Journal: Materials Today Bio

Article Title: Brain microvascular endothelial cells derived from human induced pluripotent stem cells as in vitro model for assessing blood-brain barrier transferrin receptor-mediated transcytosis

doi: 10.1016/j.mtbio.2022.100232

Figure Lengend Snippet: Competitive binding assay between 10 ​μg/mL of FGA-cys-T7 and FGA-cys-Tfr-T12 peptide and various concentrations (0; 125; 1250 ​nM) of unlabeled Tf with hiPS-BMEC (n ​= ​3). (A) Schematic illustration of the method. The medium in the bottom compartment was collected at determined time to measure fluorescence intensity. (B) Effective permeability coefficient of 10 ​μg/mL FGA-cys-T7 peptide (full bars) and FGA-cys-Tfr-T12 peptide (hatched bars) when co-incubated with various concentrations of unlabeled Tf by hiPS-BMEC after 1 ​h incubation at 37 ​°C. Data are presented as means ​± ​S.D. Statistical analysis was performed using one-way ANOVA (∗ p ​≤ ​0.05, n. s. p ​> ​0.05). The biological replicates of hiPS-BMEC are issued from the same differentiation. (C) Colocalization of FGA-cys-T7 or FGA-cys-Tfr-T12 peptide and Alexa Fluor 647- transferrin (AF 647-Tf) with hiPS-BMEC. Scale bar ​= ​10 ​μm. The biological replicates of hiPS-BMEC are issued from the same differentiation. (D) Quantification of the colocalization percentage between FGA-peptides and AF 647-Tf using hiPS-BMEC calculated with IMARIS software. Data are presented as means ​± ​S.D. Statistical analysis was performed using the Student's t -test (∗ p ​≤ ​0.05). (E) Schematic illustration of the interaction between FGA-cys-T7 or FGA-cys-Tfr-T12 peptide and Tf for the binding to TfR with hiPS-BMEC.

Article Snippet: The colocalization ratio between lysotracker deep red and FGA-peptides or AF 488-Tf, as well as the colocalization ratio of AF 647-Tf and FGA-cys-T7 or FGA-cys-Tfr-T12 peptide was quantified by IMARIS software (Oxford Instruments, Version 9.2.1, Bitplane, Belfast, UK).

Techniques: Competitive Binding Assay, Fluorescence, Permeability, Incubation, Software, Binding Assay

Journal: Autophagy

Article Title: Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6 S10F mutant

doi: 10.1080/15548627.2017.1392420

Figure Lengend Snippet: Impaired autophagy activity is associated with reduced BECN1 protein levels in HSPB6S10F hearts. (A), Representative immunoblots of LC3A/B-I and LC3A/B-II using calsequestrin (CASQ1/2) as a loading control; (B), Quantitative analysis of LC3A/B-II; n = 4 hearts for NTG and 4 hearts for HSPB6S10F; (C and D), Autophagy flux: (C), Western blots of LC3A/B-I and LC3A/B-II, as well as CASQ1/2 in mice that were i.p. injected with chloroquine (CQ, 50 mg/kg) or sterile saline (Vehicle) for 4 h before sacrifice; (D), Autophagic flux data that were analyzed and expressed as mean ± SEM. *: P < 0.05, vs NTG-Vehicle; #: P < 0.05, vs. NTG-Chloroquine; n = 6 hearts for each group; (E), Representative immunoblots of BECN1 and CASQ1/2; (F), Quantitative analysis of protein levels of BECN1; n = 6 hearts for NTG, and 6 hearts for HSPB6S10F; Values represent means ± SEM; *: P < 0.05, vs NTG; (G), Electron microscopy revealed impaired autophagy in HSPB6S10F heart tissue. White arrows denote typical autophagosomes; (H), Representative immunoblots of LC3A/B-I, LC3A/B-II, BECN1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); (I and J), Quantitative analysis of protein levels of BECN1 and LC3A/B-II; n = 6 hearts for each group; Values represent means ± SEM; *: P < 0.05, vs GFP-NTG; #: P < 0.05, vs BECN1-NTG; (K), Representative images of cardiomyocyte apoptosis by Hoechst staining in each group. The apoptotic cells were indicated by the arrows, scale bars = 50 μm; (L), Normalized percentage of apoptotic cells per total number of cardiac cells in each group. n = 4 hearts for NTG and 4 hearts for HSPB6S10F. Values represent means ± SEM; *: P < 0.05, vs GFP-NTG; #: P < 0.05, vs GFP-HSPB6S10F.

Article Snippet: 3 Briefly, equivalent amounts of recombinant GST-HSPB6 WT and GST-HSPB6 S10F proteins bound to glutathione-Sepharose 4BTM resin (Sigma-Aldrich, GE17-0756) were mixed with cardiac homogenates from NTG mice at 4°C for 16 h. The beads were washed with 10 mM Na 3 PO 4 (Sigma-Aldrich, 342483), pH 7.2, 10 mM NaN3 (Sigma-Aldrich, S2002), 120 mM NaCl (Sigma-Aldrich, S7653), 0.1% (v:v) Tween-20 (Sigma-Aldrich, P1379) and were subsequently analyzed by western blot using BECN1 primary antibody (Novus Biologicals, NB500-249) and peroxidase-conjugated anti-rabbit secondary antibodies (GE Health Life Sciences, NA934).

Techniques: Activity Assay, Western Blot, Control, Injection, Sterility, Saline, Electron Microscopy, Staining

Journal: Autophagy

Article Title: Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6 S10F mutant

doi: 10.1080/15548627.2017.1392420

Figure Lengend Snippet: BECN1 protein reduction is attributed to the ubiquitin-proteosomal degradation pathway. (A), Ventricular mRNA levels of Becn1 in HSPB6S10F hearts were not altered: n = 3 hearts for NTG and 3 hearts for HSPB6S10F; (B), Proteasomal inhibitor, Bortezomib, restored BECN1 levels in HSPB6S10F-overexpressing cells (Con: control, Bor: Bortezomib); (C), Quantification of BECN1 levels in NTG and HSPB6S10F cardiomyocytes with/without Bortezomib treatment: n = 5 hearts for NTG and 5 hearts for HSPB6S10F; (D), Ubiquitination of BECN1 was enhanced in HSPB6S10F hearts: n = 4 hearts for NTG and 4 hearts for HSPB6S10F; (E to G), Enhanced BECN1 ubiquitination is associated with reduced interaction of HSPB6 with BECN1. (E), Reduced interaction of HSPB6 with BECN1 in HSPB6S10F hearts: n = 4 hearts for NTG, and 4 hearts for HSPB6S10F; (F), left panel: SDS-gel stained with Coomassie Blue showing the purified GST-HSPB6 WT and GST-HSPB6S10F; right panel: GST affinity-isolation assay showing reduced protein levels of BECN1 associated with GST-HSPB6S10F; (G), Quantification of protein levels of BECN1 binding to GST-HSPB6 WT or GST-HSPB6S10F: 3 independent experiments were performed. Values represent means ± SEM; *: P < 0.05, vs NTG or HSPB6 WT.

