Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

doi: 10.1073/pnas.1701872114

Figure Lengend Snippet: Deletion of the NR0B1 microsatellite reduces NR0B1 expression, impairs A673 cell growth, and inhibits colony formation. (A) Sequencing results validating knockout of the NR0B1 GGAA-microsatellite about 1.5 kb upstream of the NR0B1 TSS in A673 cells. The sgRNAs targeted to either side of this region are underlined. GGAA-microsatellite is highlighted red, and CRISPR/Cas9 deleted region is highlighted blue. Gel shows deletion of NR0B1 microsatellite region compared with control (nondeleted), with densitometry quantification on Right (P < 0.01). Data are represented as mean ± SEM (n = 2). (B) NR0B1 mRNA (P < 0.05) and protein expression levels in control and CRISPR/Cas9-mediated knockout of NR0B1 microsatellite in A673 Ewing sarcoma cells, with Western blot densitometry quantification on Right. Control CRISPR/Cas9 plasmids do not contain sgRNAs. Data are represented as mean ± SEM (n = 3). (C) Growth and colony formation assay quantification of CRISPR/Cas9 control vs. NR0B1 microsatellite knockout in A673 cells (P < 0.05). Data are represented as mean ± SEM (n = 3).

Article Snippet: Mammalian expression constructs included the following: Lentiviral vectors containing CRISPR/Cas9 cDNA and sgRNA ( SI Materials and Methods ); retroviral vectors encoding Luc-RNAi and EF-2–RNAi and cDNAs for EWS/FLI, Δ22, R2L2, Mut9, and NR0B1 are previously described ( 4 , 13 , 22 , 23 ); the Mut9/R2L2 construct was ordered as a gene block (IDT) and cloned into the pMSCV hygro vector between EcoRI and HindIII restriction sites.

Techniques: Expressing, Sequencing, Knock-Out, CRISPR, Western Blot, Colony Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

doi: 10.1073/pnas.1701872114

Figure Lengend Snippet: Deletion of the NR0B1 microsatellite in other cell lines. (A) NR0B1 mRNA and protein expression levels in control and CRISPR/Cas9-mediated knockout of the NR0B1 microsatellite in TC-71 and EWS/502 Ewing sarcoma cells (P < 0.05). Data are represented as mean ± SEM (n = 3). (B) Growth curves and soft agar assay quantification for NR0B1 microsatellite deletion in two other Ewing sarcoma cell lines (TC-71 cells and EWS/502 cells). Growth curve data are represented as mean ± SEM (n = 4). Control vs. CRISPR for TC-71 and EWS/502 cells are each statistically significant (P < 0.05). Soft agar data are represented as mean ± SEM (n = 2). (C) Densitometry quantification of PCR-amplified NR0B1-microsatellite–containing region for A673 control (wild-type NR0B1-microsatellite) allele vs. CRISPR-Cas9 knockout (deleted NR0B1-microsatellite) allele at different time points for up to 3 wk postlentiviral infection. (D) NR0B1 mRNA and protein expression levels in control and CRISPR/Cas9-mediated knockout of the NR0B1 microsatellite in non-Ewing sarcoma HEK293 cells. Data are represented as mean ± SEM (n = 3). n.s., not statistically significant. (E) NR0B1 protein levels and colony formation assay quantification for A673 cells with NR0B1 cDNA rescue in CRISPR/Cas9 control vs. microsatellite knockout. n.s., not statistically significant. Data are represented as mean ± SEM (n = 2).

Article Snippet: Mammalian expression constructs included the following: Lentiviral vectors containing CRISPR/Cas9 cDNA and sgRNA ( SI Materials and Methods ); retroviral vectors encoding Luc-RNAi and EF-2–RNAi and cDNAs for EWS/FLI, Δ22, R2L2, Mut9, and NR0B1 are previously described ( 4 , 13 , 22 , 23 ); the Mut9/R2L2 construct was ordered as a gene block (IDT) and cloned into the pMSCV hygro vector between EcoRI and HindIII restriction sites.

Techniques: Expressing, CRISPR, Knock-Out, Soft Agar Assay, Amplification, Infection, Colony Assay

Journal: PLoS Pathogens

Article Title: Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

doi: 10.1371/journal.ppat.1003106

Figure Lengend Snippet: Mature ( A ), germline ( B ), chimera 1 ( C ), or chimera 2 ( D ), IgGs were immobilized to anti-human IgG FC capture BLI biosensors and Env proteins were in solution at 1.2 µM. The QH0692 and D368R Envs were in gp120 form. The remaining Envs were in gp140 form (either as trimers or as monomers).

