Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires.

doi: 10.4049/jimmunol.165.12.6716

Figure Lengend Snippet: FIGURE 1. CD8aa cells are absent from the thymus. Cytofluorometry of E14 and adult thymocytes and adult gut using anti-CD8a and CD8b Abs 11-39 and EP-42, respectively, and anti-mouse IgG-specific Abs coupled to FITC or PE. Arrows point to the locations of CD8aa cells

Article Snippet: For three-color analysis we used mouse-anti-TCR Vb1 mAb coupled to biotin (TCR2, Southern Biotechnology, Birmingham, AL), mouse antiCD8b mAb (Ep42, an IgG2a, gift from M. Ratcliffe, Montreal, Canada), and mouse anti-CD8a mAbs (11-39, an IgG1 recognizing all polymorhic forms; 11-7 and 11-13, both IgG1 recognizing only CD8 of the strain H.B15.H12; (32)).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires.

doi: 10.4049/jimmunol.165.12.6716

Figure Lengend Snippet: FIGURE 2. Identification of two chicken strains congenic for the CD8 a-chain. H.B15.H7 and H.B15.H12 strains express different CD8a alleles. A, Cytofluorometry of adult H7 and H12 chicken thymocytes using anti-CD8a Abs 11-13 and 11-39. Note that Ab 11-13 does not recognize CD8a on thy- mocytes of H7 animals. B, mAbs 11-13 and 11-39 immunoprecipitate the CD8 a-chain. The thymocytes of a 3-wk-old H12 chicken were 125I labeled. The lysate was precipitated with mAbs 11-13 and 11-39. Immunoprecipitates were analyzed by SDS-PAGE on a 10% gel under reducing conditions. The mo- lecular mass standards are indicated on the left. C, mAb 11-13 recognizes the allotypic CD8 a-chain. COS-7 cells were transfected with pCDM8 plasmids carrying CD8a from the inbred chicken lines H.B15.H7 (a) or H.B15.H12 (b), respectively. The cells were then stained with the CD8a allotypic mAb 11-13. Only H12 CD8 was recognized. Staining of the cells with mAb 11-39 served as a positive control. Abs were detected with HRP-conjugated rabbit anti- mouse-Ig.

Article Snippet: For three-color analysis we used mouse-anti-TCR Vb1 mAb coupled to biotin (TCR2, Southern Biotechnology, Birmingham, AL), mouse antiCD8b mAb (Ep42, an IgG2a, gift from M. Ratcliffe, Montreal, Canada), and mouse anti-CD8a mAbs (11-39, an IgG1 recognizing all polymorhic forms; 11-7 and 11-13, both IgG1 recognizing only CD8 of the strain H.B15.H12; (32)).

Techniques: Labeling, SDS Page, Transfection, Staining, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intestinal CD8 alpha alpha and CD8 alpha beta intraepithelial lymphocytes are thymus derived and exhibit subtle differences in TCR beta repertoires.

doi: 10.4049/jimmunol.165.12.6716

Figure Lengend Snippet: FIGURE 3. Embryonic TCRgd1 CD8aa2 thymocytes differentiate into TCRgd1 CD8aa1 iIELs. E14 H12 thymocytes (2 3 107) were in- jected i.v. into E16 H7 recipient embryos. The iIELs were analyzed 18 days after injection by cytofluorometry. CD8aa1 donor cells were detected by Ab 11-13, which specifically recognizes the H12 CD8 a-chain (donor). Total CD8a1 iIELs, including both host and donor cells from the same animal, were detected with Ab 11-39 recognizing donor and host CD8a1

Article Snippet: For three-color analysis we used mouse-anti-TCR Vb1 mAb coupled to biotin (TCR2, Southern Biotechnology, Birmingham, AL), mouse antiCD8b mAb (Ep42, an IgG2a, gift from M. Ratcliffe, Montreal, Canada), and mouse anti-CD8a mAbs (11-39, an IgG1 recognizing all polymorhic forms; 11-7 and 11-13, both IgG1 recognizing only CD8 of the strain H.B15.H12; (32)).