Article Snippet: 3 Briefly, equivalent amounts of recombinant GST-HSPB6 WT and GST-HSPB6 S10F proteins bound to glutathione-Sepharose 4BTM resin (Sigma-Aldrich, GE17-0756) were mixed with cardiac homogenates from NTG mice at 4°C for 16 h. The beads were washed with 10 mM Na 3 PO 4 (Sigma-Aldrich, 342483), pH 7.2, 10 mM NaN3 (Sigma-Aldrich, S2002), 120 mM NaCl (Sigma-Aldrich, S7653), 0.1% (v:v) Tween-20 (Sigma-Aldrich, P1379) and were subsequently analyzed by western blot using BECN1 primary antibody (Novus Biologicals, NB500-249) and peroxidase-conjugated anti-rabbit secondary antibodies (GE Health Life Sciences, NA934).

Techniques: Ubiquitin Proteomics, Control, SDS-Gel, Staining, Purification, Isolation, Binding Assay

Journal: Autophagy

Article Title: Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6 S10F mutant

doi: 10.1080/15548627.2017.1392420

Figure Lengend Snippet: Acute overexpression of HSPB6 WT and HSPB6S10F in adult rat cardiomyocytes and examination of the expression and ubiquitination levels of BECN1. (A to C), HSPB6 WT increased BECN1 protein levels and reduced its ubiquitination, whereas, expression of BECN1 was decreased and its ubiquitination was increased in HSPB6S10F cardiomyocytes: 4 independent experiments were performed using 4 adult rat hearts. Values represent means ± SEM; *: P < 0.05, vs GFP; #: P < 0.05, vs HSPB6S10F; (D and E), The ability of BECN1 to interact with HSPB6 is independent of its ubiquitination. (D), PYR-41 (ubiquitin-activating enzyme E1 inhibitor) abolished BECN1's ubiquitination (+: PYR-41 treatment, -: PBS control); (E), Decreased interaction of HSPB6 with BECN1 in HSPB6S10F cardiomyocytes in the presence of PYR-41: 4 independent experiments were performed using 4 adult rat hearts.

Article Snippet: 3 Briefly, equivalent amounts of recombinant GST-HSPB6 WT and GST-HSPB6 S10F proteins bound to glutathione-Sepharose 4BTM resin (Sigma-Aldrich, GE17-0756) were mixed with cardiac homogenates from NTG mice at 4°C for 16 h. The beads were washed with 10 mM Na 3 PO 4 (Sigma-Aldrich, 342483), pH 7.2, 10 mM NaN3 (Sigma-Aldrich, S2002), 120 mM NaCl (Sigma-Aldrich, S7653), 0.1% (v:v) Tween-20 (Sigma-Aldrich, P1379) and were subsequently analyzed by western blot using BECN1 primary antibody (Novus Biologicals, NB500-249) and peroxidase-conjugated anti-rabbit secondary antibodies (GE Health Life Sciences, NA934).

Techniques: Over Expression, Expressing, Ubiquitin Proteomics, Control

Journal: Autophagy

Article Title: Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6 S10F mutant

doi: 10.1080/15548627.2017.1392420

Figure Lengend Snippet: HSPB6 competes with BCL2 binding to BECN1. (A and B), Reciprocal coimmunoprecipitations with the HSPB6 antibody and the BECN1 antibody revealed an interaction between HSPB6 and BECN1 in both NTG and HSPB6 WT TG hearts; (C to E), Recombinant human BECN1 protein was subject to a competitive ELISA containing increasing amounts of HSPB6 and BCL2, which revealed a competitive binding of HSPB6 and BCL2 with BECN1. BSA was used as a control. (C), A diagram for competitive ELISA to detect protein-protein interaction. High-binding 96-well ELISA plates were coated overnight with recombinant human BECN1 in carbonate buffer. Purified recombinant human BCL2 (D) or HSPB6 (E), as well as increasing amounts of HSPB6 (D) or BCL2 (E), were added and incubated for 2 h followed by 3 washes in PBST. Mouse monoclonal anti-BCL2 (D) or anti-HSPB6 (E) was used as a detection antibody and incubated for 2 h. Finally, tetramethylbenzidine substrate reagent was added, and absorbance was determined by a microplate reader at 450 nm. Three independent experiments were performed, values represent means ± SEM; *: P < 0.05, vs BSA; (F and G), Immunoprecipitation studies using BCL2 antibody as the bait and probed with BECN1 antibody in heart homogenates from HSPB6S10F TG mice and HSPB6 WT TG mice respectively, with NTG mice as controls. (F), Increased interaction of BECN1 with BCL2 in HSPB6S10F hearts; (G), Abolished interaction of BECN1 with BCL2 in HSPB6 WT hearts; 4 independent experiments were performed using 4 hearts for each group.

Article Snippet: 3 Briefly, equivalent amounts of recombinant GST-HSPB6 WT and GST-HSPB6 S10F proteins bound to glutathione-Sepharose 4BTM resin (Sigma-Aldrich, GE17-0756) were mixed with cardiac homogenates from NTG mice at 4°C for 16 h. The beads were washed with 10 mM Na 3 PO 4 (Sigma-Aldrich, 342483), pH 7.2, 10 mM NaN3 (Sigma-Aldrich, S2002), 120 mM NaCl (Sigma-Aldrich, S7653), 0.1% (v:v) Tween-20 (Sigma-Aldrich, P1379) and were subsequently analyzed by western blot using BECN1 primary antibody (Novus Biologicals, NB500-249) and peroxidase-conjugated anti-rabbit secondary antibodies (GE Health Life Sciences, NA934).

