Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Proliferative response of GFP (control) and GluA2-dominant negative subunit expressing glioma cells (GluA2-DN) after 24-hour exposure to soluble extracellular neuroligin-3 (NLGN3; 100nm) in the presence and absence of AMPA-receptor blocker, NBQX (10μM). b , Western blot analysis of phospho-AKT (Ser473) and total AKT in GFP (control) glioma cells in response to 5-minute exposure of soluble extracellular neuroligin-3 (NLGN3; 100nm) in the presence and absence of AMPA-receptor blocker, NBQX (10μM); left. Quantitative analysis of the ratio of pAKT/AKT normalized to vehicle (right). c , Western blot analysis of phospho-AKT (Ser473) and total AKT in GluA2-DN expressing glioma cells in response to 5-minute exposure of soluble extracellular neuroligin-3 (NLGN3; 100nm; left). Quantitative analysis of the ratio of pAKT/AKT normalized to vehicle (right). d , Time course of evoked glioma cell EPSC block by NASPM (100 μM, duration=red bar (n=7/5 cells/mice; left); Representative trace before (black) and after (red) addition of NASPM (right). e, Quantification of (d). f , GluA2 subunit Q/R editing efficiency in SU-DIPGXIII-FL and SU-DIPGVI cells as measured by PCR and expressed as % edited. g , Expression of ADAR1, the enzyme responsible for Q/R editing of GluA2 mRNA. Plot illustrates ADAR1 enzyme mRNA expression relative to beta-actin as measured by qPCR. Analyses in a,b,c,f,g were calculated from three independent sets of cells. Data shown as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s post-hoc analysis (a,b), by two-tailed Student’s t-test (c), by two-tailed paired Student’s t-test (e). All data shown as mean ± s.e.m. *P<0.01, **P<0.001, ***P<0.001, ****P<0.0001, NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Control, Dominant Negative Mutation, Expressing, Western Blot, Blocking Assay, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a, Electrophysiological responses by model. Number of whole cell patch clamp recordings from cells in xenografted hippocampal slices separated by electrophysiological response to local electrical stimulation. b , Demonstration of depolarizing inward current in SU-DIPXIII-FL-ChR2 cells in response to single stimulation and 20Hz pulses of blue light as measured in current clamp (top) and voltage clamp (bottom). c , Proliferation index of xenografted SU-DIPGXIII-FL-YFP control glioma cells (no opsin expressed) in response to blue light stimulation or mock stimulation as measured by the proportion of GFP+/HNA+ cells expressing Ki67 24-hours after five optogenetic stimulation sessions (n=3 mice, mock stim; n=4 mice, stim). d, Quantification of cleaved caspase-3 in xenografted SU-DIPGXIII-FL-YFP control glioma cells in response to blue light stimulation or mock stimulation as measured by total number of HNA+ cells co-labeled with cleaved caspase-3 (n=3 mice/group). e , As in (d), quantification of cleaved caspase-3 in xenografted SU-DIPXIII-FL-ChR2 glioma cells (n=3 mice, mock stim; n=4 mice, stim). f , Validation of GluA2-dominative negative AMPA receptor subunit expressing construct. Representative traces of whole-cell voltage-clamp recording of WT (black) and GluA2-DN expressing (grey) SU-DIPGVI cells in response to 500μM (S)-AMPA (n=6 cells). g , Representative traces of whole-cell voltage-clamp recording in WT (black) and GluA2-DN expressing (grey) SU-DIPGXIII-FL cells in response to 500μM (S)-AMPA (n=6 cells). SU-DIPGXIII-FL cells are unable to homogeneously express the dominant construct, and therefore may be connected to WT GluA2 expressing cells, which accounts for the remaining current in the illustrated trace. Incorporation of the GluA2-DN construct thus results in a significantly abrogated AMPAR-dependent depolarization. Data shown as mean ± s.e.m for (c,d,e). All P-values determined by two-tailed Student’s t-test. NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Patch Clamp, Control, Expressing, Labeling, Biomarker Discovery, Construct, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Optogenetic paradigm for glioma depolarization. ChR2-expressing glioma (blue), region of analysis (light blue). b , Proliferation index of SU-DIPGXIII-FL-ChR2 xenograft after mock stimulation (mock stim) or blue light stimulation (stim) measured as percent of GFP+/HNA+ cells expressing Ki67 (mock stim, n=8; stim, n=9 mice). c , As in (b), but SU-DIPGVI-ChR2 xenografts (n=6 mice/group). d , Representative confocal micrographs from (c), illustrating proliferating SU-DIPGVI-ChR2. Red=human nuclei; white=Ki67. Scale bar=50μm. e-f , Kaplan-Meier survival curves of SU-DIPGXIII-P* xenografts overexpressing e, GFP-only (green) or GluA2-WT-GFP (red) and f, GFP-only (in 80% of cells, green) or GluA2-DN-GFP (in 80% of cells, blue); n=5 mice/group. g , Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in (f), determined by GFP/total human nuclei pixel intensity; (n=3 mice/group). h , Representative confocal micrographs of (f-g). White=human nuclei; green=GFP. Scale bar=50μm. i , Representative confocal images of SU-DIPGXIII-FL xenografts expressing GFP-only control (top) or GluA2-DN-GFP (bottom). Gray=MBP; White=glioma-GFP. Scale bar=500μm. j , Quantification of (i) (n=8 mice/group). k , Proliferation index of SU-DIPGVI xenografts treated with perampanel (AMPAR blocker) or vehicle control; (n=8 mice/group). l , Proliferation index of SU-DIPGXIII-FL in mice treated with meclofenamate (gap junction blocker) or vehicle control; (n=9 vehicle, n=8 treated mice). Data shown as mean±s.e.m (b,c,g,j,k,l). **P<0.01. ***P<0.001, ****P<0.0001. P-values determined by two-tailed unpaired Student’s t-test (b,c,g,k,l); two-tailed log rank analyses (e,f); two-sided Mann-Whitney test (j).

