Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 6. Lyn is essential to the activation of PI 3-kinase through phosphorylation of its p110 subunit. (A; Supplementary movies M1–M4) HEK 293–TLR2 cells were transfected with a fusion protein combining GFP and the PH domain of AKT. Transfected cells were either co-transfected with LynDK (500 ng/ml) or incubated with PP2. Cells were then stimulated with Pam3 (100 ng/ml) and the kinetics of the AKT–PH construct was observed by microvideoscopy using Zeiss Axiovert inverted microscope equipped with the Metafluor imaging system. Presented here are the images corresponding to 15 min of Pam3 stimulation. Controls correspond to cells incubated with DMSO and pcDNA empty vector, or cells incubated with LY294002 (25 mM), a specific inhibitor of PI 3-kinase. (B) HEK 293–TLR2 cells were transfected with pcDNA vector (vehicle) or LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Lysates were immunoprecipitated with anti-Flag Abs and recruitment of PI 3-kinase to TLR2 was observed by Western blot with anti-p85a Abs. (C) Tyrosine phosphorylation of the p85a subunit was evaluated by Western blot in HEK 293–TLR2 transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Anti-phosphotyrosine (4G10 clone) and anti-p85a Abs were used for immuno- precipitation and Western blot. (D) HEK 293–TLR2 were transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml) and lysates were immunoprecipitated with either 4G10 or anti-p110 Abs. Phosphorylation of p110 was then revealed by Western blot with anti-p110 or 4G10 Abs. Controls correspond to cells transfected with pcDNA empty vector. (E) THP1–CD14 cells were incubated with PP2 (25mM) or DMSO, stimulated with Pam3 (100 ng/ml) and lysed. Phosphorylation of p110 catalytic subunit of PI 3-kinase was revealed by Western blot of THP1–CD14 lysates immunoprecipitated with either 4G10 or anti-p110 Abs. Controls correspond to cells treated with DMSO. These results are representative of three independent experiments.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Transfection, Incubation, Construct, Inverted Microscopy, Imaging, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 7. Lyn controls the PI 3-kinase pathway through phosphorylation of its p110 subunit after TLR2 engagement. The presence of bacteria-derived acylated lipoproteins results in the heterodimerization of TLR2 with TLR1/6 in membrane microdomains. MyD88/ IRAK are recruited to the receptor and lead to degradation of IkB and translocation of NF-kB subunits. A Rac/PI 3-kinase-dependent signaling pathway has also been described, including CD14 and Lyn that contribute to the activation cluster. After tyrosine phos- phorylation of the cytoplasmic domain of TLR2, the p85a subunit of PI 3-kinase is recruited to the receptor and phosphorylated on tyrosine. A Lyn-dependent tyrosine-phosphorylation is required to activate the PI 3-kinase catalytic subunit p110 that allows for recruitment of AKT to the inner membrane, precluding a cascade that results in transactivation of the p65 subunit of NF-kB. This leads to the nuclear translocation of a functional p50/p65 NF-kB heterodimer that results in the gene expression of pro-inflammatory cytokines.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Bacteria, Derivative Assay, Membrane, Translocation Assay, Activation Assay, Functional Assay, Gene Expression

Journal: Advanced Science

Article Title: Therapeutic Reprogramming of Glioblastoma Phenotypic States Using Multifunctional Heparin Nanoparticles