Techniques: Binding Assay, Recombinant, Competitive ELISA, Control, Enzyme-linked Immunosorbent Assay, Purification, Incubation, Immunoprecipitation

Journal: Autophagy

Article Title: Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6 S10F mutant

doi: 10.1080/15548627.2017.1392420

Figure Lengend Snippet: Overexpression of HSPB6 WT in the murine heart activates autophagic flux. (A), Representative immunoblots for determining the expression of HSPB6, LC3A/B-I/II and BECN1 in NTG and HSPB6 WT hearts; (B), Quantitative results of immunoblots: values are expressed as the mean ± SEM, n = 3 hearts for NTG and 3 hearts for HSPB6 WT, *: P < 0.05, vs NTG; (C), Quantification of autophagic flux by cadaverine dye-binding assay. Mice were i.p. injected with chloroquine (CQ) for 4 h and cadaverine dye-binding analysis showed that autophagy levels were significantly increased in HSPB6 WT hearts treated with both saline (Vehicle) and CQ, compared to NTG hearts; (D and E), Increased autophagy flux in HSPB6 WT hearts: (D), Representative western blots of LC3A/B-I and LC3A/B-II with CASQ1/2 as loading control; (E), Autophagic flux data expressed as the means ± SEM. n = 5 hearts for each group; *: P < 0.05, vs NTG-Vehicle; #: P < 0.05, vs. NTG-CQ; (F), Electron microscopy reveals autophagosomes in myocytes isolated from NTG and HSPB6 WT hearts. Black arrows: autophagosomes; (G), Quantitative results of the number of autophagosomes in NTG and HSPB6 WT cardiomyocytes. Ten fields from cardiomyocytes of 3 different hearts for each group were counted; (HSPB6 is colocalized with LC3A/B labeled autophagosome in neonatal rat cardiomyocytes. The GFP-HSPB6 fused plasmid cotransfected neonatal cardiomyocytes with the mcherry-fused LC3A/B plasmid and 48 h later, cells were examined under confocal fluorescence microscopy. There was colocalization of HSPB6 (green) with LC3A/B (red), scale bars = 20 μm. Three independent experiments were performed and values represent means ± SEM; *: P < 0.05, vs NTG.

Article Snippet: 3 Briefly, equivalent amounts of recombinant GST-HSPB6 WT and GST-HSPB6 S10F proteins bound to glutathione-Sepharose 4BTM resin (Sigma-Aldrich, GE17-0756) were mixed with cardiac homogenates from NTG mice at 4°C for 16 h. The beads were washed with 10 mM Na 3 PO 4 (Sigma-Aldrich, 342483), pH 7.2, 10 mM NaN3 (Sigma-Aldrich, S2002), 120 mM NaCl (Sigma-Aldrich, S7653), 0.1% (v:v) Tween-20 (Sigma-Aldrich, P1379) and were subsequently analyzed by western blot using BECN1 primary antibody (Novus Biologicals, NB500-249) and peroxidase-conjugated anti-rabbit secondary antibodies (GE Health Life Sciences, NA934).

Techniques: Over Expression, Western Blot, Expressing, Binding Assay, Injection, Saline, Control, Electron Microscopy, Isolation, Labeling, Plasmid Preparation, Fluorescence, Microscopy

Journal: Autophagy

Article Title: Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6 S10F mutant

doi: 10.1080/15548627.2017.1392420

Figure Lengend Snippet: Preinhibition of autophagy dampens HSPB6-mediated cardioprotective effects. (A), A diagram of the experimental protocol; (B and C), The levels of LC3A/B-II and BECN1 were reduced in the HSPB6 WT hearts pretreated with 3-MA; (D), Post-I/R myocardial function recovery was significantly reduced in 3-MA-treated HSPB6 WT mice, compared with saline-treated HSPB6 WT mice; (E), Total LDH in coronary effluent, collected during the first 10 min of reperfusion, was significantly increased in HSPB6 WT mice treated with 3-MA, compared with saline group. n = 6 hearts for each group. Values represent means ± SEM; *: P < 0.05, vs saline group; (F to J), siRNA-mediated inhibition of autophagy abolishes the protective effects of HSPB6 WT: (F), Representative flow cytometric pseudo-color density plots illustrating the distribution of apoptosis in siRNA-control-treated neonatal rat cardiomyocytes infected with Ad-GFP, Ad-Hspb6S10F or Ad-Hspb6 WT under baseline or hypoxia (H2O2, 500 μM, 24 h) conditions. Cell apoptosis is featured by the exposure of phosphatidylserine at cell surface and dense clumping of genetic material in nucleus, which can be indicated by ANXA5-binding and eFluor-labeling, respectively. The early phase of apoptosis can be revealed by ANXA5 alone, whereas the late apoptosis appears when the cell is double positive for ANXA5 and eFluor. Q1: early apoptosis; Q2: late apoptosis; Q4: live cells; (G), Quantitative analysis of total apoptotic events in the absence (baseline) or presence of hypoxic injury; (H), Representative immunoblots of ATG7, LC3A/B-I/LC3A/B-II and HSPB6, using CASQ1/2 as loading control, in Ad-GFP, Ad-Hspb6S10F or Ad-Hspb6 WT cardiomyocytes transfected with siRNA-Control siRNA or Atg7 siRNA; (I), Representative flow cytometric pseudo-color density plots illustrating the distribution of apoptosis in infected cardiomyocytes after knockdown of Atg7; (J), Quantitative analysis of total apoptotic events after knockdown of Atg7 under basal and hypoxic conditions. Values are presented as means ± SEM, n = 6 preparations for each group. *: P < 0.05, vs GFP-baseline; #: P < 0.05, vs HSPBS10F-baseline; †: P < 0.05, vs GFP and H2O2; $: P < 0.05, vs HSPBS10F and H2O2.

Article Snippet: 3 Briefly, equivalent amounts of recombinant GST-HSPB6 WT and GST-HSPB6 S10F proteins bound to glutathione-Sepharose 4BTM resin (Sigma-Aldrich, GE17-0756) were mixed with cardiac homogenates from NTG mice at 4°C for 16 h. The beads were washed with 10 mM Na 3 PO 4 (Sigma-Aldrich, 342483), pH 7.2, 10 mM NaN3 (Sigma-Aldrich, S2002), 120 mM NaCl (Sigma-Aldrich, S7653), 0.1% (v:v) Tween-20 (Sigma-Aldrich, P1379) and were subsequently analyzed by western blot using BECN1 primary antibody (Novus Biologicals, NB500-249) and peroxidase-conjugated anti-rabbit secondary antibodies (GE Health Life Sciences, NA934).