Article Snippet: The constant IgG splice variant cDNA (containing the two exons which encode the transmembrane and cytoplamic domains of human IgG) was synthesized by Genscript (Piscataway, NJ).

Techniques:

Journal: PLoS Pathogens

Article Title: Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

doi: 10.1371/journal.ppat.1003106

Figure Lengend Snippet: ( A ) Cell-surface expression of b12 mature, germline and chimeric BCRs on the surface of B cells. ( B ) Intracellular Ca 2+ flux mediated by the mature, germline and chimeric BCRs following BCRs cross-linking by goat anti-human IgG (H+L) F(ab′) 2 in A20 cells. ( C ) Binding of mature, germline and chimeras to SF162 gp140 trimer. ( D ) Intracellular Ca 2+ flux upon addition of SF162 gp140 trimers to B cells (DG-75) expressing the indicated b12 BCRs. ( E ) Binding of mature, germline and chimeras to QH0692 gp140 trimer. ( F ) Intracellular Ca 2+ flux upon addition of QH0692 gp140 trimers to B cells expressing the indicated b12 BCRs.

Article Snippet: The constant IgG splice variant cDNA (containing the two exons which encode the transmembrane and cytoplamic domains of human IgG) was synthesized by Genscript (Piscataway, NJ).

Techniques: Expressing, Binding Assay

Journal: Brain

Article Title: Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A 2A receptor

doi: 10.1093/brain/awz288

Figure Lengend Snippet: Conditional (Tet-Off) transgenic mouse model of neuronal A 2A R overexpression. ( A ) Conditional overexpression of A 2A R in neurons is achieved by crossing of CaMKII-tTA mice, expressing the transactivator protein tTA, under the control of a neuronal forebrain promoter (CaMKII) with the TRE-A 2A R strain, in which murine A 2A R is under the control of a Tet-responsive element. A 2A R expression is elicited in CaMKII-expressing neurons by the binding of the tTA protein to the TRE promoter. Transgene expression is maintained off from mating until offspring weaning (P28) by doxycycline (0.2 mg/ml in drinking water) to avoid potential perinatal effects linked to early A 2A R overexpression. ( B ) Representative western blots of A 2A R in the hippocampus of double CaMKII-tTA/TRE-A 2A R (A 2A R mice) and littermate controls (WT, wild-type). In absence of doxycycline, at P28 (P28 w/o Dox, left ), double transgenic A 2A R animals exhibited receptor immunoreactivity while its level remained undetectable in the hippocampus of wild-type animals. Doxycycline treatment from mating to P28 (P28 w/ Dox, middle ) abolished A 2A overexpression. Doxycycline removal from P28 promoted hippocampal A 2A R overexpression in the latter animals as exemplified in 6 month-old animals i.e. 5 months after doxycycline removal ( right ). ( C ) A 2A R immunostaining by immunohistochemistry under the same experimental conditions showing expression of the receptor in animals treated ( middle ) or not with doxycycline ( left ) as well as receptor re-expression following doxycycline withdrawal ( right ). Upper panels represent immunostainings at the level of the striatum and lower panels at the level of the hippocampus and cortex. Scale bar = 1 mm. ( D ) Co-immunostainings with A 2A R (red) and either neuronal (NeuN), microglial (Iba1) or astrocytic (GFAP and S100β) markers (green) showing the neuronal-specificity of A 2A R overexpression in CaMKII-tTA/TRE-A 2A R mice. DAPI (blue) represents cell nuclei. Scale bar = 20 µm. ( E ) Co-immunostainings between A 2A R (red), NeuN (as marker of mature neurons, white) and doublecortin (DCX, as marker of immature neurons, green) in CaMKII-tTA/TRE-A 2A R mice (A 2A R). A 2A R was not expressed in immature neurons. Scale bar = 100 µm. ( F ) Averaged time course of field excitatory postsynaptic potentials (fEPSP) after perfusion with SCH58261 (50 nM) for 30 min on hippocampal slices from wild-type and double CaMKII-tTA/TRE-A 2A R transgenic mice (* P < 0.05, n = 5 per group). A 2A R blockade significantly inhibited fEPSPs in double transgenic mice suggesting a gain of function of A 2A R upon their overexpression, whereby A 2A R exerts a tonic control on basal synaptic transmission, a phenomenon that is not observed in wild-type animals.

