Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: Generation of hsa-miR-24-3p is dependent on CR3 and TLR4. (a) The level of hsa-miR-24-3p 1 h after infection was higher in MEV Ca than in MEV. Blocking of CR3 or TLR4 on blood monocytes had a minor effect on hsa-miR-24-3p content in vesicles. However, when both CR3 and TLR4 were blocked, hsa-miR-24-3p was absent from vesicles. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. EVs were isolated from 10 6 human monocytes, and hsa-miR-24-3p levels were determined using qPCR. (b) Mmu-miR-24-3p content was significantly higher in REV Ca (vesicles from opsonized C. albicans -induced RAW 264.7 cells) than in REV (vesicles from untreated RAW 264.7 cells). The level of mmu-miR-24-3p was not elevated in CR3- or TLR4 -silenced RAW 264.7 cells. Data represent mean values ± SD, P = 0.0245 and P = 0.0166, unpaired two-tailed t test, n = 3 or 4 different experiments. (c) Candida -treated CD11b- and TLR4 -silenced RAW 264.7 cells generated the same amount of REV as nonsilenced cells. EVs isolated from the same numbers of cells were counted by DLSM. (d) CD11b and TLR4 colocalized on blood monocytes upon incubation with soluble β-glucan (sβG) and mannan but not on untreated cells, as observed by CLSM. Green, CD11b; red, TLR4. Bars, 10 μm. Data are representative of n = 3 independent experiments. (e) Colocalization of CD11b and TLR4 in the presence of soluble β-glucan (sβG) and mannan on monocytes, as confirmed by PLA. Red, CD11b–TLR4 complexes; blue, DNA. Bars, 10 μm. Data are representative of n = 3 experiments. (f) EVs isolated from sβG and mannan-treated human monocytes for 1 h (MEV sβG+mannan ) contained significantly more hsa-miR-24-3p than MEV. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. REV Ca but not REV Ca(TLR4 and CD11b silenced) induced significant growth (g) and hyphal filamentation (h) in C. albicans . EVs were isolated from opsonized C. albicans -infected or control RAW 264.7 cells (REV and REV Ca , respectively). EVs were also isolated from opsonized C. albicans - infected or control TLR4- and CD11b-silenced RAW 264.7 cells [REV (TLR4 and CD11b silenced) and REV Ca(TLR4 and CD11b silenced) , respectively]. Isolated EVs were counted by DLSM, and C. albicans was incubated with identical amounts of vesicles obtained from the indicated treatments.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Infection, Blocking Assay, Two Tailed Test, Isolation, Generated, Incubation

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: hsa-miRNA-24-3p acts across species boundaries (cross-kingdom) as it regulates C. albicans gene expression. C. albicans induces the release of miRNA-containing vesicles in human monocytes upon binding of mannan and soluble β glucan to TLR4 and CR3, respectively, followed by receptor colocalization. Subsequently, EVs are released from multivesicular bodies transporting miRNAs. EVs attach via CR1 on the vesicle to opsonized C. albicans . hsa-miRNA-24-3p but not hsa-miRNA-21-5p inhibits sol1 translation in C. albicans , leading to enhanced growth and filamentation of the fungus. Graphic created with BioRender.com.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Expressing, Binding Assay

Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 1. Expression of TLR4 mRNA in HPDLCs. Four passage HPDLCs were obtained as described in the ‘Materials and methods’. Total RNA was extracted, and the mRNA expression of TLR4 was analyzed by reverse transcriptase PCR (a). Levels of b-actin mRNA served as internal controls (b). Lanes: M, molecular size marker; 1, sample A; 2, sample B; 3, sample C; 4, negative controls. The results were representative of three different experiments.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Expressing, Reverse Transcription, Marker

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 2. Expression of TLR4 in HPDLCs detected by immunofluorescence staining. Four passage HPDLCs from three individual healthy subjects were obtained as described in the ‘Materials and methods’. After fixation, the cells were treated with monoclonal antibody to TLR4 (anti- TLR4 mAb) (green), and the nuclei were counterstained with PI (red) (a). The other cells were treated with PBS, which was substituted for anti- TLR4 mAb, and served as controls (b). Scale bars = 20 mm.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Expressing, Staining

