (a gift from D.T.S.

Techniques: Over Expression, Transfection, Immunostaining, Control, Cell Culture, Infection, Western Blot

Journal: Scientific Reports

Article Title: Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

doi: 10.1038/srep12624

Figure Lengend Snippet: ( a , b ) SEP-GluA1 was imaged before and after glycine stimulation. Arrows indicate spines showing the changes of SEP-GluA1 intensity during glycine-induced LTP. Pseudocolor intensity scale bar is shown. Scale bars, 1 μm each. See also for more images. ( c ) Data represent means ± SEM of ΔF/ F 0 from spines. n = 32, 41, 31, 31 spines from n = 7, 5, 6, 6 neurons for control, CCNY-WT, shCCNY, and shCCNY + rescue, respectively. Bonferroni’s post-hoc . ( d ) The number of SEP-GluA1 inserted and accumulated per 100 μm of dendrite. n = 11, 10, 10, 6 neurons from left to right. * p < 0.01 relative to control, ** p < 0.001 relative to control, ## p < 0.005 relative to shCCNY, student’s t test. ( e ) Total expression level of CCNY is unchanged during glycine-induced LTP. Cultured hippocampal neurons infected with lentivirus overexpressing CCNY-WT or CCNY shRNA were applied to immunoblot analysis before and 20 minutes after glycine stimulation. ( f ) Data represent means ± SEM of CCNY level. n = 12, 5, 7 for control, CCNY-WT, and shCCNY, respectively. NS, not significant. ( g ) CCNY regulates phosphorylation of GluA1 at Ser845 during glycine-induced LTP. Cultured hippocampal neurons infected with lentivirus overexpressing CCNY shRNA or scrambled shRNA were immunoblotted with anti-phospho-GluA1 (S845) antibodies before and 15–20 min after glycine stimulation. ( h ) Data represent means ± SEM of phosphorylated levels of GluA1 at S845. n = 7. * p < 0.05, ** p < 0.005, student’s t test.

Article Snippet: Primary antibodies against CCNY (Proteintech group), GFP (Roche), GluA1 (a gift from Michael Ehlers, Pfizer Neuroscience), phospho-GluA1 (S845) (Thermo scientific), PSD-95 (Thermo scientific, 7E3-1B8), Synaptophysin (Synaptic Systems), Prox1 (Proteintech group), Ctip2 (Genetex), Py (a gift from D.T.S.

Techniques: Control, Expressing, Cell Culture, Infection, shRNA, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: Cyclin Y inhibits plasticity-induced AMPA receptor exocytosis and LTP

doi: 10.1038/srep12624

Figure Lengend Snippet: LTP stimulus induces AMPA receptor insertion to the surface (middle panel). Overexpression of CCNY (CCNY OE) inhibits plasticity-induced AMPA receptor exocytosis in the spine, therefore blocking LTP (left panel). Knockdown of CCNY (CCNY KD) significantly increases plasticity-induced phosphorylation of AMPA receptors (p845-GluA1) and their delivery to the plasma membrane in the spine, therefore enhancing LTP (right panel).

Article Snippet: Primary antibodies against CCNY (Proteintech group), GFP (Roche), GluA1 (a gift from Michael Ehlers, Pfizer Neuroscience), phospho-GluA1 (S845) (Thermo scientific), PSD-95 (Thermo scientific, 7E3-1B8), Synaptophysin (Synaptic Systems), Prox1 (Proteintech group), Ctip2 (Genetex), Py (a gift from D.T.S.

Techniques: Over Expression, Blocking Assay, Knockdown, Phospho-proteomics, Clinical Proteomics, Membrane