Article Snippet: The overexpression of mouse A 2A R in forebrain neurons was achieved by crossing the in-house developed TRE-A 2A R transgenic strain (in which mouse receptor cDNA is under the control of a Tet-responsive element) and the transgenic CaMKII-tTA line, expressing the tetracycline-controlled transactivator protein (tTA) under regulatory control of the forebrain-specific calcium-calmodulin-dependent kinase II (CaMKII) promoter [B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ; SN 7004; The Jackson Laboratory; A].

Techniques: Transgenic Assay, Over Expression, Expressing, Control, Binding Assay, Western Blot, Immunostaining, Immunohistochemistry, Marker, Transmission Assay

Journal: Brain

Article Title: Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A 2A receptor

doi: 10.1093/brain/awz288

Figure Lengend Snippet: Neuronal overexpression of A 2A R favours spatial memory deficits in THY-Tau22 transgenic mice. Effects of neuronal overexpression of A 2A R on spontaneous activity, anxiety-like behaviour, spatial learning and memory of THY-Tau22 mice. ( A and B ) No change of either spontaneous locomotion or velocity was observed using actimetry. ( C ) Anxiety-like behaviour evaluated using elevated plus maze. Double transgenic mice overexpressing A 2A R performed as wild-type controls. As expected, tau transgenic mice spent more time in the open arms, a change similarly observed in triple tau/A 2A R transgenic mice. *** P < 0.001 versus wild-type mice using one-way ANOVA followed by Tukey’s post hoc test . ( D ) Evaluation of the spatial learning using the Barnes maze task revealed that all groups of animals learned the position of the escape box in a time-dependent manner during the four days of training. ( E ) During the probe test, while displaying a preference, A 2A R mice spent significantly less time in the target quadrant (T) than wild-type controls. At the early age tested (5–6 months old), tau transgenic mice did not exhibit significant memory impairments with a strong preference for the target quadrant. In contrast, triple tau/A 2A R mice did not show preference for the target quadrant (T) over the other quadrants (O), supporting significant spatial memory deficits. $ P < 0.05 versus wild-type mice; ° P < 0.05, °° P < 0.01, °°° P < 0.001 versus Target quadrant using one-way ANOVA followed by Tukey’s post hoc test. ( F ) In agreement, the latency to reach the target hole was significantly increased for tau/A 2A R mice as compared to the other experimental groups. °° P < 0.01 versus wild-type mice using one-way ANOVA followed by Tukey’s post hoc test. n = 7–22 per group. Results are expressed as mean ± SEM.

Article Snippet: The overexpression of mouse A 2A R in forebrain neurons was achieved by crossing the in-house developed TRE-A 2A R transgenic strain (in which mouse receptor cDNA is under the control of a Tet-responsive element) and the transgenic CaMKII-tTA line, expressing the tetracycline-controlled transactivator protein (tTA) under regulatory control of the forebrain-specific calcium-calmodulin-dependent kinase II (CaMKII) promoter [B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ; SN 7004; The Jackson Laboratory; A].

Techniques: Over Expression, Transgenic Assay, Activity Assay, Mouse Assay

Journal: Brain

Article Title: Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A 2A receptor

doi: 10.1093/brain/awz288

Figure Lengend Snippet: Impact of neuronal A 2A R overexpression on hippocampal tau pathology. Human tau expression, phosphorylation and aggregation in the hippocampus of triple transgenic mice (tau/A 2A R) versus tau transgenic controls were evaluated by immunohistochemistry, bidimensional electrophoresis (2D) and western blots. ( A ) Co-immunostainings with A 2A R (red) and human tau (TauE1E2 antibody, human total tau, green) in the CA1 and dentate gyrus (DG) regions of triple tau/A 2A R transgenic mice. Neurons expressing human tau transgene (arrows) were found to overexpress A 2A R. DAPI (blue) represents cell nuclei. Scale bar = 50 µm. ( B ) 2D profile of total human tau (Cter antibody) in triple tau/A 2A R mice and littermate tau controls, shows an increase of tau isovariants in the acidic range of PI (arrow). ( C ) Quantification of tau phosphorylation at T181, S199, S212/T214 (AT100), S262, S396 and S404 epitopes, as well as dephosphorylated tau (tau-1) in triple tau/A 2A R animals and littermates tau controls. Analysis revealed tau hyperphosphorylation in tau/A 2A R mice signed by increased pS396 and reduced tau-1 (dephosphorylated tau). # P < 0.05, ## P < 0.01 versus tau mice using Student’s t -test. n = 6–7 per group. ( D ) Conformational tau immunostaining using MC1 antibody in triple tau/A 2A R animals and littermates tau controls revealed no difference between groups. n = 5–11 per group. Scale bar = 500 µm. Results are expressed as mean ± SEM.