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 3. Gene expression changes in HPDLCs stimulated with lipopolysaccharide analyzed by real-time PCR. HPDLCs were cultured in DMEM with or without 1 mg mL1 lipopolysaccharide for 6 h. cDNA was prepared. Real-time PCR was used to quantify TLR4 (a), IL-6 (b), Fos (c) and LY64 (d) mRNA expression levels. For each transcript, conventional PCR-amplified products were used as standards (from 1 100 to 1 106) to generate a standard curve for absolute real-time quantification. The absolute mRNA levels of all the genes were then normalized to b-actin levels of individual samples. Three different experiments were performed, and the representative results were shown. HPDLCs from three individual persons were pooled for each experiment. Lipopolysaccharide induced the expression of TLR4 and IL-6, while it repressed the expression of Fos and LY64.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Expressing

Journal: FEMS Immunology & Medical Microbiology

Article Title: Toll-like receptor 4 signaling plays a role in triggering periodontal infection

doi: 10.1111/j.1574-695x.2008.00386.x

Figure Lengend Snippet: Fig. 4. Secretion of IL-6 in HPDLCs stimulated with lipopolysaccharide in the absence or presence of anti-TLR4 mAb. (a) HPDLCs were plated at a density of 5 104 cells mL1 in 96-well plates. After 2 days, the cells were exposed to Escherichia coli lipopolysaccharide (100 ng mL1, 1 mg mL1, 10 mg mL1). Following incubation for various periods of time (6, 12, 24, 48 h) at 37 1C, the supernatants were harvested and assayed via ELISA for the concentration of IL-6. (b) HPDLCs (5 104 cells mL1 in 96-well plates) were incubated with 1 mg mL1 lipopolysaccharide for 24 h in the absence or presence of various titers of anti-TLR4 mAb (500–25-fold dilutions). The levels of IL-6 in the culture supernatants were measured by ELISA. P o 0.05, significantly different from levels in the absence of anti-TLR4 mAb. Three different experiments were performed, and each data point represents the mean SD. HPDLCs from three individual people were pooled for each experiment.

Article Snippet: The endogenous peroxidase signal was quenched by incubation in 1.5% H2O2 for 15 min. After 30 min of blocking with normal goat serum, the cells were incubated with mouse anti-TLR4 mAb (Serotec) at a dilution of 1 : 200 overnight at 4 1C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of cellular physiology

Article Title: Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression.

doi: 10.1002/jcp.21050

Figure Lengend Snippet: Fig. 1. EORP increases surface expression of TLR4 and CD14 within J774A.1 cells. A: J774A.1 cells were incubated with one of EORP (25 mg/ml), LPS (1 mg/ml), fucoidan (25 mg/ml), F1 fraction of Reishi (25 mg/ml) or F2 fraction of Reishi (25 mg/ml) for 24 h, fixed with 2% paraformaldehyde, followedbystainedwithPE-conjugatedTLR4orCD14antibodyfor30min,thenanalyzedbyflowcytometry.Dottedline:isotypecontrolantibody; shade histograms: treatment as indicated. The histograms were quantified and represent as mean fluorescence intensity (MFI). B: J774A.1 cells were incubated with medium or EORP (25 mg/ml) for 24 h, followed by stained with PE-conjugated TLR4 or CD14 antibody, then analyzed by confocal microscope. C: J774A.1 cells were labeled with BODIPY TR C5-ceramide (1 mM) at 4-C for 30 min followed by fixed with 2% paraformaldehydeandpermeatedwith0.1%TritonX-100,cellswerestainedwithFITC-conjugatedTLR4orCD14antibody,andthenanalyzedby confocal microscope. Merged images of Golgi marker and TLR4 or CD14 are indicated by arrows. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Article Snippet: Anti-rabbit IgG-HRP, anti-mouse IgG-HRP, and anti-IL-1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-mouse TLR4 blocking antibody and PE-conjugated anti-mouse TLR4 antibody were obtained from IMGENEX Corporation (Carlsbad, CA); PE-conjugated antimouse CD14 antibody was obtained from BD Biosciences (Mountain View, CA); anti-mouse macrophage scavenger receptor (MSR) antibody (2F8) and FITC-conjugatedMSR antibodywere obtained from Serotec, Inc. (Oxford, UK).

Techniques: Expressing, Incubation, Staining, Microscopy, Labeling, Marker

Journal: Journal of cellular physiology

Article Title: Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression.

doi: 10.1002/jcp.21050

Figure Lengend Snippet: Fig. 8. The proposed mechanism of Ganoderma lucidum polysaccharides enhances the modulation of CD14/TLR4-mediated endocytosis and signaling in IL-1 cytokine expression.

