Journal: Journal of Neuroscience

Article Title: Astrocytes Control Glutamate Receptor Levels at Developing Synapses through SPARC- -Integrin Interactions

doi: 10.1523/jneurosci.4757-10.2011

Figure Lengend Snippet: Figure 1. SPARC is expressed by hippocampal astrocytes during development and is regulated by neural activity. A, Immuno- labeling for SPARC and either GS, IBA-1, or NeuN (P14 hippocampus) demonstrates that SPARC is expressed in astrocytes, some microglia but not neurons. SP, Stratum pyramidale; SR, stratum radiatum. B, Developmental time course for the expression of SPARCinthehippocampusrevealedbyimmunoblot.SPARClevelspeakduringthefirstcoupleofpostnatalweeks(ANOVAwithpost hoc Holm–Sidak test vs adult time point, *p 0.05). C, Treatment of organotypic hippocampal slices with the GABAA receptor antagonistbicuculline(20M,24h)toelevateactivityresultsinsignificantlyincreasedSPARCexpression[3.58-foldincreasewith bicucullineascomparedtocontrol;2-tailedttest,*p 0.0054,(n 4)].GFAPisshownasacontrolforrelativeastrocytenumber. D, Increased SPARC protein can be detected in media taken from bicuculline-treated (20 M, 24 h) neuron–astrocyte feeder cultures (see Fig. 4A), but not in bicuculline-treated astrocyte cultures. Immunoblot analysis of SC1/Hevin and GAPDH was per- formed as a control for protein level. E, F, Treatment of dissociated astrocytes with glutamate (20 M, 6 h) or DHPG (10 M, 6 h) leads to a significant increase in SPARC expression [2-tailed t test, *p 0.0057 for glutamate, *p 0.0297 for DHPG (n 3)]. Error bars indicate SEM. Scale bars: A, 5 m; C, 20 m.

Article Snippet: Recombinant mouse SPARC protein was purchased from R and D Systems.

Techniques: Activity Assay, Immunolabeling, Expressing, Western Blot, Control

Journal: Journal of Neuroscience

Article Title: Astrocytes Control Glutamate Receptor Levels at Developing Synapses through SPARC- -Integrin Interactions

doi: 10.1523/jneurosci.4757-10.2011

Figure Lengend Snippet: Figure2. SPARCKOmiceshowenhancedsynapticstrengthandalterationsinNMDAR/AMPARratios.A,NeuNandMAP2immunolabeling(P27hippocampus)indicatingthathippocampalarchitectureand dendriticstructureissimilarinWTandKOmice.B,RepresentativeimagesofCA1apicaldendritesexpressingmembrane-targetedmCherryinWTandKOorganotypicslices.C,Quantificationofspinedensity betweenWTandSPARCKOneurons(Mann–WhitneyU,p 0.5362)D,SampletracesofmEPSCrecordingsfromhippocampalpyramidalneuronsofWT(top)andSPARCKO(bottom)organotypichippocampal slices.E,GraphofaveragemEPSCamplitudesshowingsignificantincreasesinamplitudesinSPARCKOneurons(2-tailedttest,*p 0.0073).F,CumulativedistributionofmEPSCamplitudesfromcellsshown in the histogram (K–S test, p 0.001). G, Graph of average mEPSC frequencies showing a significant increase in SPARC KO neurons (2-tailed t test, *p 0.0375). H, Representative current traces of AMPAR-mediatedinwardcurrents(60mV)andmixedNMDARAMPAR-mediatedoutwardcurrents(40mV)recordedfromhippocampalneuronsinacuteWTandSPARCKOslices.I,SPARCKOmiceshow asignificantreductionintheratioofNMDAR/AMPARcurrents(2-tailedttest,*p 0.0154).Scalebars:A,toppanels,100m;bottompanels,20m;B,3m.

Article Snippet: Recombinant mouse SPARC protein was purchased from R and D Systems.

Techniques:

Journal: Journal of Neuroscience

Article Title: Astrocytes Control Glutamate Receptor Levels at Developing Synapses through SPARC- -Integrin Interactions

doi: 10.1523/jneurosci.4757-10.2011

Figure Lengend Snippet: Figure 3. SPARC KO mice show defects in long-term plasticity. A, fEPSPs were recorded from the stratum radiatum of CA1 in acutehippocampalslicesfromWTandKOmice.RepresentativeaveragedtracesoffEPSPsrecordedbefore(1)and55minafter(2) high-frequency stimulation (bottom left). Note that significant LTP, as shown in the graph (bottom right), was only expressed in slices from WT mice at 55–60 min after high-frequency stimulation (2-tailed t test, *p 0.0127). B, LTD was induced by low-frequencystimulation(LFS).RepresentativeaveragedtracesoffEPSPrecordedbefore(1)andat45minafter(2)LFS(bottom left).ThegraphshowsthepercentagedepressionoffEPSPsat45–50minafterLFS.Nodifferencesbetweengenotypeswerefound (2-tailed t test, p 0.7564).