Techniques: Saline, Inhibition, Control, Infection, Binding Assay, Labeling, Western Blot, Transfection, Knockdown

Journal: Molecular cell

Article Title: Lipid droplet-derived monounsaturated fatty acids traffic via PLIN5 to allosterically activate SIRT1

doi: 10.1016/j.molcel.2019.12.003

Figure Lengend Snippet: A) Saturation plot of SIRT1 activity towards FOXO3a and the effects of 18:1 and resveratrol (n=4). B) Lineweaver-Burk reciprocal plots were generated to determine Km, Vmax, and Kcat for the FOXO3a peptide substrate. C-D) Km and Kcat/Km fold change for each concentration of 18:1 on FOXO3a. E) Kcat/Km fold change for resveratrol (Res; 10 μM). F) Saturation plot of SIRT1 activity towards H3 and the effects of 18:1 and resveratrol. G) Lineweaver-Burk reciprocal plots for the H3 peptide substrate. H-I) Km and Kcat/Km fold change for each concentration of 18:1 with H3. J-K) Kcat/Km fold change for each concentration of resveratrol and fatty acids for the H3 peptide substrate. L) Competition assay of SIRT1 activity towards FOXO3A, PGC-1α and H3 acetylated peptide substrates.

Article Snippet: Antibodies were purchased or obtained from the following sources; Total-Plin5 (Progen; Heudelberg, Germany), Histone H3, SIRT1, Acetylated Lysine (Cell Signaling Technologies; Danvers, MA), PGC-1α (MilliporeSigma; Burlington, MA), phospho-PLIN5 (NeoBioLab targeting; Cys-LARRGRRW(pS)VELK), PLIN2 [Barbara Atshaves developed in ( Atshaves et al., 1999 )].

Techniques: Activity Assay, Generated, Concentration Assay, Competitive Binding Assay

Journal: Molecular cell

Article Title: Lipid droplet-derived monounsaturated fatty acids traffic via PLIN5 to allosterically activate SIRT1

doi: 10.1016/j.molcel.2019.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies were purchased or obtained from the following sources; Total-Plin5 (Progen; Heudelberg, Germany), Histone H3, SIRT1, Acetylated Lysine (Cell Signaling Technologies; Danvers, MA), PGC-1α (MilliporeSigma; Burlington, MA), phospho-PLIN5 (NeoBioLab targeting; Cys-LARRGRRW(pS)VELK), PLIN2 [Barbara Atshaves developed in ( Atshaves et al., 1999 )].

Techniques: Recombinant, Reporter Assay, Mutagenesis, Software, Derivative Assay

Journal: Nature cancer

Article Title: Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling

doi: 10.1038/s43018-019-0018-6

Figure Lengend Snippet: a, Drug sensitivity profiles of tepoxalin and seven additional compounds where ABCB1 RNA expression was the top predictive genomic feature of the PRISM activity profile (n = 489 cell lines). Tepoxalin was the only compound tested where high ABCB1 expression was associated with sensitivity rather than resistance to tepoxalin. Cell line viability is depicted with ABCB1 gene expression from CCLE RNAseq log2 RPKM data. Cell lines are ranked by mean viability with a ceiling at 100%. b, ABCB1 knockout is the top hit that rescues tepoxalin activity in a genome-wide CRISPR/Cas9 gene knockout screen. LS1034-Cas9 cells (pXPR_311) were infected with the Brunello sgRNA library, selected with puromycin, and treated with 16 μM tepoxalin versus vehicle control with two replicates in independent flasks. Cells were passaged every 3–4 days over a 30-day period. Two-sided p-values are computed using MAGeCK-MLE and shown versus gene-level log fold-changes. c, ABCB1 overexpression sensitizes to tepoxalin activity in a genome-wide CRISPR/dCas9 gene activation screen. LS1034-dCas9 cells (pXPR_109) were infected with the Calabrese sgRNA library, selected with puromycin, and treated with 16 μM tepoxalin versus vehicle control with two replicates in independent flasks. Cells were passaged every 3–4 days over a 14-day period. Two-sided p-values are computed using MAGeCK-MLE and shown versus gene-level log fold-changes. d, Tepoxalin cellular competition assay following ABCB1 knockout. LS1034-Cas9 cells (pXPR_311) were stably infected with Firefly luciferase and parental LS1034 cells (without Cas9) were stably infected with Renilla luciferase. Cells were mixed in 1:1 ratio an infected with the indicated sgRNA construct against ABCB1 or an intergenic region on chromosome 2 (negative control). Following puromycin selection, cell mixtures were treated with 16 μM tepoxalin. Firefly to Renilla luminescence ratio is plotted as log fold-change over time. Mean of three technical replicates is shown and results are representative of three independent experiments.

Article Snippet: Antibodies and western immunoblotting The following antibodies were used: polyclonal rabbit anti-PDE3A from Bethyl Laboratory (A302–740A, 1:1000 dilution), monoclonal mouse anti-V5 from Life Technologies (R960–25, 1:5000 dilution), monoclonal rabbit anti-ABCB1 (D3H1Q) from Cell Signaling Technology (12683, 1:1000 dilution), monoclonal mouse anti-β-Actin (8H10D10) from Cell Signaling Technology (3700, 1:1000 dilution), and monoclonal rabbit anti-SLC26A2 Antibody (3F6) from Novus Biologicals (H00001836-M04, 1:1000 dilution).

Techniques: RNA Expression, Activity Assay, Expressing, Knock-Out, Genome Wide, CRISPR, Gene Knockout, Infection, Over Expression, Activation Assay, Competitive Binding Assay, Stable Transfection, Luciferase, Construct, Negative Control, Selection

Journal: Nature cancer

Article Title: Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase

doi: 10.1038/s43018-020-00115-2

Figure Lengend Snippet: (A) Quantitative PCR validation of top ranked genes from RNA-Seq in n=5 patient derived xenografts with t(12;21) ETV6-RUNX1 (TEL-AML1) translocation or t(4;11) MLL-AFF1 (MLL-AF4)] translocation. Results are normalized to 36B4 human housekeeping gene and presented as Log2 fold change enrichment comparing CNS to spleen. p (two tailed) = One sample T and Wilcoxon test. (B) Gene expression of SCD1 from RNAseq data deposited in public databases. Left: Samples of BM of patients at diagnosis and relapse and CNS at relapse, unpaired analysis. Right: Patient-derived xenograft samples established by transplantation of patients’ ALL cells onto NSG mice. SCD1 expression in cells isolated from paired CNS and spleen is shown in the graph. FDR – False discovery rate. (C) Intracellular staining of SCD1 in cells from the BM (green) and CNS (grey) of a mouse transplanted with 018z cells. The peaks are relative to the percentage of human CD19+ cells normalized to mode.