Article Snippet: Anti-rabbit IgG-HRP, anti-mouse IgG-HRP, and anti-IL-1 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-mouse TLR4 blocking antibody and PE-conjugated anti-mouse TLR4 antibody were obtained from IMGENEX Corporation (Carlsbad, CA); PE-conjugated antimouse CD14 antibody was obtained from BD Biosciences (Mountain View, CA); anti-mouse macrophage scavenger receptor (MSR) antibody (2F8) and FITC-conjugatedMSR antibodywere obtained from Serotec, Inc. (Oxford, UK).

Techniques: Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Mapping of the TLR4/MD2/RAGE-interacting motifs of HMGB1 or PTX3. (A, B) Schematic diagram of the domain of HMGB1 (A) and PTX3 (B) (top). GST-tagged truncates covering TLR4-, RAGE-, or MD2-binding domain of HMGB1 or PTX3 were constructed. Bottom panel (A, B) depicts GST-pull down of the indicated tagged receptors (HA-TLR4, Myc-RAGE, and His-MD2) with transiently expressed GST-tagged truncates. HEK293T cells transfected with the GST-tagged constructs in combination with HA-TLR4, Myc-RAGE or His-MD2 are indicated followed by GST-pull down. Representative western blots of WCLs and eluates after GST-pull down are shown, using GAPDH as a loading control in WCLs. Based on pulldown binding results, TLR4-, MD2-, and RAGE-targeted peptides were constructed and designated as H-T, H-R, P-T, or P-M peptides (indicated in panels A, B). (C) BMDMs were incubated with H-T, H-R, P-T, or P-M peptide for the indicated concentrations for 48 h, and then cell viability was measured with cell viability assay. Data shown are the means ± SD of three experiments (D) Cell-free immunoprecipitation assay using H-T, H-R, P-T, or P-M peptides. Mix 5 μg of alarmin protein (rHMGB1 or rPTX3) with 5 μg of each indicated tagged receptors protein (HA-TLR4, Myc-RAGE, and His-MD2), and mix as indicated to confirm the competitive inhibition effect of peptides was treated for 4 h. They were immunoprecipitated with α Flag for 2 h, separated by SDS-PAGE, and evaluated by immunoblotting. (E) Competition between rHMGB1 (top) or PTX3 (bottom) and the constructed peptides for their respective receptors. After treatment with rHMGB1 (top) or PTX3 (bottom) (10 μg/mL, 3 h) together with increasing amounts of the indicated peptides, BMDMs were used for immunoprecipitation (IP) with α TLR4 or α RAGE and IB with indicated antibodies. (F) Decrease of LPS-induced cytokine production by H-T, P-T, or P-M peptide and rHMGB1-induced cytokine production by H-R peptide. TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) together with increasing amounts of the indicated peptides-treated BMDMs were determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), bars denote mean ± SEM (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001), and significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay, Construct, Transfection, Western Blot, Control, Incubation, Viability Assay, Immunoprecipitation, Inhibition, SDS Page, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Extracellular HMGB1 and PTX3 contribute to inflammatory responses by interacting with TLR4 or RAGE in murine macrophages. (A, B) Release of HMGB1 (A) or PTX3 (B) from murine macrophages after stimulation with LPS. LPS-induced HMGB1 (A) or PTX3 (B) release from murine macrophages. Immunoblots (IB) for HMGB1 and PTX3 in the supernatant and cell lysates in LPS-treated BMDMs at indicated time points after LPS (100 ng/mL). (C, D) Interaction between HMGB1 (C) or PTX3 (D) with its receptors. HEK293T cells were transfected with constructs, and cell lysates were immunoprecipitated with a flag-specific antibody and were analyzed by immunoblotting with the indicated antibodies. BMDMs were incubated with the rFlag-HMGB1 (C) or rFlag-PTX3 (D) 10 μg/mL for 3 h, and cell lysates were immunoprecipitated with α TLR4 or α RAGE (C, right), and α TLR4 or α MD-2 (D, right). Immunoprecipitants and WCL were analyzed by immunoblotting with the indicated antibodies. (E) TNF- α , IL-6, IL-12p40, and IL-10 levels in the supernatant of LPS (100 ng/mL, 18 h) or recombinant alarmins (rHMGB1 or rPTX3, each (2, 5 μg/mL))-treated BMDMs determined by ELISA. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: Inhibitory effects of TMR-Lipo on TLR4/RAGE-mediated responses. (A) LPS-primed BMDMs (100 ng/mL LPS with 12 h) were treated with TMR-Lipo (0.1, 1, 5 μmol/L) for 12 h, followed by co-IP with α TLR4, α RAGE, or α MD2 and IB with indicated antibodies. (B) Schematic representation of TLR4 and RAGE signaling pathway and therapeutic targeting of TMR-Lipo (C) BMDMs were treated with TMR-Lipo as indicated concentrations (2 h), followed by treatment with LPS (100 ng/mL, 8 h). The amounts of HMGB1, PTX3, MyD88, IRAK, TRAF6, NF- κ B, KRAS, and p-p38 were measured by IB in the whole cell lysates. GAPDH was used as a loading control. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant. (D) BMDMs were treated with TMR-Lipo (0.1, 0.5, 5, 20, or 100 μmol/L) in the presence LPS for 18 h. The secretion levels of TNF- α , IL-6, IL-12p40 and IL-10 were measured by ELISAs. The data shown represent at least three independent experiments ( n ≥ 3), and bars denote mean ± SEM (∗∗ P < 0.01, ∗∗∗∗ P < 0.001), significance was measured by two-way ANOVA. n.s = not significant.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dual alarmin-receptor-specific targeting peptide systems for treatment of sepsis