Article Snippet: Recombinant mouse SPARC protein was purchased from R and D Systems.

Techniques:

Journal: Journal of Neuroscience

Article Title: Astrocytes Control Glutamate Receptor Levels at Developing Synapses through SPARC- -Integrin Interactions

doi: 10.1523/jneurosci.4757-10.2011

Figure Lengend Snippet: Figure4. NeuronsgrownwithSPARCKOastrocytesdevelopnormallybuthaveincreasedGluR1andGluR2surfaceexpression.A,DiagramillustratingtheculturingofWTneuronsaboveafeeder layerofWTorKOastrocytes.B,Immunostainingshowingthecolocalizationofpresynapticsynapsin(magenta)andpostsynapticPSD-95(green)torevealsynapticpunctainWTandSPARC-deficient cultures.C,D,Quantificationofsynapticpunctumdensityandsize.TherewerenodetectabledifferencesbetweenWTandKOcultures[2-tailedttest,p 0.0912fordensity,p 0.1563forsize(n 4)].E,F,RepresentativeimagesofsurfaceGluR1expressionshowingincreasedintensityofpunctainSPARC-deficientculturescomparedtoWTcultures.Theboxedregionismagnifiedbeloweach image.RecombinantSPARCapplication(SPARC;0.5g/ml,48h)rescuessurfaceGluR1levelsinSPARC-deficientcultures[ANOVAwithposthocHolm–Sidaktest,*p 0.00025(n 3)].G,H, Representative images of surface GluR2 expression showing increased intensity of puncta in SPARC-deficient cultures compared to WT cultures. The boxed region is magnified below each image. Application of recombinant SPARC (0.5 g/ml, 48 h) restores surface GluR2 levels to WT levels [ANOVA with post hoc Holm–Sidak test, *p 0.00246 (n 4)]. I, J, Representative immunoblots showingcellsurface(leftpanel)andtotalGluR1,GluR2,andNR1(rightpanel)levelsinWT,SPARC-deficient,andSPARC-deficientculturestreatedwithSPARC(0.5g/ml,48h).Cellsurfacereceptor levelsweredeterminedbycellsurfacebiotinylation.NeuronsculturedwithSPARCKOastrocyteshadsignificantlyhighersurfaceGluR1[ANOVAwithposthocHolm–Sidaktest(n 3),*p 0.003] andGluR2(*p 0.008)butnotNR1( p 0.472)levelswithoutanincreaseintotalreceptorlevels.AbnormallevelsofGluR1andGluR2intheSPARCKOconditioncouldbecompletelyrescuedwith recombinant SPARC. Scale bars: B, 40 m; E, G, 20 m.

Article Snippet: Recombinant mouse SPARC protein was purchased from R and D Systems.

Techniques: Expressing, Recombinant, Western Blot

Journal: Journal of Neuroscience

Article Title: Astrocytes Control Glutamate Receptor Levels at Developing Synapses through SPARC- -Integrin Interactions

doi: 10.1523/jneurosci.4757-10.2011

Figure Lengend Snippet: Figure5. ApplicationofSPARCproteinorpriorsynapticdepressiontotheKOconditionrestoressynapsestoapermissiblerangeforsynapticplasticity.A,B,SynapticstrengthinSPARC-deficient culturesisnotincreasedfurtherbychronicactivityblockade.WTculturesdisplaysignificantincreasesinsurfaceGluR1levelsinresponsetoTTXtreatment(1M,48h).Incontrast,SPARC-deficient culturesdonotexhibitanychangesinsurfaceGluR1levels[ANOVAwithposthocHolm–Sidaktest,*p 0.0055,forKOculturesp 0.865(n 5)].C,RescueofKOcultureswithrecombinantSPARC allowsTTX-inducedincreasesinsynapticstrengthtooccur[ANOVAwithposthocHolms–SidaktestversusSPARCtreatment,*p0.05(n 4)].SPARCprotein(0.5g/ml)wasappliedfor24hand thenincubatedwithTTX(1M)forasubsequent48h.D,SynapticstrengthinSPARC-deficientculturesisdecreasedbybicucullineapplication[2-tailedttest,*p 0.00283(n 5)](20M,48h) Error bars indicate SEM. Scale bars, 20 m. E, F, Synapses in acute slices from SPARC KO mice can show potentiation when given a prior LTD-inducing stimulus. The graph shows the average percentagechangeinfEPSPsat55–60minafterhigh-frequencystimulation(HFS)(2)comparedto25–30minafterLFS(1)inbothWT[2-tailedttest,p 0.0388(n 6)]andKOslices[2-tailed t test, *p 0.042 (n 5)]. Post-HFS potentiation was not significantly different between WT and KO slices (2-tailed t test, p 0.29). Scale bars, 20 m.