Article Snippet: Fifty μg of total protein lysate were loaded on a 12% Polyacrylamide gel and incubated overnight with rabbit anti-human monoclonal SCD1 antibody 1:2000 (SCD1 (C 12 H 5 ) Rabbit mAb #2794, Cell Signaling) and vinculin (Vinculin Antibody #4650, Cell Signaling) at 4°C.

Techniques: Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Derivative Assay, Translocation Assay, Two Tailed Test, Expressing, Transplantation Assay, Isolation, Staining

Journal: Nature cancer

Article Title: Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase

doi: 10.1038/s43018-020-00115-2

Figure Lengend Snippet: (A) SCD1 gene expression levels following SCD1 overexpression in 018z cells. (B) Western blot of SCD1 protein after overexpression in 018z cells. (C)I-II Ratio of relative levels of oleoyl-CoA/stearoyl-CoA and of palmitoleoyl-CoA/palmitoyl-CoA in control (CTL) and SCD1-high 018z cells. p=Student’s t-test. (D) Human leukemia burden in 018z xenograft model. Total amount of leukemic cells in CNS, BM and spleen of NSG mice xenografted with human 018z ALL cell line overexpressing SCD1 (SCD1-high) or control (CTL) GFP+ at the time of sacrifice. (E) Human leukemia burden in REH xenograft model. Total amount of leukemic cells in CNS, BM and spleen of NSG mice xenografted with human REH ALL cell lines overexpressing SCD1 (SCD1-high) or control (CTL) GFP+ at the time of sacrifice. p=paired Student’s t-test. (F) Competition assay in vivo: FACS plots of cells injected (INPUT) and cells isolated from the CNS of the mice at sacrifice (OUTPUT). Top: GFP+ control cells transduced with a GFP-carrying lentiviral backbone and mCherry+ control cells transduced with a mCherry carrying lentiviral backbone injected in a ratio of 1:1. Bottom: GFP+ SCD1 overexpressing (SCD1-high) cells and mCherry+ control cells transduced with a mCherry carrying lentiviral backbone injected in a ratio of 1:1. (G) Ratio of total number of SCD1-high 018z cells (GFP+) to control 018z cells (mCherry+) in CNS, BM and spleen of NSG mice transplanted with a mixture of the two cell types in a ratio of 1:1. The backbone vector for SCD1 overexpression was used as control vector. p=paired Student’s t-test.

Article Snippet: Fifty μg of total protein lysate were loaded on a 12% Polyacrylamide gel and incubated overnight with rabbit anti-human monoclonal SCD1 antibody 1:2000 (SCD1 (C 12 H 5 ) Rabbit mAb #2794, Cell Signaling) and vinculin (Vinculin Antibody #4650, Cell Signaling) at 4°C.

Techniques: Expressing, Over Expression, Western Blot, Competitive Binding Assay, In Vivo, Injection, Isolation, Transduction, Plasmid Preparation

Journal: Nature cancer

Article Title: Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase

doi: 10.1038/s43018-020-00115-2

Figure Lengend Snippet: (A) Western blot analysis of SCD1 after CRISPR-Cas9 gene ablation in 018z cells. Each lane represents a different gRNA used for the initial screening. gRNA4 decreased SCD1 protein expression while gRNA1-3 did not affect its levels. (B) Gene expression level of SCD1 after CRISPR-Cas9 knockout in 018z cells by gRNA4. (C)I-II Ratio of relative levels of oleoyl-CoA/stearoyl-CoA and of palmitoleoyl-CoA/palmitoyl-CoA in control (CTL) and SCD1-low 018z cells. p=Student’s t-test (D) In vitro proliferation of SCD1-low and control (CTL) 018z cells after 96 hours in in medium supplemented with 10% lipidated or delipidated FBS. The dotted line represents the initial number of cells plated at T0. p=two-way ANOVA. (E) Human leukemia burden in 018z xenograft model. Total amount of leukemic cells in CNS, BM and spleen of NSG mice xenografted with human 018z ALL cell lines SCD1-low or control GFP at the time of sacrifice. p=paired Student’s t-test. (F) Intracellular staining of SCD1 in human cells isolated from the BM of mice transplanted with SCD1-low and control (CTL) 018z cells. The scramble vector for SCD1 downregulation was used as control vector. The peaks are relative to the percentage of human CD19+ cells normalized to mode.

Article Snippet: Fifty μg of total protein lysate were loaded on a 12% Polyacrylamide gel and incubated overnight with rabbit anti-human monoclonal SCD1 antibody 1:2000 (SCD1 (C 12 H 5 ) Rabbit mAb #2794, Cell Signaling) and vinculin (Vinculin Antibody #4650, Cell Signaling) at 4°C.

Techniques: Western Blot, CRISPR, Expressing, Knock-Out, In Vitro, Staining, Isolation, Plasmid Preparation

Journal: Nature cancer

Article Title: Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase

doi: 10.1038/s43018-020-00115-2

Figure Lengend Snippet: In vitro proliferation of “SCD1-high”, “SCD1-low” and matching control (CTL) 018z cells after seeding 0.5x106 cells/well for 96 hours in medium supplemented with 10% lipidated (A-B) or delipidated (C-D) FBS treated with 1μM of the SCD1 inhibitor SW203668 or vehicle (DMSO).The dotted line represents the initial number of cells plated at T0. p=two-way ANOVA. (D) GFP+mCherry+FFLuc+ 018z cells were injected intravenously to NSG mice and treated from day 1 to day 10 with the SCD1 inhibitor SW203668 or vehicle (n=5 group). Representative bioluminescence of the tumor load at the time of sacrifice in three different pairs of mice, top: vehicle treated mice; bottom: drug treated mice. Decrease in the tumor load is clear in the spine and the skull area (CNS – marked with white box). Total amount of leukemic cells in CNS (E) and BM (F) of NSG mice xenografted with human GFP+mCherry+FFLuc+ 018z and treated with SW203668 for 10 days at the time of sacrifice. The backbone vector for SCD1 overexpression was used as control vector for SCD1-high and the scramble vector was used as control for SCD1-low. BM – bone marrow. p=Student’s t-test.