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Expressing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Kaplan-Meier survival curves of second cohort of mice orthotopically xenografted with control GFP-only or GluA2-DN-GFP over-expressing cells (SU-DIPGXIII-P* xenograft model; n=5 mice per group). b , Representative coronal sections of mouse brains bearing SU-DIPGXIII-FL xenografts either expressing control GFP construct (left) or GluA2-DN-GFP construct; right). Gray, MBP; White, glioma-GFP. c , Proliferation indices of SU-DIPGXIII-FL cells at baseline in neuronal medium, in response to 10μM NBQX, in co-culture with neurons, or in co-culture with neurons in the presence of 10μM NBQX (n=3 biological replicates/group, except n=4 for baseline). d, Representative images of neuron-glioma co-cultures in the presence and absence of NBQX. Green = neurofilament (neuronal processes); Red = nestin (glioma cell processes); White = Ki67. Scale bar = 50μm. e , in vitro growth analysis of control GFP or GluA2-DN-GFP cells monitored over 3 days. f, in vitro apoptosis analysis of control GFP or GluA2-DN-GFP as measured by % of total cells co-stained with cleaved-caspase. g , 3D Matrigel invasion assay in WT (GFP) and GluA2-DN (GluA2-DN-GFP) expressing SU-DIPGXIII-FL cells 72 hours after seeding. h , Representative images of (g) at time 0 hr (left) and 72 hr (right) in control GFP-expressing (top) and GluA2-DN-GFP expressing cells (bottom). Scale bar = 1000μm. i , 3D migration assay in WT (GFP) and GluA2-DN (GluA2-DN-GFP) expressing SU-DIPGXIII-FL cells 72 hours after seeding. j , Representative images of (i) at time 0 hr (left) and 72 hr (right) in control GFP-expressing (top) and GluA2-DN-GFP expressing cells (bottom). Scale bar =1000μm. k, Representative confocal micrographs illustrating proliferating SU-DIPGVI cells in vehicle or perampanel-treated mice (n=8 mice/group). Red = human nuclei; white = Ki67. Scale bar = 50μm. l , IVIS bioluminescence analysis of overall tumor growth in SU-DIPGXIII-FL xenografts treated with vehicle or meclofenamate over a two-week period. Data represented as fold change in total flux; n=5 mice/group. Data shown as mean ± s.e.m. for (c,e,f,g,i,l). For analyses in (d-j), n=3 biological replicates. P-values determined by two-tailed log rank analyses (a), by one-way ANOVA with post-hoc analysis (c), by two-tailed unpaired Student’s t-test (f,g,i,l). *P<0.05, **P< 0.01,****P<0.0001. NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Control, Expressing, Construct, Co-Culture Assay, In Vitro, Staining, Invasion Assay, Migration, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits.