Article Snippet: Recombinant mouse SPARC protein was purchased from R and D Systems.

Techniques:

Journal: Journal of Neuroscience

Article Title: Astrocytes Control Glutamate Receptor Levels at Developing Synapses through SPARC- -Integrin Interactions

doi: 10.1523/jneurosci.4757-10.2011

Figure Lengend Snippet: Figure 6. SPARC regulates 3-integrin complexes to control surface AMPARs. A, Representative images of surface 3-integrin expression showing increased intensity of surface 3-integrin punctainSPARC-deficientculturescomparedtoWTcultures.RecombinantSPARCapplication(0.5g/ml,48h)restoressurface3-integrinlevelsinSPARC-deficientcultures.Theboxedregionis magnifiedbeloweachimage.B,Quantificationofsurface3-integrinexpression[ANOVAwithposthocHolm–Sidaktest,*p 0.000158(n 4)].C,Surface1-integrinlevelsaresimilarinWT andSPARC-deficientcultures[27.405arbitraryunitsforWTversus27.794arbitraryunitsforSPARC-deficientcultures;2-tailedttest,p 0.845(n 3)].D,HEK293Tcellsexpressing3-integrin havereducedattachmenttovitronectininthepresenceofSPARC[ANOVAwithposthocHolm–Sidaktest,*p 0.0036,(n 4)].E,DiagramillustratingthedomainstructureofSPARCandlocation ofPeptide2.3.Peptide2.3reducessurface3-integrin(F),GluR2(G),andGluR1(H)levelsinSPARC-deficientculturestoasimilarextentasthefull-lengthprotein[ANOVAwithposthocHolm–Sidak test,*p 0.0081for3-integrin(n 5),*p 0.0127forGluR2(n 5),and*p 0.00511forGluR1(n 4)vscontrol].Apeptidecontainingascrambledsequence(scPep2.3)hadnoeffect. I,CoapplicationofthedisintegrinEchistatin(300nM)preventsSPARCrescue(0.5g/ml,48h)ofsurfaceGluR1levelsinSPARC-deficientcultures(ANOVAwithposthocHolm–Sidaktest,*p 0.003, SPARC-treated vs SPARC-treated and Echistatin condition). J, The 3-integrin function-blocking antibody Clone 2C9.G2 (10 g/ml, 48 h) blocks SPARC-mediated rescue of surface GluR1 in SPARC-deficient cultures (ANOVA with post hoc Holm–Sidak test, *p 0.006). An isotype-matched control antibody had no effect. Error bars indicate SEM. Scale bars, 20 m.

Article Snippet: Recombinant mouse SPARC protein was purchased from R and D Systems.

Techniques: Control, Expressing, Blocking Assay

Journal: Critical Care Explorations

Article Title: Surfactant Protein D Influences Mortality During Abdominal Sepsis by Facilitating Escherichia coli Colonization in the Gut

doi: 10.1097/CCE.0000000000000699

Figure Lengend Snippet: Surfactant protein D (SPD) is synthesized by the gallbladder and promotes colonization of both the cecum and colon with Escherichia coli . A , SPD +/+ gut organs were harvested at baseline or post-cecal ligation and puncture (CLP) with Western blots performed for SPD or β -actin (loading control). Blots represent pooled samples, n = 2/group. Representative gel shown from three experiments. Lungs from SPD −/− and SPD +/+ mice were used as negative and positive controls, respectively. B , Gallbladder was isolated from SPD +/+ mice after CLP or sham surgery and from SPD −/− mice after CLP with Western blots performed for SPD or glyceraldehyde 3-phosphate dehydrogenase antibody (GAPDH; loading control). Blots represent pooled samples, n = 5–7/group. Lungs from SPD −/− mice and SPD +/+ were used as negative and positive controls, respectively. C , SPD −/− mice ( n = 9) were gavaged with recombinant surfactant protein D (rSPD), followed by gavage with green fluorescent protein (GFP)-labeled E. coli , and compared with SPD −/− mice gavaged only with GFP-labeled E. coli ( n = 9). After 24 hr, cecum and colon were harvested. GFP-labeled E. coli were then detected by culture (Mann-Whitney * p < 0.05, ** p < 0.01). CFU = colony forming units.