Article Snippet: Fifty μg of total protein lysate were loaded on a 12% Polyacrylamide gel and incubated overnight with rabbit anti-human monoclonal SCD1 antibody 1:2000 (SCD1 (C 12 H 5 ) Rabbit mAb #2794, Cell Signaling) and vinculin (Vinculin Antibody #4650, Cell Signaling) at 4°C.

Techniques: In Vitro, Injection, Plasmid Preparation, Over Expression

Journal: Nature cancer

Article Title: Metabolic adaptation of acute lymphoblastic leukemia to the central nervous system microenvironment is dependent on Stearoyl CoA desaturase

doi: 10.1038/s43018-020-00115-2

Figure Lengend Snippet: Human leukemia burden in PDXs. Cells from 4 different PDXs were injected intravenously into NSG mice. Xenografted mice were treated daily from day 7 with the SCD1 inhibitor SW203668 or vehicle (n=5 group) at 20 mg/kg. The total amounts of leukemic cells in CNS and BM of NSG mice at the time of sacrifice are plotted in the graphs (A-D). p=Student’s t-test.

Article Snippet: Fifty μg of total protein lysate were loaded on a 12% Polyacrylamide gel and incubated overnight with rabbit anti-human monoclonal SCD1 antibody 1:2000 (SCD1 (C 12 H 5 ) Rabbit mAb #2794, Cell Signaling) and vinculin (Vinculin Antibody #4650, Cell Signaling) at 4°C.

Techniques: Injection

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , b , Binding enzyme-linked immunosorbent assay (ELISA) of spike to fibrinogen ( a ) or fibrin ( b ). K d , dissociation constant. A 450 , absorbance at 450 nm. c , Fibrinogen immunoprecipitation (IP) with spike. d , Spike and fibrinogen immunoreactivity in the lungs at 3 d.p.i. Representative of five Beta-infected WT mice. Scale bar, 300 μm. e , Peptide array of fibrinogen chains Aα, Bβ and γ blotted with spike. The binding signal intensity is shown (white to orange). f , Scanning electron microscopy (SEM) images and quantification of the fibrin clot fibre radius in human plasma with spike. The fibre radius distribution was determined in n = 25 (plasma) and n = 28 (plasma with spike) images from three biologically independent experiments (generalized linear mixed-effects model with Holmes multiple correction; ) and the fibre radius proportion (<0.05 µm) was determined from n = 3 biologically independent experiments (two-sided paired t -test; ). Scale bar, 1 µm. FOV, field of view. g , The turbidity of fibrin polymerization with spike in human plasma. h , Immunoblot (IB) analysis of fibrin degradation by plasmin representative from five (0, 2 and 4 h) or three (1 and 6 h) biologically independent experiments. i , ROS in BMDMs stimulated with fibrin and/or spike. n = 6 (unstimulated and spike) and n = 3 (fibrin or fibrin with spike) biologically independent experiments. a.u., arbitrary units. j , Fibrin γC domain and spike-binding epitope γ 364–395 (red). Alanine scanning of γ 377–395 blotted with His–spike. The binding of spike to Ala-substituted peptides is shown. The residues that are required for binding are indicated in yellow. k , Competitive ELISA of 5B8-huFc (5B8 with human IgG1 Fc region) or huIgG1 versus spike for binding to fibrin. n = 3 biologically independent experiments. l , ROS in BMDMs stimulated with fibrin and/or spike treated with 5B8 or IgG2b. n = 3 biologically independent experiments. Representative data of n = 3 ( a – c ) or n = 4 ( g ) biologically independent experiments. For i and l , statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. Data are mean ± s.e.m. Gel source data are provided in Supplementary Fig. .

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Infection, Peptide Microarray, Electron Microscopy, Clinical Proteomics, Western Blot, Competitive ELISA, Comparison

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Binding ELISA of Spike S1(N501Y) to fibrin. Dissociation constants (K d ). Representative curvefits from two independent biological experiments in duplicates. b , Spike overlap with perivascular fibrin(ogen) deposition in lung of Beta-infected WT mice at 3 d.p.i. The 51% of the calculated proportion of fibrin that colocalizes with Spike protein is significantly higher than the 23% predicted if the correlation were random. Fisher’s exact test (two-tailed); n = 78 images from 5 mice (Methods). Representative confocal images are shown. Scale bar, 200 μm. c , Scatter plot of positive correlation of fibrinogen and Spike immunoreactivity in n = 78 images from 5 mice, Pearson correlation two-tailed (Methods). d . 3D reconstruction of light sheet acquisitions of whole lung tissue from an Alexa546-fibrinogen and Alexa647-Spike S1(N501Y)-injected WT mouse following 3DISCO tissue clearing. Two representative focal fibrinogen deposits from n = 3 mice were selected for 3D visualization. Volumetric rendering reveals close interactions between fibrinogen deposits (green) and trimeric spike (magenta), confirming colocalization. Scale bars, 100 μm (top), 300 μm (bottom). e , Fibrinogen crystal structure (PDB: 3GHG) with mapped peptides (red). Proximity of peptides γ 163-181 and γ 364-395 (inset). f , Peptide array mapping with immobilized peptides of SARS-CoV-2 Spike blotted with fibrinogen and fibrinogen γ chain. Heatmap of signal intensity showing binding sites (white-orange) within the S1-NT Spike domain. Key indicates fluorescence intensities signal values from low (white) to high (orange). Schematic indicating Spike domains and amino acid sequence.

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Injection, Peptide Microarray, Fluorescence, Sequencing

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Predicted computational complex model using crystal structure of fibrinogen (PDB ID: 3GHG) and cryoEM structure of Spike (PDB ID: 6VSB). b , Surface of this complex. c , Schematic representation of intermolecular interface of this predicted model. d , 3D representation of interface shown by side chain of the few residues involved in interaction. e , Intermolecular interface showing atom-wise interaction with all the residues involved in the interaction including a hydrogen bond highlighted in green dashed line using LigPlot +, v.2.2. https://www.ebi.ac.uk/thornton-srv/software/LigPlus/ .