Techniques: Injection

Journal: Cancers

Article Title: Cotargeting of XPO1 Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Acute Myeloid Leukemia

doi: 10.3390/cancers12061574

Figure Lengend Snippet: Midostaurin CRISPR knockout screen. ( a ) Screen design. ( b ) Volcano scatter plot showing significant positive and negative selection results. ( c ) Changes in levels of the two most efficient sgRNAs targeting XPO1 in four replicate screens. ( d ) Enriched gene pathway analysis. ( e ) Schematic of nuclear pore complex (NPC) components with log-fold change (LFC) values from the screen indicated.

Article Snippet: Cultures were then exposed to 30 nM midostaurin (Selleck), or dimethyl sulfoxide (DMSO, 0.00012%) as a vehicle control for three days.

Techniques: CRISPR, Knock-Out, Selection

Journal: Cancers

Article Title: Cotargeting of XPO1 Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Acute Myeloid Leukemia

doi: 10.3390/cancers12061574

Figure Lengend Snippet: Genetic validation of XPO1 as a cotarget with FLT3. ( a ) Design of three guides targeting XPO1 used to knock down XPO1 expression in MOLM-13 cells. ( b ) Immunoblot analysis showing protein expression of XPO1 in MOLM-13 cells, XPO1 knockdown (KD) cell lines, and HG3 (positive control). ( c ) MOLM-13 parent cells and XPO1 knockdown cells were treated with 10 nM midostaurin or 8 nM gilteritinib for 48 h and proliferation changes compared. For each knockdown experiment, conditions were compared with each clone separately and then the results pooled together across clones to get an average effect ( p < 0.001 where noted).

Article Snippet: Cultures were then exposed to 30 nM midostaurin (Selleck), or dimethyl sulfoxide (DMSO, 0.00012%) as a vehicle control for three days.

Techniques: Biomarker Discovery, Knockdown, Expressing, Western Blot, Positive Control, Clone Assay

Journal: Cancers

Article Title: Cotargeting of XPO1 Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Acute Myeloid Leukemia

doi: 10.3390/cancers12061574

Figure Lengend Snippet: In vitro pharmacologic validation of XPO1 as a cotarget with FLT3. ( a ) MOLM-13 or MV4-11 cells were treated with a range of doses of midostaurin or gilteritinib plus selinexor for 48 h and proliferation changes measured. Regions of synergy were determined using highest single agent analysis shown here; mathematical synergy was calculated in . ( b ) Similar proliferation and synergy analysis was applied to primary FLT3 -ITD AML patient samples, AML1 and AML2, which were cocultured with HS5 stromal cells for 96 h. For each drug ( n = 2 patient samples), blast cells were separated from stroma prior to development with MTS and results averaged prior to analysis against a highest single agent model shown here; mathematical synergy was calculated in .

Article Snippet: Cultures were then exposed to 30 nM midostaurin (Selleck), or dimethyl sulfoxide (DMSO, 0.00012%) as a vehicle control for three days.

Techniques: In Vitro, Biomarker Discovery

Journal: Cancers

Article Title: Cotargeting of XPO1 Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Acute Myeloid Leukemia

doi: 10.3390/cancers12061574

Figure Lengend Snippet: In vivo pharmacologic validation of XPO1 as a cotarget with FLT3. ( a ) Design of murine experiment where NSG mice were engrafted with MOLM-13 cells expressing luciferase and treated with vehicle, 50 mg/kg midostaurin daily, 30 mg/kg gilteritinib daily, 15 mg/kg selinexor twice weekly, or combinations of midostaurin or gilteritinib plus selinexor. ( b ) IVIS imaging of two mice per group and Kaplan-Meier analysis of survival of the entire cohort. ( c ) Estimated mean survival for each group with the 95% confidence interval (CI). ( d ) Representative images of spleens from each group ex vivo. ( e ) Differences in spleen weights between groups were estimated using analysis of variance (ANOVA) methods. p -values were adjusted for multiple comparisons within each drug combination using Holm’s procedure.

Article Snippet: Cultures were then exposed to 30 nM midostaurin (Selleck), or dimethyl sulfoxide (DMSO, 0.00012%) as a vehicle control for three days.

