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: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. b Western blots show Plexin-B2 expression in SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. c IF images show Plexin-B2 expression in SD2 GSCs, with Hoechst nuclear counterstain. d Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of SD2; right, box plots of cell stiffness, showing 25–75% quartiles, median (line), and mean (plus sign). n = 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. e Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. f Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively (figure modified after ref. ). Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in Ctrl and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. g Live-cell imaging of SD3 GSCs labeled with SPY-actin show differences of cortical F-actin (arrows) between Ctrl and PB2 KO GSCs. NucSpot for nuclear staining. Right, box plots of SPY-actin cortical intensity, with 25–75% quartiles, median (line), and mean (plus sign). For Ctrl, n = 37 cell cortical areas; for PB2 KO, n = 28 cell cortical areas. Two-sided unpaired t-test. h Model of Plexin-B2 regulation of cortical contractility and membrane tension. Source data are provided as a Source Data file.

Article Snippet: The mutations I436C & S993C (“Lock1”) and I436C & T1051C (“Lock2”) were introduced into a CRISPR-resistant PLXNB2 cDNA plasmid by site directed mutagenesis (Thermo).

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Modification, Fluorescence, Live Cell Imaging, Staining

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Dextran uptake assay of SD2 GSCs labeled with SPY-Actin. Enlarged images of boxed areas are shown below. Box plots show areas of dextran + puncta per cell, with 25–75% quartiles, median (line), and mean (plus sign). n = 85 cells for Ctrl, n = 44 cells for PB2 KO. Mann–Whitney–Wilcoxon test, two-sided. b Top, live cell confocal plane images of SD2 GSCs with side views of z-stacks show intracellular localization of diffuse dextran-Alexa 488 in PB2 KO cells, whereas Ctrl cells contained only dextran + endocytic vesicles. Bottom, histograms of fluorescence profiles show bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n = 177 cells for Ctrl, n = 161 cells for PB2 KO. Mann–Whitney–Wilcoxon test, two-sided. c , d Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell images at 72 hr after transfection show fluorescent probes on endomembranes (arrow) in control GSCs, but membrane retention of the probes (arrowhead) in PB2 KO GSCs. For myr-palm-GFP: n = 21 cell membranes for WT; n = 13 cell membranes for PB2 KO. For myr-palm-CFP: n = 6 cell membranes for WT; n = 5 cell membranes for PB2 KO. Two-sided unpaired t-test. Bar graphs show means ± SEM. e Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow and NucSpot. Middle, TauSTED live-cell images show reduced endocytic vesicles (arrowheads) in PB2 KO cells compared to Ctrl. Right, box plots show areas of MemGlow + clusters per cell. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 26 cells for Ctrl, n = 13 cells for PB2 KO. Two-sided unpaired t-test. f Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis in GSCs. Source data are provided as a Source Data file.

Article Snippet: The mutations I436C & S993C (“Lock1”) and I436C & T1051C (“Lock2”) were introduced into a CRISPR-resistant PLXNB2 cDNA plasmid by site directed mutagenesis (Thermo).

Techniques: Labeling, MANN-WHITNEY, Fluorescence, Membrane, Transfection, Control, Super-Resolution Microscopy

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Illustration of voltage sensitive membrane dye FluoVolt, with fluorescence quenched by electron transfer from electron-rich donor (high inner membrane surface charge), mediated by “molecular wire” in plasma membrane (figure modified after ref. ). b Left, live-cell images show reduced FluoVolt fluorescence in plasma membrane of PB2 KO cells (higher negative charges of inner membrane). Right, box plots of membrane FluoVolt intensity. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 25 cells for Ctrl, n = 27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. c Top, still images from live-cell imaging show higher FluoVolt fluorescence at rear zone (arrowhead) in Ctrl GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Calcium chelator BAPTA-AM disrupted polarized FluoVolt pattern. White arrow denotes migration direction. Bottom, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n = 15 cells per group for Ctrl. vs KO, one-way ANOVA followed by Tukey’s multiple comparison test. n = 21 cells for vehicle and n = 16 cells for BAPTA-AM, two-sided unpaired t-test. Bar graphs represent mean ± SEM. d Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in Ctrl or PB2 KO GSCs. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. e Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, and asymmetry of FluoVolt and R( +8)-pre-GFP. Source data are provided as a Source Data file.

Article Snippet: The mutations I436C & S993C (“Lock1”) and I436C & T1051C (“Lock2”) were introduced into a CRISPR-resistant PLXNB2 cDNA plasmid by site directed mutagenesis (Thermo).

Techniques: Membrane, Fluorescence, Modification, Live Cell Imaging, Migration, Comparison, Dominant Negative Mutation