doi: 10.1002/advs.202509590

Figure Lengend Snippet: Preclinical characterization of HP‐NPs in IDH WT glioblastoma patient avatars. A) representative live bioluminescence imaging of BT12 patient derived GB stem cells expressing luciferase, implanted in the mouse brain striatum. Imaging performed seven days post implantation (D7) and five days prior to the treatment initiation (T‐5). B) representative live bioluminescence imaging of BT12 patient derived GB stem cells expressing luciferase, implanted in the mouse brain striatum. Imaging performed nineteen days post‐implantation (D19) after three rounds of caudal vein infusions (T7) with vehicle saline (n = 3, 200 µL), doxorubicin (n = 3, 200 µL, 1.5 mg kg −1 ), heparin nanoparticles (HP‐NP, n = 6, 200 µL, 5 mg kg −1 ) or doxorubicin‐loaded heparin nanoparticles (HP‐DOX‐NP, n = 6, 200 µL, 5 mg kg −1 ). C) representative live bioluminescence imaging of BT12 patient derived GB stem cells expressing luciferase, implanted in the mouse brain striatum. Imaging performed after twenty‐eight days (D28) and six rounds of caudal vein infusions (T16) with vehicle saline (n = 3, 200 µL), doxorubicin (n = 3, 200 µL, 1.5 mg kg −1 ), heparin nanoparticles (HP‐NP, n = 6, 200 µL, 5 mg kg −1 ) or doxorubicin‐loaded heparin nanoparticles (HP‐DOX‐NP, n = 6, 200 µL, 5 mg kg −1 ). D) quantification of the intravital photon counts on day 28. Vehicle patient avatars (n = 3), doxorubicin (DOX) 1.5 mg kg −1 (n = 3), HP‐NPs (5 mg kg −1 , n = 6) and HP‐DOX‐NP (n = 6). E) tumor volume determined from histological sections (ten per mouse) of mouse brain avatars treated with vehicle (n = 3), DOX (n = 3), HP‐NP (n = 5) and HP‐DOX‐NP (n = 6). F) representative immunofluorescence pathology micrographs of Vehicle, DOX, HP‐NP, and HP‐DOX‐NP treated patient avatars brain section labelled for human vimentin (hVIM, white), mouse endothelial podocalyxin (PODXL, magenta) and DAPI (blue). G) close‐up micrograph of the typical pathology for BT12 patient avatars, featuring enlarged tumor blood vessels (top right insert) devoid of necrotic features (bottom image) or nuclear atypia (arrow‐pointing insert). H) close‐up micrograph of the typical pathology for BT12 patient avatars treated with doxorubicin, featuring cell and blood‐vessel‐free intratumoral areas (top right insert) and presenting necrotic features (bottom image) with pyknotic‐like nuclei (arrow‐pointing yellow insert) when compared to non‐necrotic tumor areas (arrow‐pointing white insert). I) close‐up micrograph of the typical pathology for BT12 patient avatars treated with HP‐NP, featuring smaller tumors devoid of cellular atypia cell and blood‐vessel‐free intratumoral areas (top right insert) and presenting necrotic features (bottom image) with nuclear atypia (arrow‐pointing yellow insert) when compared to non‐necrotic tumor areas (arrow‐pointing white insert). J) close‐up micrograph of the typical pathology for BT12 patient avatars treated with HP‐DOX‐NP, featuring histological discontinuity associated with pyknotic nuclei (yellow insert) compared to unaffected tumor areas (white insert)and blood‐vessel‐free intratumoral areas (top right insert) and presenting necrotic features (bottom image) with nuclear atypia (arrow‐pointing yellow insert) when compared to non‐necrotic tumor areas (arrow‐pointing white insert). K) quantification of necrotic features, data presented as necrotic area (µm 2 ) averaged from two brain sections per vehicle, DOX, HP‐NP, and HP‐DOX‐NP treated patent avatars. Panels D, E, and K: statistical significance was determined by one‐way ANOVA with Kruskal‐Wallis post hoc test for multiple comparisons of treatment versus vehicle. L) Representative fluorescent micrographs for doxorubicin autofluorescence (470/595 nm) in Vehicle, DOX, HP‐NP, and HP‐DOX‐NP treated patient avatars brain section counterstained with DAPI.

Article Snippet: Monoclonal mouse anti‐human vimentin Cy3 conjugate (C9080, MilliporeSigma, 1:900); monoclonal rat anti‐mouse podocalyxin (MAB1556, R&D Systems, Bio‐Techne, 1:800) and mouse monoclonal anti human HBEGF (MAB2591, R&D Systems 1:200)

Techniques: Imaging, Derivative Assay, Expressing, Luciferase, Saline, Immunofluorescence

Journal: Biology

Article Title: A Replication-Competent Flavivirus Genome with a Stable GFP Insertion at the NS1-NS2A Junction

doi: 10.3390/biology15030220

Figure Lengend Snippet: Western blot analysis of intracellular proteins in cells harboring YFV or YFV-derived replicons. ( a ) The photograph shows a membrane from a two-color immunoblot of cell lysates stained with a polyclonal mouse anti-YFV antiserum (red channel, 700 nm) and an anti-GFP antibody (green channel, 800 nm). Imaging was performed on a LI-COR Odyssey system. Lane designations: 1, Naive BHK-21 cells (uninfected control); 2, BHK-21 cells infected with wild-type YFV; 3, Packaging cells harboring the YFrep/GFP/prME replicon, expressing the 25C-GFP-2A fusion protein; 4, Packaging cells harboring the YFrep/NS1-GFP replicon, expressing the NS1-GFP fusion protein; 5, Protein marker (Precision Plus Protein Kaleidoscope, Bio-Rad #1610375); only marker bands detected in the 700 nm channel are visible. Molecular weights (in kDa) of the protein marker are indicated on the right. Key findings: YFV-specific proteins (red bands) detected in lanes 2–4: NS5 (104 kDa), NS3 (69.2 kDa), NS1 (39.7 kDa) and prM (18.7 kDa). GFP-containing fusion proteins (green bands) were detected in lanes 3 and 4. Lane 3: The band corresponding to the 25C-GFP-2A fusion protein (~31.5 kDa) is indicated by an asterisk (★). Lane 4: The band corresponding to the full-length NS1-GFP fusion protein (~68.4 kDa) is indicated by a double green arrow (⇒). In lanes 3 and 4, circles (◯) mark unidentified GFP-containing bands at approximately 20 kDa and 60 kDa, respectively, likely representing degradation or proteolytic fragments. ( b ) Positions of potential proteolytic cleavage products should the NS1-GFP fusion protein (from the YFrep/NS1-GFP replicon) be cleaved, releasing full-length NS1 (39.7 kDa, red triangle) and the C-terminal fragment (marked ‘GFP-10aa’, 28.7 kDa, green triangle).