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Software

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Topographic visualization of fibrin fibre surface in SEM images of fibrin clots in healthy human donor plasma in the presence of Spike. b , SEM of fibrin clots in human plasma in the presence of Spike. x4000 magnification. Images representative of n = 3 independent biological replicates quantified in ( c ) and Fig. . c , Fibre radius proportion less than 0.05 µm (boxplot) and intersection density (bar plot) in plasma or plasma with Spike. Generalized linear mixed effects model (boxplot) and two-sample two-sided Welch t-test (bar plot). n = 25 (plasma), n = 28 (plasma with Spike) images from n = 3 biologically independent experiments quantified in Fig. . Images from biologically independent experiments are indicated by different colour dots in boxplot. Box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. d , Alanine scan mutagenesis peptide array. Fibrin peptide γ 377-395 was subjected to double-alanine scanning mutagenesis and incubated with His-tagged recombinant Spike. Signal intensity bar graph of the binding of Spike to sequential Ala-Ala substituted peptides (red). Control signal is shown in blue. Residues with low signal intensity upon Ala-Ala substitution are required for binding and highlighted in yellow. e . ELISA of 5B8-huFc or huIgG1 isotype control pre-incubated with fibrin versus the Spike for binding to fibrin. Data are mean ± s.e.m from three biologically independent experiments. f . Iba-1 immunoreactivity in brain following stereotaxic co-injection of fibrinogen with PBS or Spike in WT mice. Scale bar, 50 µm. Data are from n = 6 mice per group. One-way ANOVA with Tukey’s multiple comparisons test. All data are mean ± s.e.m.

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Clinical Proteomics, Mutagenesis, Peptide Microarray, Incubation, Recombinant, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Injection

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Lung pathology of Beta-infected WT, Fga −/− and Fgg γ390–396A mice. b , Microscopy analysis of Mac2 (macrophages) and fibrin/fibrinogen in uninfected (UI) ( n = 4) and Beta-infected WT ( n =10), Fga −/− ( n = 10) and Fgg γ390–396A ( n = 9) mice; gp91 phox in uninfected ( n = 3) and Beta-infected WT ( n = 10), Fga −/− ( n = 10) and Fgg γ390–396A ( n = 9) mice; and Trichrome (collagen, blue; fibrin, red) in uninfected ( n = 4) and Beta-infected WT ( n = 5), Fga −/− ( n = 5), Fgg γ390–396A ( n = 4) mice. Data are from mice infected in two independent experiments. c , Gene set enrichment analysis (GSEA) of pathways significantly altered in Beta-infected lungs of Fga −/− mice compared with WT mice. NES, normalized enrichment score. d , Significant genes and pathways. Uninfected: n = 4 (WT) and n = 3 ( Fga −/− ) mice; Beta: n = 4 (WT) and n = 5 ( Fga –/– ) mice. e , Microscopy analysis of NKp46, granzyme and spike in lung after infection. NKp46: uninfected, n = 8 (WT); infected, n = 10 (WT), n = 10 ( Fga −/− ) and n = 9 ( Fgg γ390–396A ) mice; granzyme: uninfected, n = 4 (WT); infected, n = 5 mice per group; spike: uninfected, n = 4 (WT); infected: n = 10 (WT), n = 10 ( Fga −/− ) and n = 9 ( Fgg γ390–396A ) mice. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple-comparison test ( b and e ) and two-sided quasi-likelihood F -test implemented in edgeR ( d ). In d , bold font indicates adjusted P < 0.05 (Benjamini–Hochberg). Each lane represents the average scaled z -score for each genotype. Data are mean ± s.e.m. Scale bars, 100 μm ( b and e ). The diagram in a was created with BioRender.

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Infection, Microscopy, Comparison

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Spike PV production (Methods). b , Immunoblot of Spike expression in PVs blotted with anti-Spike, anti-p24 Gag (detecting p55) and anti-Vpr. Spike PVs expressed S1, S2, cleaved S1 and Spike multimeric forms. PVs express comparable levels of the proviral backbone indicated by HIV Env VPs (Vpr). c , Fibrinogen immunoprecipitation with PVs blotted with anti-Spike or anti-fibrinogen. d , ROS production in fibrin-stimulated BMDMs (24 h) with PVs. n = 3 biologically independent experiments. e , Fibrin(ogen) from lungs of n = 6 WT mice per group 24 h after PV injection. Scale bars, 200 µm and 50 µm (inset). Welch two-sample t-test (two-tailed) followed by Holm multiple correction testing. f . Confocal microscopy of Spike (green) and fibrin(ogen) (red) in lung 24 h after Spike PVs injection; orthogonal views of the y/z and x/z planes show the localization of fibrinogen and Spike. Scale bar, 50 μm. Scatter plot shows correlation of fibrinogen and Spike in n = 24 images from three mice, Pearson correlation (Methods). g , gp91-phox (red) and Mac-2 (green) in lungs 24 h after injection of n = 6 mice (Bald, Spike or HIV-ENV PVs) and n = 3 uninjected controls (UI). Scale bars, 100 μm. h , Mac-2 (green) and gp91-phox (red) in lungs from WT and Fga –/– mice after Bald PVs injection. Scale bar, 70 μm. Representative images from n = 6 mice per group. Quantification in Fig. . i , Iba-1 in corpus callosum after stereotactic co-injection of fibrinogen with PBS, Bald PVs or Spike PVs. Scale bar, 50 µm. n = 6 mice per group. Representative immunoblots from two ( b ) or three ( c ) biologically independent experiments. d, g, i , One-way ANOVA with Tukey’s multiple comparisons test. All data are mean ± s.e.m. For gel source data, see Supplementary Fig. .