Article Snippet: SPD +/+ and SPD −/− mice were gavaged with 10 μL/g of ampicillin-resistant GFP-labeled E. coli (ATCC 25922GFP, 3.74 × 10 8 colony forming units/mL) with or without recombinant human surfactant protein D (rSPD) (Cat 1920-SP-050, R&D systems).

Techniques: Synthesized, Ligation, Western Blot, Control, Isolation, Recombinant, Labeling, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

doi: 10.1371/journal.pone.0050138

Figure Lengend Snippet: Cells were pretreated with various of MAP kinase inhibitors SB203580 (10 µM), PD98059 (20 µM) or SP600125 (10 µM) for 30 min followed by stimulation with desipramine (20 µM; A) or fluoxetine (20 µM; D) for another 24 h. Whole cell lysates were subjected to Western blot for detection of HO-1 expression. Cells were incubated with desipramine or fluoxetine for the indicated time periods, and cell lysates were subjected to immunoblots with antibodies against phospho-JNK (B and C) and phospho-ERK (D and E). Results are the representatives of three or four independent experiments.

Article Snippet: SP600125 was obtained from Tocris Bioscience (Ellisville, MO).

Techniques: Western Blot, Expressing, Incubation

Journal: PLoS ONE

Article Title: Desipramine Protects Neuronal Cell Death and Induces Heme Oxygenase-1 Expression in Mes23.5 Dopaminergic Neurons

doi: 10.1371/journal.pone.0050138

Figure Lengend Snippet: (A) Cells were treated with desipramine (20 µM) for indicated time periods (60 or 120 min) and nuclear extracts were collected, and the binding activity of Nrf2 to Nrf2-DNA binding element was examined by EMSA analysis. The DNA binding activity of Nrf2 is significantly different between desipramine treatment group and control group (one-way ANOVA followed by Bonferroni’s post hoc test). Cells were pretreated with PD98059 or SP600125 with desipramine (20 µM), and nuclear extracts were examined by EMSA analysis. Lane 1 was loaded without nuclear extracts (probe only). Results are expressed as the means ± S.E.M. from three independent experiments. *, p <0.05 as compared with the vehicle control group. #, p<0.05 as compared with the desipramine treatment group. (B) Cells were transfected with Control siRNA (100 nM) or Nrf2 siRNA (50 and 100 nM) for 24 h followed by stimulation with desipramine (20 µM) for another 24 h, and the protein levels of Nrf2 and HO-1 were determined by Western blot. The HO-1 expression is significantly different between Nrf2 siRNA group and control siRNA group (one-way ANOVA followed by Bonferroni’s post hoc test). Results are expressed as the means ± S.E.M. from three independent experiments. *, p <0.05 as compared with the control group. #, p<0.05 as compared with the desipramine treatment alone group.

Article Snippet: SP600125 was obtained from Tocris Bioscience (Ellisville, MO).

Techniques: Binding Assay, Activity Assay, Control, Transfection, Western Blot, Expressing

Journal: Biomedicines

Article Title: Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism

doi: 10.3390/biomedicines11112908

Figure Lengend Snippet: Effect of recombinant human SPARC on glucose and fatty acid metabolism in myotubes. Human myoblasts were differentiated to myotubes for 7 days and incubated the last 2 days with or without recombinant human SPARC (8 µg/mL). Oxidation (trapped CO 2 ) and cell-associated radioactivity (CA) of [ 14 C]glucose or [ 14 C]oleic acid was measured after incubation for 4 h and normalized to Ctr (3 independent experiments, with 4 biological replicates from each experiment). ( A ) Cellular uptake of glucose (CO 2 + CA). ( B ) Complete oxidation of glucose. ( C ) Cellular uptake of oleic acid (CA + CO 2 ). ( D ) Complete oxidation of oleic acid. Data are shown as mean ± SEM. * p < 0.05 vs. Ctr, unpaired t -test. Absolute values for Ctr: ( A ) 42.1 ± 4.5 nmol/mg protein, ( B ) 24.7 ± 3.3 nmol/mg protein, ( C ) 105.6 ± 2.8 nmol/mg protein, ( D ) 18.0 ± 1.8 nmol/mg protein.

Article Snippet: Recombinant human SPARC was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Recombinant, Incubation, Radioactivity