Article Snippet: The overexpression of mouse A 2A R in forebrain neurons was achieved by crossing the in-house developed TRE-A 2A R transgenic strain (in which mouse receptor cDNA is under the control of a Tet-responsive element) and the transgenic CaMKII-tTA line, expressing the tetracycline-controlled transactivator protein (tTA) under regulatory control of the forebrain-specific calcium-calmodulin-dependent kinase II (CaMKII) promoter [B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ; SN 7004; The Jackson Laboratory; A].

Techniques: Over Expression, Expressing, Phospho-proteomics, Transgenic Assay, Immunohistochemistry, Electrophoresis, Western Blot, Immunostaining

Journal: Brain

Article Title: Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A 2A receptor

doi: 10.1093/brain/awz288

Figure Lengend Snippet: Neuronal overexpression of A 2A R promotes the upregulation of a microglial transcriptomic signature in the hippocampus of tau transgenic mice. RNA sequencing analysis of the hippocampus of wild-type, double A 2A R, tau and triple tau/A 2A R animals at the age of 6 months ( n = 4 per genotype). ( A ) Volcano plot showing the 505 genes differentially regulated genes between tau/A 2A R mice and tau mice. Red dots represent significantly dysregulated genes with log2 fold change > 0.32 and adjusted P -value < 0.05. Sixty-four genes were found significantly upregulated ( right ) and 441 significantly downregulated ( left ). ( B ) Functional annotation of the 64 upregulated genes in the triple tau/A 2A R mice versus tau was performed with DAVID for GOTERM_Biological Process and showed a significant association with immune system processes, innate immune response and phagocytosis engulfment. ( C ) Known and predicted protein interaction (STRING) of the genes belonging to the significant GO term processes shown in C . ( D ) Heat map representing the cellular enrichment of each upregulated gene based on a transcriptome database of purified populations of neurons, astrocytes, oligodendrocyte precursor cells (OPC), newly formed oligodendrocytes (NFO), myelinating oligodendrocytes (MO), microglia and endothelial cells ( Zhang et al. , 2014 ). Relative cellular enrichment of each gene is given as the percentage of highest expression. Expression of 54 out of the 64 genes upregulated was knowledgeable in the database. Among these 54 genes, 33 were particularly enriched in microglial cells, contrasting with the lack of neuronal enrichment. ( E ) Cell number and cell morphology of Iba1-immunolabeled microglia (green) were analysed in confocal images using custom-written ImageJ plugins. A representative confocal image, the 3D reconstruction, visualization of spanned volume and cell skeleton derived from one representative cell in the confocal image are shown. ( F and G ) Quantification of microglia cell number and the morphological parameters ramification index, spanned volume and total tree length of cell skeleton revealed no difference between the mouse groups in the CA1 ( F ) or dentate gyrus ( G ) regions. n = 5–6 mice per genotype. Results are expressed as mean ± SEM. Scale bar = 20 μm.

Article Snippet: The overexpression of mouse A 2A R in forebrain neurons was achieved by crossing the in-house developed TRE-A 2A R transgenic strain (in which mouse receptor cDNA is under the control of a Tet-responsive element) and the transgenic CaMKII-tTA line, expressing the tetracycline-controlled transactivator protein (tTA) under regulatory control of the forebrain-specific calcium-calmodulin-dependent kinase II (CaMKII) promoter [B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ; SN 7004; The Jackson Laboratory; A].