doi: 10.1016/j.apsb.2024.08.015

Figure Lengend Snippet: TMR-Lipo-Abs protects mice from CLP-induced polymicrobial sepsis. (A–C) Schematic design of mid-grade (A) or high-grade (B, C) sepsis mouse model induced by cecal ligation and punctuation (CLP) depending on the position of the cecal ligation (top). The survival of CLP mice treated with the indicated therapy was monitored for 10 days (A) or 72 h (B, C); mortality was measured for n = 10 mice per group (bottom). Statistical differences compared with the PBS or TMR-Lipo-Abs-treated mice are indicated (log-rank test). The data are representative of three independent experimental replicates with similar results. (D) Serum cytokine levels. The data shown represent at least three independent experiments ( n ≥ 3), significance was measured by Ordinary one-way ANOVA (TNF- α , ∗∗ P = 0.0010 and ∗∗∗ P = 0.0002, IL-6, ∗∗ P = 0.0057 and ∗∗∗ P = 0.0004 and IL12p40, both ∗∗∗∗ P < 0.0001). n.s = not significant. (E) IB identification and comparison of HMGB1 and PTX3 expression in spleen, lung, and liver cells of CLP mice after different treatment as a DAMP indicator for tissue damage. (F) Representative H&E staining of the lung, spleen, and liver from 3 mice per group. Scale bar, 100 μm.

Article Snippet: Flag-HMGB1 (MG50913-CF), Flag-PTX3 (HF12082-CF), HA-TLR4 (MG50657-NY), Myc-RAGE (MG50489-CM), and His-MD2 (MG51098-NH) plasmids were purchased from Sino Biological (Beijing, China).

Techniques: Ligation, Comparison, Expressing, Staining

Journal: Journal of Innate Immunity

Article Title: Macrophage-Biomimetic Nanoparticles Ameliorate Ulcerative Colitis through Reducing Inflammatory Factors Expression

doi: 10.1159/000519363

Figure Lengend Snippet: Inflammation-targeting MM-PLGA-TAS in RAW264.7 cells and colitis mice. a RAW264.7 cells show increased effective uptake of nanoparticles in the inflammatory state. After treating RAW 264.7 cells with LPS and TNFα (or DMSO as negative controls) for 12 h, cells were treated with PLGA-TAS/DIR or MM-PLGA-TAS/DIR for 6 h and photographed using fluorescence microscopy. DAPI was used to label cell nuclei. Scale bars, 20 μm. PLGA-TAS/DIR or MM-PLGA-TAS/DIR were orally administrated to healthy controls and colitis mice for 12 h, and then mice ( b ) or gastrointestinal tissues ( c ) images were obtained using an IVIS. d Representative fluorescent of normal mice and DSS-induced mice, which were treated with PLGA-TAS/DIR or MM-PLGA-TAS/DIR. The percent of positive cells was measured. Scale bars, 100 μm. Two-tailed unpaired t test. * p < 0.05, ** p < 0.01, *** p < 0.001. NC, negative control; TAS, tasquinimod; PLGA, poly-lactic acid-glycolic acid; MM, macrophage membrane; DIR, infrared dye; DSS, dextran sulfate sodium; LPS, lipopolysaccharide; DAPI, 4-, 6-diamidino-2-phenylindole; IVIS, in vivo imaging system.

Article Snippet: For extracellular staining, RAW264.7 cells were incubated with anti-TLR4 (FAB2759P; R&D Systems) antibody or an isotype control antibody combination at 4°C for 30 min, followed by washing and analysis on a BD FACSCalibur.

Techniques: Fluorescence, Microscopy, Two Tailed Test, Negative Control, Membrane, In Vivo Imaging