Techniques: In Vivo, Biomarker Discovery, Expressing, Luciferase, Imaging, Ex Vivo

Journal: Journal of neurochemistry

Article Title: Linoleic acid-derived metabolites constitute the majority of oxylipins in the rat pup brain and stimulate axonal growth in primary rat cortical neuron-glia co-cultures in a sex-dependent manner

doi: 10.1111/jnc.14818

Figure Lengend Snippet: Flowchart of the study designed. Pregnant rats were maintained in a temperature and light controlled animal facility until they gave birth. The pups were decapitated on postnatal day (PND) 0 or 1 and their brains excised. Brains were subjected to fatty acid or oxylipin analysis. Neuro-glia were also isolated from pup brains and exposed to LA, 13-HODE or PGE2.

Article Snippet: LA (NuChek, Elysian, MN, USA), 13-HODE (Cayman Chemical, Ann Arbor, MI, USA), or PGE2 (Cayman Chemical) were first dissolved as 1000X stocks in absolute ethanol and then diluted at 1:1000 directly into cultures.

Techniques: Isolation

Journal: Journal of neurochemistry

Article Title: Linoleic acid-derived metabolites constitute the majority of oxylipins in the rat pup brain and stimulate axonal growth in primary rat cortical neuron-glia co-cultures in a sex-dependent manner

doi: 10.1111/jnc.14818

Figure Lengend Snippet: Axonal length of primary rat cortical neurons incubated with different concentrations of A.) linoleic acid (LA), B.) 13- hydroxyoctadecadienoic acid (13-HODE), and C.) prostaglandin-E2 (PGE2). Data are presented as mean ± SD (n = 7–9 wells from four independent dissections). Data were analyzed by two-way ANOVA. For subsequent analyses, data for each sex were analyzed separately using a one-way ANOVA followed by Dunnett’s multiple comparison post hoc test to determine concentration-specific effects. An unpaired t-test was used to determine differences in axonal length in cultures treated with the positive control Y-27632 vs vehicle control. Results were considered significant at p<0.05. Asterisk (*) denotes significance at p<0.05, ** at p<0.01; **** at p<0.0001. Dagger (+) reflects p=0.06 between positive control (Y-27632) and vehicle for males treated with 13-HODE.

Article Snippet: LA (NuChek, Elysian, MN, USA), 13-HODE (Cayman Chemical, Ann Arbor, MI, USA), or PGE2 (Cayman Chemical) were first dissolved as 1000X stocks in absolute ethanol and then diluted at 1:1000 directly into cultures.

Techniques: Incubation, Concentration Assay, Positive Control

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Characteristics of MAbs against the HGE agent

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques:

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Western immunoblot analysis of HGE agent 13, E. chaffeensis, E. canis, E. sennetsu, and their uninfected host cells with three MAbs. (A) Proteins were separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis, hybridoma (3E65, 5C11, and 5D13) culture supernatants were used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Western Blot, Staining, Molecular Weight

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Effect of PMSF on the molecular size of major antigens recognized by MAb 5C11. Uninfected HL-60 cells, purified HGE agent 13, 1 mM PMSF-treated HGE agent, and the OMP fraction of the HGE agent were separated on a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with a 1:50 dilution of MAb 5C11 in ascitic fluid. Molecular weight (MW) standards were from Bio-Rad.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Purification, Incubation, Molecular Weight

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Comparisons of six purified HGE agent isolates with three MAbs on a 10% polyacrylamide gel (A) and by Western blot analysis (B). (A) Proteins from purified HGE agent isolates (13, 2, 11, 3, 6, and USG) were separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis, hybridoma (3E65, 5C11, and 5D13) culture supernatants were used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Purification, Western Blot, Staining, Molecular Weight

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Western blot analysis of the OMP fractions from six HGE agent isolates with three MAbs. (A) The OMP fractions from the six HGE agent isolates (13, 2, 11, 3, 6, and USG) were separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis, hybridoma (3E65, 5C11, and 5D13) culture supernatants were used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Western Blot, Staining, Molecular Weight

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Western blot analysis of rP44 with three MAbs. (A) Purified HGE agent 13 and affinity-purified rP44 were separated on a 10% polyacrylamide gel and stained with Coomassie blue. (B) For Western blot analysis, hybridoma (3E65, 5C11, and 5D13) culture supernatants were used at 1:10 dilutions. Molecular weight (MW) standards were from Bio-Rad.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Western Blot, Purification, Affinity Purification, Staining, Molecular Weight