Techniques: Over Expression, Transgenic Assay, RNA Sequencing, Functional Assay, Purification, Expressing, Immunolabeling, Derivative Assay

Journal: The FASEB Journal

Article Title: Activation of JAK/STAT3 restores NK‐cell function and improves immune defense after brain ischemia

doi: 10.1096/fj.201700962r

Figure Lengend Snippet: Figure 4. Genetic activation of STAT3 preserved NK-cell–derived IFN-g expression after MCAO. C57BL/6 mice were subjected to sham operation or MCAO surgery. A, B) Real-time PCR and flow cytometry plots showed STAT3 gene (A) and protein levels (B) in splenic NK cells 24 h after MCAO; n = 4 per group. C) The schematic graph of the experimental design. Splenic NK cells isolated from wild-type mice were treated with STAT3-CRISPR activation plasmid or control vector. After transfected for 48 h, 3 3 106 NK cells were transferred intraveniously into groups of Rag22/2gc2/2 mice before MCAO. D) Flow cytometry plots and bar graph showed STAT3 phosphorylation in NK cells 2 h after MCAO; n = 4/group. E, F) Flow cytometry plots (E) and bar graph (F) show the effect of STAT3 activation on functional marker expression (CD69, perforin, and IFN-g) of splenic NK cells at d 1 after MCAO. Plots represent the results from 3 independent experiments. Error bars represent mean 6 SEM. **P , 0.01 by 2-tailed, unpaired Student’s t test.

Article Snippet: CRISPR activation plasmid cDNA transfection STAT3 clustered regularly interspaced short palindromic repeats (CRISPR) activation plasmid (sc-423176-ACT; Santa Cruz Biotechnology, Dallas, TX, USA) was used to activate and overexpress STAT3 inNK cells.

Techniques: Activation Assay, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Cytometry, Isolation, CRISPR, Plasmid Preparation, Control, Transfection, Flow Cytometry, Phospho-proteomics, Functional Assay, Marker

Journal: The FASEB Journal

Article Title: Activation of JAK/STAT3 restores NK‐cell function and improves immune defense after brain ischemia

doi: 10.1096/fj.201700962r

Figure Lengend Snippet: Figure 5. STAT3 activation in NK cells improves survival and reduces lung bacterial burden in MCAO mice. Splenic NK cells isolated from C57BL/6 mice targeting STAT3 were established by introducing a STAT3 activation plasmid. STAT3-activated (STAT3-CRISPR) NK cells were then passively transferred via intravenious injection to Rag22/2gc2/2 transgenic mice, followed by MCAO surgery. A) Representative MRI images and summarized data showed stroke lesion volume after adoptive transfer with STAT3-CRISPR NK cells; n = 5 mice/group. B) The neurodeficit score was assessed by modified neurological severity score (mNSS) after STAT3-CRISPR NK-cell transfer and MCAO surgery. P . 0.05 by 2-way ANOVA (A, B). C) The survival of the mice was noted at 0 to 8 d after cell transfer and MCAO surgery; n = 15 mice/group. *P , 0.05 by 2-way ANOVA. D) Lung tissues from MCAO mice transferred with STAT3-CRISPR NK cells were collected for bacteriologic analysis at 3 d after MCAO surgery. Data summarized in D graphically illustrate the quantification of bacteria burden in lung after passive transfer with NK cells. Data are presented in colony-forming units (CFU) per organ lung tissue homogenate. **P , 0.01 by 2-tailed, unpaired Student’s t test. E) Hematoxylin and eosin–stained lung sections exhibited typical signs (thickening of alveolar walls and neutrophilic infiltrates) of bacterial burden in MCAO mice. The lung tissues from wild-type mice adoptively transferred with control vector transfected-NK or STAT3-CRISPR NK cells were collected at d 3 after MCAO for histologic examination. Scale bars, 50 mm. F) ELISA measurement of IFN-g protein levels in serum from the control or STAT3-CRISPR NK groups at d 3 after MCAO. **P , 0.01 by 2-tailed, unpaired Student’s t test. Mean 6 SEM.

Article Snippet: CRISPR activation plasmid cDNA transfection STAT3 clustered regularly interspaced short palindromic repeats (CRISPR) activation plasmid (sc-423176-ACT; Santa Cruz Biotechnology, Dallas, TX, USA) was used to activate and overexpress STAT3 inNK cells.

Techniques: Activation Assay, Isolation, Plasmid Preparation, CRISPR, Injection, Transgenic Assay, Adoptive Transfer Assay, Bacteria, Staining, Control, Transfection, Enzyme-linked Immunosorbent Assay