tlr2 plasmid tlr2 plasmid  (Addgene inc)

Journal: PLOS ONE

Article Title: ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation

doi: 10.1371/journal.pone.0292340

Figure Lengend Snippet: Raw264.7 macrophages were transfected with TLR2-YFP (green) for 48 h. After that, the transfected cells were stimulated with Pam2CSK4 (1 μg/ml) for 1 h. Acidification of endosomes/lysosomes was then determined by adding the Lysotracker Red (LTR) dye to these cells in the presence or absence of ECDD-S16 and incubation was continued for 2 h. The treated cells were then fixed and processed for CLSM image analysis. (A) Confocal microscopy showing the colocalization of TLR2-YFP and LTR. Scale bar is 10 μm. (B) The co-localization of TLR2-YFP and LTR was quantified. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001.

Article Snippet: TLR2 plasmid (pcDNA3-TLR2-YFP) was a gift from Doug Golenbock (Addgene plasmid # 13016; http://n2t.net/addgene:13016 ; RRID:Addgene_13016).

Techniques: Transfection, Incubation, Confocal Microscopy, Comparison

Journal: PLOS ONE

Article Title: ECDD-S16 targets vacuolar ATPase: A potential inhibitor compound for pyroptosis-induced inflammation

doi: 10.1371/journal.pone.0292340

Figure Lengend Snippet: Raw264.7 macrophages were transfected with TLR2-YFP (green) for 48 h. After that, the transfected cells were stimulated with Pam2CSK4 (1 μg/ml) for 1 h. Acidification of endosomes/lysosomes was then determined by adding the Lysotracker Red (LTR) dye to these cells in the presence or absence of ECDD-S16 and incubation was continued for 2 h. The treated cells were then fixed and processed for CLSM image analysis. (A) Confocal microscopy showing the colocalization of TLR2-YFP and LTR. Scale bar is 10 μm. (B) The co-localization of TLR2-YFP and LTR was quantified. All data were determined by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001.

Article Snippet: Raw264.7 cells were transiently transfected with TLR2 plasmid (5 μg) using Nucleofector solution kit V (Amaxa, London, UK) according to the manufacturer’s instructions.

Techniques: Transfection, Incubation, Confocal Microscopy, Comparison

Journal: bioRxiv

Article Title: Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

doi: 10.1101/2023.09.06.556449

Figure Lengend Snippet: ( a ) Principal component analysis of lipid content from phagosomes containing Pam3csk4-beads (Pam3-phag, blue) relative to phagosomes containing uncoupled-beads (control-phag, grey) isolated from immortalized bone marrow derived macrophages (iBMDM) in a representative experiment, n=4. ( b ) Fold change of MS/MS values of different lipid species in phagosomes containing Pam3-phag relative to control-phag isolated from iBMDM. Lipid species determined by lipidomics were aggregated per lipid class and each dot represents the cumulative value in n=4 independent experiments. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; Cer, Ceramide; DG, diacylglycerol; TG, triacylglycerol. ( c , d ) RAW264.7 cells stably expressing the PS-probes Venus-LACT-C2 or Venus-FVIII-C2 were fed BSA-beads, BSA-beads coupled with anti-BSA antibody (IgG-BSA-beads), or Pam3csk4-beads (Pam3-beads) for 30min and enrichment of PS-probe to phagosome membranes relative to cytosolic signal was determined by immunofluorescence. Each dot represents a phagosome (n>10) in a representative experiment, n=2. ( e ) Cumulative MS/MS peak area of PC and PS of Pam3-phag from wild-type (WT) and TLR2-KO iBMDM determined by lipidomic analysis. Replicates in a representative experiment, n=2. ( f ) Scheme of mouse TLR2 (Uniprot: Q9QUN7) depicted in a membrane. Transmembrane domain is highlighted in yellow; acidic and basic amino acid are colored in red and blue, respectively; and Toll-Interleukin receptor (TIR) domain is boxed in magenta. Numbers indicate amino acid position showcasing the basic patch ( 628 KRKPKK 6 ). ( g - j ) RAW264.7-sg-TLR2-KO cells stably expressing the PS-probe Venus-LACT-C2 ( g , h ) or Venus-LC3 ( i, j ) were transduced to express TLR2 full length or lacking the TIR domain (TLR2ΔTIR), in either wild-type (WT), K628E-R629D-K630E-K632E-K633E (ACID), P631H (corresponding to human SNP rs5743704), or P681H mutant TLR2. Transduction with empty vector (e.v.) served as a negative control. Cells were fed Pam3csk4-beads (30min, g, h; 1h, i, j) and Venus-LACT-C2 or Venus-LC3 translocation to phagosomes was determined by immunofluorescence. (g, i) Representative confocal images and (h, j) violin-plots of PS-probe enrichment at the phagosome membrane relative to cytosolic signal (n>20 phagosomes) in one representative experiment, n=2. ** P < 0.01, **** P < 0.001, *** P < 0.0001 by two-sided Student’s t test.