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: Fig. 4 | Glioma membrane depolarization promotes glioma progression. a, Optogenetic model for glioma depolarization. Blue dots represent ChR2-expressing glioma cells; light blue rectangle denotes region of analysis. P, postnatal day. b, Proliferation index of SU-DIPG- XIII-FL-ChR2 xenografts after mock stimulation or blue light stimulation, measured as percentage of GFP+/HNA+ cells expressing Ki67 (mock stim, n = 8; stim, n = 9 mice). c, As in b but for SU-DIPG-VI-ChR2 xenografts (n = 6 mice per group). d, Representative confocal micrographs from c, illustrating proliferation of SU-DIPG-VI-ChR2 xenografts. Red denotes human nuclei staining by HNA; white denotes Ki67. Scale bar, 50 µm. e, f, Kaplan–Meier survival curves of SU-DIPG-XIII-P* (P denotes pontine tumour) xenografts that overexpress GFP-only (green) or GluA2- WT-GFP (red) (e) and GFP-only (in 80% of cells, green) or GluA2-DN- GFP (in 80% of cells, blue) (f) (n = 5 mice per group). g, Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in f, determined by

Article Snippet: We introduced SpeI (5′-TAAGCAACTAG TATGCAAAAGATTATGCAT-3′) and XmaI (5′-TGCTTACCCGGGC TAAATTTTAACACTTTCGAT-3′) restriction sites in full-length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Membrane, Expressing, Staining

Journal: Nature Communications

Article Title: VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

doi: 10.1038/ncomms11020

Figure Lengend Snippet: ( a ) Increased leucine concentrations in the medium induce the phosphorylation of S6 ribosomal proteins in the cultured neurons. The basal leucine concentration in Neurobasal medium is 0.8 mM. Full-size blots are shown in . ( b – g ) Cultured neurons were transfected with the indicated plasmids at 12 DIV, treated with different concentrations of leucine at 15 DIV and analyzed for ( b , c ) AHA incorporation, ( d , e ) ER distribution and ( f , g ) dendritic spine density at 18 DIV. Full-size cell images of ( f ) are shown in . In ( b ), the transfected neurons in the images are either outlined or indicated by arrows. ( g ) Rapamycin (Rapa; 10 nM) and MNK1 inhibitor CGP57380 (CGP; 10 μM) were added 6 h before harvesting to reduce the beneficial effect of leucine on dendritic spine density. Full-size cell images of ( g ) are shown in . Scale bars, ( b , d ) 20 μm; ( d ), enlarged and ( f ) 2 μm. The data from three independent experiments are presented as the mean+s.e.m. (error bars). The sample sizes ( n ) of the examined neurons ( c , e ) and dendrites ( f , g ) are indicated. * P <0.05; ** P <0.01; *** P <0.001; NS, non-significant. One-way ANOVA ( a ); two-way ANOVA ( c , e , f , g ).

Article Snippet: The antibodies and reagents used in this study were as follows: eIF2 α (ref. ) (sc-133,132, mouse, 1:1,000), Santa Cruz Biotechnology; VCP (612,182, mouse, 1:1,000), BD Transduction Laboratories; AHA (C10102), reaction buffer kits (C10269 and C10276), detection reagent Alexa Fluor 488 (A10267), GFP (A6455, rabbit, 1:1,000), Invitrogen; TAMRA (MA1-041, mouse, 1:1,000), P47 (PA5-21633, rabbit, 1:500), Calreticulin (PA3-900, rabbit, 1:200) and phospho-eIF2 α (MA5-15133, rabbit, 1:1,000), Thermo; HA (3F10, rat, 1:500), Roche; MYC (9B11, mouse, 1:1,000), Phospho-S6 ribosomal protein (4856, rabbit, 1:1,000), S6 ribosomal protein (2217, rabbit, 1:1,000), Cell Signaling; GFP (ref. ) (ab13970, chicken, 1:5,000), Abcam; puromycin (12D10, mouse, 1:1,000), GRIN2A (NR2A, 06-313, rabbit, 1:1,000) (ref. ), GRIN2B (NR2B, 06-600, rabbit, 1:1,000) (ref. ), GRIA1 (GluR1, MAB2263, mouse, 1:1,000), GRIA2/3 (GluR2/3, 07-598, rabbit, 1:1,000), GRM5 (mGluR5, AB5675, rabbit, 1:1,000), PSD-95 (MABN68, mouse, 1:2,000), Millipore; beta-actin (AC-74, mouse, 1:5,000), Sigma-Aldrich; CASK (mouse, 1:500) (ref. ), rabbit polyclonal UFD1L (generated by immunization with full-length mouse UFD1L protein, 1:1,000); L-leucine, tunicamycin, cycloheximide and CGP57380 (Sigma-Aldrich); Rapamycin (LC Laboratories).