Article Snippet: GFP was detected using a DyLight800-conjugated goat anti-GFP antibody (Rockland Immunochemicals, 600-145-215) at a 1:5000 dilution.

Techniques: Western Blot, Derivative Assay, Membrane, Staining, Imaging, Control, Infection, Expressing, Marker

Journal: PLoS ONE

Article Title: Roadmap for the use of base editors to decipher drug mechanism of action

doi: 10.1371/journal.pone.0257537

Figure Lengend Snippet: A . Western Blot for Cas9 expression levels in three cell lines: Wild-type HEK293T, HEK293T + dCas9-AID*Δ as a pool, and the single cell clone of HEK293T + dCas9-AID* selected for the highest expression of Cas9. Housekeeping protein vinculin is used as reference. Blots were cropped where indicated by the arrow. Full uncropped blots are provided in . B . Clone HEK293T dCas9-AID*Δ was infected with 6xCRE-mCherry reporter system and single cell clones were evaluated. The most homogeneous clone for mCherry expression was selected. mCherry red fluorescence was measured with high content imaging using a cell incubator imaging system. Cells treated with and without isoproterenol were monitored for 30 hours after stimulation. C . mCherry fluorescence intensity of the same single cell clone as panel B, 24 hours after isoproterenol treatment or in its absence. D . B2AR and B1AR mRNA expression 72h after transfection of 10pmol of siRNA. The mean of 4 replicates is shown. E , Left, mCherry fluorescence intensity of cells transfected with control siRNA or siRNA against B2AR. Right, mCherry fluorescence intensity of cells transfected with control siRNA or siRNA against B1AR. In both cases, cells were stimulated with isoproterenol after siRNA treatment. F . mCherry fluorescence intensity of knock-out cells for B2AR, with and without stimulation of isoproterenol.

Article Snippet: Clones were tested for Cas9 expression by Western Blot in Peggy Sue (Protein Simple).

Techniques: Western Blot, Expressing, Infection, Clone Assay, Fluorescence, Imaging, Transfection, Control, Knock-Out

Journal: Frontiers in Oncology

Article Title: Guanylate cyclase-C Signaling Axis as a theragnostic target in colorectal cancer: a systematic review of literature

doi: 10.3389/fonc.2023.1277265

Figure Lengend Snippet: GUCY2C-associated studies included in the systematic review.

Article Snippet: • Vaccine: Investigating a prime-boost approach involving a chimeric adenoviral vector (Ad5.F35) engineered to overcome pre-existing immunity, followed by recombinant Lm to enhance immune response toward the gastrointestinal cancer antigen GUCY2C. , • Vaccines: Ad-GUCY2C • Adenovirus expressing mouse GUCY2C1-429 fused to the influenza HA107-119 CD4+ T-cell epitope • Lm-GUCY2C and Lm-LacZ • mouse macrophage cell line J774A.1 (ATCC) • BALB/cJ mice , • In vitro infections • Immunizations • Ad5-neutralizing immunity studies • IFNγ ELISpot assay • Intracellular cytokine staining • In vivo tumor studies • Safety studies • Blood chemistry and cytokine analyses • Western blot , • Immunization with both heterologous Ad-GUCY2C and Lm-GUCY2C enhances CD8+ T-cell responses specific to GUCY2C and boosts antitumor immune activity. • Previous exposure to Ad5 restricts the effectiveness of Ad5.F35+Lm immunization protocols, whereas prior Lm exposure does not impose such limitations. • Alterations in the qualitative characteristics of the CD8+ T-cell population after a prime-boost vaccination regimen. • Lm-GUCY2C could potentially be employed to enhance GUCY2C-specific immune responses in patients undergoing clinical trials with adenovirus-based GUCY2C vaccines, aiming to prevent or manage recurrent GI cancer. , ( ) .

Techniques: Amplification, Expressing, Quantitative RT-PCR, Radioactivity, Activity Assay, Modification, Purification, Labeling, In Vivo, Mouse Assay, In Vitro, Clinical Proteomics, Viability Assay, Release Assay, Fluorescence, Staining, Immunofluorescence, Enzyme-linked Immunospot, Luciferase, Cell Culture, Activation Assay, Histopathology, Immunohistochemistry, Drug discovery, Western Blot, Isolation, Sequencing, Transduction, Variant Assay, Generated, Ex Vivo, Conjugation Assay, Flow Cytometry, Cytotoxicity Assay, Injection, Imaging, Mutagenesis, Protein-Protein interactions, Knock-Out, Transformation Assay, Plasmid Preparation, Control, Neutralization, Vaccines, Marker, Cell Isolation, Adoptive Transfer Assay, Transgenic Assay, Binding Assay, Cytotoxic T Lymphocyte Assay, Animal Model, Digital PCR, Gene Expression, Positron Emission Tomography, Comparison, Stability Assay, Immunopeptidomics, Recombinant, RNA Sequencing, Cloning, CRISPR, Chromatin Immunoprecipitation