Article Snippet: TLR2 cDNA was a kind gift from Dr. Ruslan Medzhitov (Addgene#13083).

Techniques: Control, Isolation, Derivative Assay, Tandem Mass Spectroscopy, Stable Transfection, Expressing, Immunofluorescence, Membrane, Mutagenesis, Transduction, Plasmid Preparation, Negative Control, Translocation Assay

Journal: bioRxiv

Article Title: Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

doi: 10.1101/2023.09.06.556449

Figure Lengend Snippet: ( a ) PS-bead pull-down of TLR2 intracellular domain (TLR2-ID). Uncoupled-beads (Ctrl-beads), PS-beads, in combination with wild-type (WT), K628A-R629A-K630A-K632A-K633A (ALA), and K628E-R629D-K630E-K632E-K633E (ACID) versions were used as indicated, blots representative of n=4. ( b - f ) Glass-supported lipid bilayer resembling plasma membrane composition were incubated with TLR2-ID (b-c), cytosolic tails of mCD16 (d-f) or mTIM4 (d) as indicated, and top-Fluor-PS clustering was determined by immunofluorescence. (b, e) Representative images or (c, d, f) area of clustered PS quantified in different fields from representative experiments, n=2. (b-d) Anti-FLAG or streptavidin allows peptide manipulation and disrupts lipid clustering. **** P < 0.001 by two-sided Student’s t test.

Article Snippet: TLR2 cDNA was a kind gift from Dr. Ruslan Medzhitov (Addgene#13083).

Techniques: Clinical Proteomics, Membrane, Incubation, Immunofluorescence

Journal: bioRxiv

Article Title: Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis

doi: 10.1101/2023.09.06.556449

Figure Lengend Snippet: ( a, b ) RAW264.7 cells were treated with ionomycin (10μM, 30min) combined with anti-phosphatidylserine (PS) antibody (1:50) or isotype control (anti-FLAG, 1:50) and fed Zymosan (30min). Representative confocal images (a) and violin-plots (b) of endogenous Rubicon enrichment at the phagosome membrane relative to cytosolic signal (n>40 phagosomes). ( c ) RAW264.7-sg-TLR2-KO cells were transduced to express full length TLR2 or TLR2 lacking the TIR domain (TLR2τιTIR), in either wild-type (WT), K628E-R629D-K630E-K632E-K633E (ACID), P631H (corresponding to human SNP rs5743704), or P681H mutant TLR2. Transduction with empty vector (e.v.) serves as a negative control. Cells were fed Pam3csk4-beads (30min) and Rubicon translocation was determined by immunofluorescence. Violin-plots showing the enrichment of Rubicon at the phagosome membrane relative to the cytosolic level (n>20 phagosomes). ( d, e ) RAW264.7-Rubicon-KO cells stably expressing the PS-probe Venus-FVIII-C2 and mCherry-Rubicon were fed Zymosan and analyzed by stochastic optical reconstruction microscopy (STORM). (d) Super-resolution image showing a representative membrane portion of a Zymosan-containing phagosome and (e) statistical index in super-resolution images assessing the proximity of PS-probe and Rubicon at phagosome membranes over time. Data are means ± SD of >6 biological replicates (phagosomes) in one representative experiment. ( f ) Lipid strip showing the lipid-binding specificity of full-length Rubicon recombinantly produced in insect cells, n=2. ( g ) In vitro binding competition assays of proteins in Pam3csk4-beads containing phagosomes isolated from RAW264.7-Rubicon-KO cells. Phagosomes were incubated with Lactadherin-FITC (Lact-FITC) and/or GST-Rubicon D1-644 as indicated. After anti-GST-Cy3 staining, phagosomes were quantitively analyzed by microscopy for green (Lactadherin binding) or red signal (GST-Rubicon Δ1-644 binding). Violin plots depict n>30 phagosomes. ** P <0.01, *** P <0.001, **** P <0.0001 by (b, g) two-sided Student’s t test or (c) ANOVA test (pairwise comparations Fisher’s LSD).

Article Snippet: TLR2 cDNA was a kind gift from Dr. Ruslan Medzhitov (Addgene#13083).

Techniques: Control, Membrane, Mutagenesis, Transduction, Plasmid Preparation, Negative Control, Translocation Assay, Immunofluorescence, Stable Transfection, Expressing, Microscopy, Stripping Membranes, Binding Assay, Produced, In Vitro, Isolation, Incubation, Staining