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: IFA staining of HGE agent 13 in HL-60 cells with three MAbs. Infected HL-60 cells were fixed in Diff-Quik fixative and incubated with MAbs (3E65, 5C11, and 5D13) in ascitic fluid at 1:100 dilutions and with positive and negative control (NS) mouse sera at 1:50 dilutions. Note the ring-like labeling of the HGE agent with all three MAbs. Magnification, ×1,600.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Staining, Infection, Diff-Quik, Incubation, Negative Control, Labeling

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: Transmission electron micrograph of HGE agent 13 in HL-60 cells immunogold labeled with MAbs. Infected cells were embedded in LR Gold, and ultrathin sections on grids were incubated with MAbs (3E65 and 5C11) and positive and negative control mouse sera and with goat anti-mouse IgG + IgM conjugated with 10-nm gold particles (Amersham). Bars, 0.4 μm.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Transmission Assay, Labeling, Infection, Incubation, Negative Control

Journal:

Article Title: Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

doi:

Figure Lengend Snippet: PCR detection of the HGE agent 16S rRNA gene in the blood of HGE agent-inoculated mice passively immunized with MAbs. Three mice each were inoculated with HGE agent 13-infected HL-60 cells and each MAb or positive or negative control mouse serum. Each group of mice was inoculated again on the 2nd day with the same antibody. DNAs were prepared from the blood of all mice at 5 days post-HGE agent inoculation. One microgram of DNA was used as the template for PCR. As a positive control, 10 pg of DNA extracted from purified HGE agent 13 was used, while distilled water without template was used as a negative control. The arrow shows the HGE agent-specific 16S rRNA gene fragment (287 bp) obtained by PCR amplification. N1 to N3, negative control mouse serum-treated mice; P1 to P3, positive control (polyclonal anti-HGE agent) mouse serum-treated mice; E1 to E3, MAb 3E65-treated mice; C1 to C3, MAb 5C11-treated mice; D1 to D3: MAb 5D13-treated mice. Numbers on the left indicate molecular sizes of φX174 RF DNA/HaeIII fragments (GIBCO). The experiment was repeated twice.

Article Snippet: Five isolates of the HGE agent (isolates 13, 2, 11, 3, and 6) ( 11 , 16 ) and a tick isolate (USG) ( 5 ) were propagated in the human promyelocytic leukemia cell line HL-60 (American Type Culture Collection [ATCC], Manassas, Va.) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, Ga.), 1% minimal essential medium (MEM) nonessential amino acid mixture (GIBCO, Grand Island, N.Y.), 1 mM MEM sodium pyruvate (GIBCO), and 2 mM l -glutamine (GIBCO) ( 11 , 16 ).

Techniques: Infection, Negative Control, Positive Control, Purification, Amplification

Journal: Nature Communications

Article Title: Apoptotic tumor cell-derived microRNA-375 uses CD36 to alter the tumor-associated macrophage phenotype

doi: 10.1038/s41467-019-08989-2

Figure Lengend Snippet: Coculture with breast cancer cells increases miR-375 in human MΦ. a – l Human PBMC-derived MΦ were used. a Heatmap of differentially expressed miRs from control and MΦ cocultured with MCF-7 cells ( n = 4). b Representative differentially expressed miRs in control MΦ vs. cocultured MΦ and c MA -plot. d MiR-375 abundance was measured in control, 48 h cocultured, polarized (LPS + IFNγ for 24 h, IL-4 for 48 h) and resolution-phase (resolvin D1 for 6 h) MΦ via qPCR, and normalized to untreated MΦ ( n ≥ 3). e Primary human MΦ were treated for 3 h with actinomycin D (Act D) or a DMSO control. Cells were washed and cocultured with MCF-7 cells for 24 h. MiR-375 abundance was quantified via qPCR and normalized to MΦ control ( n = 8). f PPARgamma mRNA expression was measured as a positive control ( n ≥ 5). g – j MΦ were transfected with nonspecific control (ns siRNA) or DICER siRNA for 24 h and cocultured with MCF-7 cells for another 48 h ( n = 5–6). g DICER mRNA expression in MΦ. h Endogenous miR-21-5p and miR-142-3p were measured by qPCR as a control. i MiR-375 abundance and j pre-miR-375 expression were measured by qPCR in control and cocultured MΦ. k MΦ were cocultured with indicated cell lines for 24 h. MiR-375 levels were measured by qPCR and normalized to MΦ control ( n ≥ 5). l MΦ were cocultured with MCF-7 control (empty vector transfected) or decoy (miR-375 decoy transfected) cells for 24 h. MiR-375 expression was measured by qPCR and normalized to MΦ control ( n = 27) using different MCF-7 cell passages. Data of b and d – I are mean ± SEM and p -values were calculated using two-tailed Student’s t -test ( d , f , g – k ) and one-sample t -test ( e , l ). * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., not significant