Techniques: Phospho-proteomics, Cell Culture, Concentration Assay, Transfection

Journal: Hippocampus

Article Title: N-cadherin regulates molecular organization of excitatory and inhibitory synaptic circuits in adult hippocampus in vivo

doi: 10.1002/hipo.22282

Figure Lengend Snippet: (A and B) Representative immunoblots of synaptoneurosomes isolated from whole-hippocampus prepared from adult floxed and cKO mice showing diminished levels of GluA1 and its scaffolding protein PSD95, but no changes in levels of GluA2, its scaffolding protein S-SCAM/MAGI-2, or NMDAR subunits GluN1 or GluN2. Tubulin was used as a loading control. Numbers (on left) indicate approximate positions of molecular mass markers (kDa).

Article Snippet: table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host, isotype Immunogen Source Cat. # Clone # N-cadherin Ms, IgG 1 aa 802-819 from Ms BD Transduction Labs 610921 32/Ncadherin β-catenin Rb, IgG fusion protein, full-length Hu Millipore AB19022 n/a GluA1 Rb, IgG extracellular domain, rat Millipore ABN241 n/a GluA2 Ms, IgG 2a aa 175-430 from rat BD Pharmingen 556341 6C4 GluN1 Rb, IgG C-terminal peptide from Hu Millipore AB9864R 1.17.2.6 GluN2B Rb, IgG aa 1437-1456 from Ms Millipore 06-600 n/a PSD-95 Ms, IgG 1 purified rat PSD-95 Thermo Scientific MA1-046 7E3-1B8 pan-PSD-95 Ms, IgG 2a purified rat PSD-95 Thermo Scientific MA1-045 6G6-1C9 S-SCAM Rb, IgG aa 554-571 Sigma-Aldrich SAB4503718 n/a SynCAM-1 Chk, IgY recombinant Fc-fusion MBL International CM004-3 3E1 gephyrin Rb, IgG synthetic peptide from rat Millipore AB5725 n/a GAD65 Rb, IgG synthetic peptide from Hu Millipore ABN101 n/a VGAT Ms, IgG 3 aa 75-87 from rat Synaptic Systems 131 011 117G4 Synaptophysin Ms, IgG 1 rat retina synaptosome Sigma-Aldrich S5768 SVP-38 vGlut 1 GP, IgG peptide to vGlut1 Millipore AB5905 n/a vGlut2 GP, IgG peptide to C-terminus Millipore AB2251 n/a tubulin Rb, IgG aa 426-450 from Hu Abcam ab125267 n/a actin Ms, IgG 1 aa 50-70 from Chk Millipore MAB1501 C4 GAPDH Rb, IgG synthetic peptide from Hu Trevigen 2275-PC-1 n/a Open in a separate window Chk, chicken; GP, guinea-pig; Hu, human; Ms, mouse; Rb, rabbit; aa, amino acid Primary Antibodies lists the antibodies or sera used in this study.

Techniques: Western Blot, Isolation, Scaffolding

Journal: Hippocampus

Article Title: N-cadherin regulates molecular organization of excitatory and inhibitory synaptic circuits in adult hippocampus in vivo