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Western Blot, Expressing, Immunoprecipitation, Injection, Two Tailed Test, Confocal Microscopy

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Beta infection of 5B8-treated WT mice. b , c , Lung pathology in WT mice prophylactically treated with 5B8 or IgG2b ( n = 5 (Trichrome, N protein); n = 10 (Mac2, gp91 phox , spike, granzyme)) at 3 d.p.i. ( b ) or therapeutically treated with 5B8 ( n = 11) or IgG2b ( n = 12) (Mac2 and gp91 phox ) at 7 d.p.i. ( c ). d , Beta infection of WT, Fg a −/− and Fgg γ390–396A mice or 5B8-treated WT mice at 7 d.p.i. e , Fibrinogen and IBA1 in the cortex, representative of four Beta-infected WT mice. f , IBA1 in the hippocampus. UI: n = 6 mice; Beta infected, prophylactic: n = 10 (prophylactic 5B8 or IgG2b) mice per group; Beta infected, therapeutic: n = 12 (IgG2b) and n = 11 (5B8) mice. g , IBA1 and CD68 in the hippocampus. Uninfected: n = 6 WT mice; Beta infected, n = 6 (WT), n = 6 ( Fga −/− ) or n = 5 ( Fgg γ390–396A ) mice. h , Delta infection of 5B8-treated K18-hACE2 mice. i , Fibrinogen and IBA1 in various brain regions of uninfected and Delta-infected mice at 3 d.p.i. Uninfected: n = 4 (hippocampus (Hippo)) and n = 5 (corpus callosum (Cc), striatum (Str) and frontal cortex (FCtx)) mice; Delta infected: n = 4 (frontal cortex) and n = 5 (hippocampus, corpus callosum, striatum) mice. j , k , IBA1, CD68, calbindin and NeuN in the cortex ( j ) and hippocampus ( k ). Uninfected: n = 5 mice; Delta infected, prophylactic, 3 d.p.i.: n = 5 (IgG2b) or n = 4 (5B8) mice; Delta infected, therapeutic, 9 d.p.i.: n = 6 mice per group. l , Mouse survival and weight. n = 12 mice per group (therapeutic, 5B8 or IgG2b, Delta infected). Statistical analysis was performed using log-rank tests (survival) and a mixed-effects model (weight). m , Significantly altered genes in the hippocampus of Delta-infected mice given 5B8 or IgG2b. n = 6 mice per group. Statistical analysis was performed using two-sided unpaired t -tests (unadjusted P < 0.05; ). For a – f and h – m , 5B8 or IgG2b was given intraperitoneally at a dose of 30 mg per kg body weight, prophylactically (at 0 d.p.i.) or therapeutically (at 1 d.p.i.). Statistical analysis was performed using two-tailed Mann–Whitney U -tests ( b (all except for granzyme) and c ), two-tailed Welch t -tests with Holm multiple-comparison correction ( b (granzyme) and i ) and one-way ANOVA Tukey’s multiple-comparison test ( f , g , j and k ). Data are mean ± s.e.m. Scale bars, 100 μm ( b , c , e , j and i ) or 50 μm ( f , g and k ). The diagrams in a , d and h were created using BioRender.

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Infection, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Microscopy of fibrinogen and NK1.1 in lungs from prophylactic 5B8- and IgG2b-treated Beta-infected WT mice 3 d.p.i. Scale bars, 100 μm. Infected, n = 10 mice per group; uninfected (UI), n = 4 mice. Two-tailed Mann-Whitney test (fibrin(ogen)), one-way ANOVA with Tukey multiple comparisons (NK1.1). b , Box-and-whisker plots showing the number of PFUs propagated from lung homogenates of infected animals on Vero cells at 3 d.p.i. PFU/ml from lung homogenates of Beta-infected WT mice given prophylactically 5B8 or IgG2b. n = 10 per group. Two-tailed Mann-Whitney. Box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. c , Microscopy of 4-HNE in lungs from therapeutic 5B8- and IgG2b-treated WT mice after Beta infection at 7 d.p.i. Scale bars, 100 μm. n = 12 (IgG2b) or n = 11 (5B8). Two-tailed Mann-Whitney test. d , Microscopy of brain sections from Beta-infected WT (left) and UI control (right) given i.p. injection of 30 mg/kg 5B8-huFc showing the spatial co-localization (yellow) between 5B8-huFc detected with FITC-human IgG (green), and fibrin deposition detected with antibody to fibrin(ogen) (red) at 7 d.p.i. The 5B8-huFc antibody was used in target engagement studies to enable in vivo detection in the mouse by human FITC-IgG. Scale bars, 80 μm. Data are representative of n = 3 mice. All data are mean ± s.e.m.

Article Snippet: Retro-orbital injections of 0.1 ml of PBS solution containing 20 μg Alexa-647-conjugated spike S1(N501Y) and 30 μg Alexa-546-labelled human fibrinogen (Invitrogen) were performed under isoflurane anaesthesia (1 ml insulin syringe with a 30-gauge needle).

Techniques: Microscopy, Infection, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Control, Injection, Drug discovery, In Vivo

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Effect of a Neuropilin-1-Derived Virus Receptor Trap on Enterovirus A71 Infection In Vitro

doi: 10.1128/AAC.00695-20

Figure Lengend Snippet: Functional study of NRP1-derived decoy receptor fusion proteins to EVA71. (A) NRP1-ECD-(a1b2) and NRP1-ECD-(b1b2) cDNAs were cloned into an expression vector in frame with and upstream from cDNA that encodes hFc, resulting in NRP1-ECD-(a1b2)-hFc (a) and NRP1-ECD-(b1b2)-hFc(b). (B) Purified NRP1-ECD-(a1b2)-hFc and NRP1-ECD-(b1b2)-hFc fusion proteins were electrophoresed using SDS-PAGE under nonreducing [DTT (dithiothreitol)(−)] and reducing [DTT(+)] conditions. (C) An indirect ELISA was performed by coating wells with 20 μg/ml formalin-inactivated EVA71, and the bound fusion proteins were detected using HRP-conjugated goat anti-hFc antibody. Both NRP1-ECD-(a1b2)-hFc and NRP1-ECD-(b1b2)-hFc bound to EVA71 with similarly high affinities (10 to 11 nM). All data were calculated from the averaged duplicate values and analyzed using GraphPad Prism. (D) An ELISA-based competitive binding assay was performed by coating wells with 10 μg/ml formalin-inactivated EVA71. Then, 0.2 μM recombinant scavenger receptor B2 (SCARB2) fusion protein was competitively bound with or without NRP1-ECD-hFc (0.04 to 1 μM), and binding was detected by using an HRP assay. (E) An EVA71 VP3 peptide library containing 47 peptides was used in an overlapping-peptide ELISA for epitope mapping, and bound antibodies were detected using an HRP assay.

Article Snippet: Binding of fusion proteins was revealed with a goat anti-human IgG-Fc fragment cross-adsorbed antibody (Bethyl Laboratories, Inc.) conjugated with horseradish peroxidase (HRP) and HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) solution (U.S.

Techniques: Functional Assay, Derivative Assay, Clone Assay, Expressing, Plasmid Preparation, Purification, SDS Page, Indirect ELISA, Enzyme-linked Immunosorbent Assay, Competitive Binding Assay, Recombinant, Binding Assay, Peptide ELISA