Article Snippet: Primary human monocyte-derived MΦ were cocultured with MCF-7 breast carcinoma cells for 48 h or 100 nM resolvin D1 (Cayman Chemical, Michigan, USA) for 6 h. After 48 h, residual MCF-7 cells were removed from plates and cocultured, as well as resolvin D1-treated MΦ were collected for RNA isolation using the mirVana TM miRNA isolation kit (ThermoFisher Scientific, MA, USA) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Positive Control, Transfection, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

doi: 10.1101/2021.09.23.461605

Figure Lengend Snippet: (A) Superposition of the monomeric unit of M pro (in gray, PDB code 7K40) with FXa (on the left in violet, PDB code 2P16) and thrombin (on the right in purple, PDB code 1KTS). The crystallographic structure of apixaban into FXa structure, dabigatran into thrombin structure, and catalytic dyad of M pro (His-41 and Cys-145 residues) are as spheres in pink, cyan, and orange, respectively. For better interpretation the catalytic water (H 2 O cat ) of M pro is not shown. The enzymatic inhibition profile for apixaban, rivaroxaban, and dabigatran (0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, and 10 mM) into (B) PL pro (8.19 nM) and (C) M pro (88.8 nM) velocity. The positive controls GRL0617 (PL pro ) and GC376 (M pro ) were used under the same condition of anticoagulants. (D) Michaelis-Menten enzymatic mechanism for M pro without and in the presence of a fixed apixaban or atazanavir concentration (2.5 mM) for different substrate concentrations (0, 0.76, 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, and 100 mM). (E) Enzymatic scheme for the experimental mechanism of M pro inhibition by anticoagulants. Best docking pose (ChemPLP function) for the interaction between M pro (F) substrate, and (G) substrate-apixaban into the active site of protease. Best docking pose (ChemPLP function) for the interaction between the dimer interface of M pro (H) apixaban and rivaroxaban, while (I) shows the selected amino acid residues which interact with apixaban. Substrate, rivaroxaban, dabigatran, and apixaban are in stick representation in beige, green, cyan, and pink, respectively, while the catalytic water (H 2 O cat ) is in sphere. Elements’ color: hydrogen, nitrogen, oxygen, sulfur, and chloro are in white, dark blue, red, yellow, and dark green, respectively.

Article Snippet: Nevertheless, apixaban was about 5- and 60-fold less potent in vitro in comparison to the positive control atazanavir and remdesivir, respectively , indicating that apixaban shows an interestingly scaffold for the design of novel compounds to increase its antiviral action.

Techniques: Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

doi: 10.1101/2021.09.23.461605

Figure Lengend Snippet: Antiviral activity of anticoagulants, atazanavir, and remdesivir in Calu-3 cells (densities of 2.0 × 10 5 cells/well) infected with SARS-CoV-2 (MOI 0.1) in 96-well plates. The data is presented as (A) virus production (PFU/mL) and (B) percentage of viral replication inhibition. The data represent means ± SEM of three independent experiments.

Article Snippet: Nevertheless, apixaban was about 5- and 60-fold less potent in vitro in comparison to the positive control atazanavir and remdesivir, respectively , indicating that apixaban shows an interestingly scaffold for the design of novel compounds to increase its antiviral action.

Techniques: Activity Assay, Infection, Inhibition