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. b Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO for both SD2 and SD3. β-actin serves as a loading control. c Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels by wildtype PB2 rescue construct, but not PB2 lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. d Box plots show velocity through constrictions (Rescue, n = 20 cells; dECTO, n = 20 cells; Lock1, n = 21 cells; Lock2, n = 17 cells), stalling time at constrictions (Rescue, n = 20 cells; dECTO, n = 14 cells; Lock1, n = 28 cells; Lock2, n = 22 cells), and sum of forward and backward movements constrictions (Rescue, n = 20 cells; dECTO, n = 20 cells; Lock1, n = 21 cells; Lock2, n = 17 cells), with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). One-way ANOVA followed by Dunnett’s multiple comparisons test. e Still images from videography show wildtype but not PB2 mutants restored localization of SPY-actin (arrowhead) at cell rear and MemGlow + endocytic vesicles (arrow) at cell front in GSC traversing 3 µm constrictions (chevrons). f Bar graphs show fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. For SPY-actin: n = 11 cells for Recue; n = 15 cells for dECTO; n = 10 cells for Lock1; n = 10 cells for Lock2. For MemGlow: n = 15 cells for Recue; n = 18 cells for dECTO; n = 10 cells for Lock1; n = 10 cells for Lock2. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. g Model of Plexin-B2 signaling in regulating mechano-electrical coupling of membrane tension and membrane surface charge during polarized confined migration. Note regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone and asymmetry of FluoVolt and R( + 8)-pre-GFP membrane probes. Source data are provided as a Source Data file.

Article Snippet: The mutations I436C & S993C (“Lock1”) and I436C & T1051C (“Lock2”) were introduced into a CRISPR-resistant PLXNB2 cDNA plasmid by site directed mutagenesis (Thermo).

Techniques: Western Blot, Expressing, Control, Construct, Fluorescence, Migration, Membrane

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: Cell reports

Article Title: Structural and Functional Studies of the RBPJ-SHARP Complex Reveal a Conserved Corepressor Binding Site

doi: 10.1016/j.celrep.2018.12.097

Figure Lengend Snippet: (A) The wild-type SHARP RBPID (RBPID WT ), but not the RBPJ-interacting defective SHARP RBPID (RBPID LI/AA ) mutant, causes upregulation of Notch target genes in mouse mature T (MT) cells by outcompeting endogenous SHARP for RBPJ binding. MT cells were infected with plasmids encoding GFP-tagged SHARP (2,776–2,833), either wild-type (GFP-SHARP/RBPID WT , black bars) or the RBPJ-interacting defective mutant L2791A/I2811A (GFP-SHARP/RBPID LI/AA , gray bars) or an empty vector control (Control, white bars). Left: total RNA from MT cells was analyzed by qPCR using primers specific for Tbp , Hes1 , or Hey1 . Data shown represent the mean ± SD of triplicate experiments (**p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: nuclear extracts (NEs) were prepared from MT cells and analyzed by western blotting, with TBP used as a loading control. (B–D) GFP-SHARP/RBPID WT derepresses Notch target genes via histone deacetylation. MT cells were infected with plasmids encoding GFP-SHARP/RBPID WT , GFP-SHARP/RBPID LI/AA , or an empty vector control and analyzed by chromatin immunoprecipitation (ChIP) using antibodies against H3K9ac (B), H3K27ac (C), or H3 (D). The enrichment was analyzed by qPCR on the enhancers of Hes1 and Hey1 , located at approximately +0.6 kb and −0.8 kb relative to the transcription start site, respectively. Gene desert was used as a negative control. Data were normalized to the positive control ( Gapdh 0kb ), and, in the case of the H3K9ac and H3K27ac ChIP, data were further normalized to histone occupancy (H3). Shown is the mean ± SD of two experiments measured twice each (NS, not significant; *p < 0.05; ***p < 0.001; unpaired Student’s t test). See also . (E) RBPJ is required to repress the Notch target genes Hes1 and Hey1 in MT cells, as revealed by CRISPR/Cas9 depletion of RBPJ. Left: total RNA from wild-type (control) or RBPJ-depleted (clones sgRbpj 2–12 and sgRbpj 2–14 ) MT cells was analyzed by qPCR. Shown is the mean ± SD of triplicate experiments (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: whole-cell extracts (WCEs) were prepared from MT cells and analyzed by western blotting using an anti-RBPJ antibody. GAPDH was used as loading control. (F) Hes1 and Hey1 Notch target genes are upregulated upon shRNA-mediated Rbpj knockdown but not with the SCR control shRNA. Left: total RNA from MT cells infected with shRNAs targeting Rbpj ( Rbpj sh1 or Rbpj sh4 ) or scrambled shRNA control (SCR) was analyzed by qPCR using primers specific for Tbp , Hes1 , or Hey1 . Shown is the mean ± SD of quadruplicate experiments (**p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: WCE was prepared from MT cells and analyzed by western blotting using an RBPJ antibody. GAPDH was used as a loading control. (G) Expression of RBPJ WT but not RBPJ FL/AA in the RBPJ-depleted background rescues the repression of Notch target genes. Left: total RNA from sgRbpj 2–12 MT cells infected with empty vector (control), RBPJ WT , or RBPJ FL/AA was analyzed by qPCR using primers specific for Tbp , Hes1 , or Hey1 . Shown is the mean± SD of three independent experiments measured twice each (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t test). Right: NEs were prepared from sgRbpj 2–12 MT cells infected with empty vector (control), RBPJ WT , or RBPJ FL/AA and analyzed by western blotting using anti-RBPJ antibodies. Histone H3 was used as a loading control.

Article Snippet: An engineered CRISPR/Cas9 resistant mouse RBPJ cDNA was synthetized at GENEART/Life Technologies and inserted into the pcDNA3.1 Flag2 via NotI digestion.

Techniques: Mutagenesis, Binding Assay, Infection, Plasmid Preparation, Western Blot, Chromatin Immunoprecipitation, Negative Control, Positive Control, CRISPR, Clone Assay, shRNA, Expressing

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