doi: 10.1002/hipo.22282

Figure Lengend Snippet: Primary Antibodies

Article Snippet: table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host, isotype Immunogen Source Cat. # Clone # N-cadherin Ms, IgG 1 aa 802-819 from Ms BD Transduction Labs 610921 32/Ncadherin β-catenin Rb, IgG fusion protein, full-length Hu Millipore AB19022 n/a GluA1 Rb, IgG extracellular domain, rat Millipore ABN241 n/a GluA2 Ms, IgG 2a aa 175-430 from rat BD Pharmingen 556341 6C4 GluN1 Rb, IgG C-terminal peptide from Hu Millipore AB9864R 1.17.2.6 GluN2B Rb, IgG aa 1437-1456 from Ms Millipore 06-600 n/a PSD-95 Ms, IgG 1 purified rat PSD-95 Thermo Scientific MA1-046 7E3-1B8 pan-PSD-95 Ms, IgG 2a purified rat PSD-95 Thermo Scientific MA1-045 6G6-1C9 S-SCAM Rb, IgG aa 554-571 Sigma-Aldrich SAB4503718 n/a SynCAM-1 Chk, IgY recombinant Fc-fusion MBL International CM004-3 3E1 gephyrin Rb, IgG synthetic peptide from rat Millipore AB5725 n/a GAD65 Rb, IgG synthetic peptide from Hu Millipore ABN101 n/a VGAT Ms, IgG 3 aa 75-87 from rat Synaptic Systems 131 011 117G4 Synaptophysin Ms, IgG 1 rat retina synaptosome Sigma-Aldrich S5768 SVP-38 vGlut 1 GP, IgG peptide to vGlut1 Millipore AB5905 n/a vGlut2 GP, IgG peptide to C-terminus Millipore AB2251 n/a tubulin Rb, IgG aa 426-450 from Hu Abcam ab125267 n/a actin Ms, IgG 1 aa 50-70 from Chk Millipore MAB1501 C4 GAPDH Rb, IgG synthetic peptide from Hu Trevigen 2275-PC-1 n/a Open in a separate window Chk, chicken; GP, guinea-pig; Hu, human; Ms, mouse; Rb, rabbit; aa, amino acid Primary Antibodies lists the antibodies or sera used in this study.

Techniques: Transduction, Purification, Recombinant

Journal: Hippocampus

Article Title: N-cadherin regulates molecular organization of excitatory and inhibitory synaptic circuits in adult hippocampus in vivo

doi: 10.1002/hipo.22282

Figure Lengend Snippet: (A,D,G,J) Representative confocal microscope images of sections through stratum radiatum of CA1 of adult floxed control mice (top row) or cKO mice (bottom row) immunofluorescently labeled for AMPAR subunits GluA1 (A), GluA2 (D), NMDAR subunit GluN1 (G), or the scaffolding protein PSD95 (J). Bars= 5 μm.

Article Snippet: table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Host, isotype Immunogen Source Cat. # Clone # N-cadherin Ms, IgG 1 aa 802-819 from Ms BD Transduction Labs 610921 32/Ncadherin β-catenin Rb, IgG fusion protein, full-length Hu Millipore AB19022 n/a GluA1 Rb, IgG extracellular domain, rat Millipore ABN241 n/a GluA2 Ms, IgG 2a aa 175-430 from rat BD Pharmingen 556341 6C4 GluN1 Rb, IgG C-terminal peptide from Hu Millipore AB9864R 1.17.2.6 GluN2B Rb, IgG aa 1437-1456 from Ms Millipore 06-600 n/a PSD-95 Ms, IgG 1 purified rat PSD-95 Thermo Scientific MA1-046 7E3-1B8 pan-PSD-95 Ms, IgG 2a purified rat PSD-95 Thermo Scientific MA1-045 6G6-1C9 S-SCAM Rb, IgG aa 554-571 Sigma-Aldrich SAB4503718 n/a SynCAM-1 Chk, IgY recombinant Fc-fusion MBL International CM004-3 3E1 gephyrin Rb, IgG synthetic peptide from rat Millipore AB5725 n/a GAD65 Rb, IgG synthetic peptide from Hu Millipore ABN101 n/a VGAT Ms, IgG 3 aa 75-87 from rat Synaptic Systems 131 011 117G4 Synaptophysin Ms, IgG 1 rat retina synaptosome Sigma-Aldrich S5768 SVP-38 vGlut 1 GP, IgG peptide to vGlut1 Millipore AB5905 n/a vGlut2 GP, IgG peptide to C-terminus Millipore AB2251 n/a tubulin Rb, IgG aa 426-450 from Hu Abcam ab125267 n/a actin Ms, IgG 1 aa 50-70 from Chk Millipore MAB1501 C4 GAPDH Rb, IgG synthetic peptide from Hu Trevigen 2275-PC-1 n/a Open in a separate window Chk, chicken; GP, guinea-pig; Hu, human; Ms, mouse; Rb, rabbit; aa, amino acid Primary Antibodies lists the antibodies or sera used in this study.

Techniques: Microscopy, Labeling, Scaffolding