Journal: International Journal of Molecular Sciences

Article Title: Modeling Neuroimmune Interactions in Human Subjects and Animal Models to Predict Subtype-Specific Multidrug Treatments for Gulf War Illness

doi: 10.3390/ijms22168546

Figure Lengend Snippet: Human blood cytokine levels in PTSD-based GWI subtypes as compared to HC at rest (T0) and peak exercise during a maximal graded exercise challenge (T1). Individual cytokine values are normalized to the HC values, and bars represent the mean ratios ± SEM for each cytokine. Significance was set at * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared to HC. The false discovery rate, as calculated via Storey et al. , was < 0.1 for all significant measures. Values are found in .

Article Snippet: Briefly, serum samples were analyzed using Q-PlexTM Mouse Cytokine Screen (16-plex), Quansys Imager, and Quansys reagents (Quansys Biosciences, Logan, Utah).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Modeling Neuroimmune Interactions in Human Subjects and Animal Models to Predict Subtype-Specific Multidrug Treatments for Gulf War Illness

doi: 10.3390/ijms22168546

Figure Lengend Snippet: Cytokine expression in a long-term mouse model of Gulf War Illness. ( A ) Graphical representation of the animal dosing paradigm. Mice were exposed to corticosterone (CORT; 200 mg/L in 0.6% ethanol) for 4 days at a time every other week for a total of 11 weeks. DFP (4 mg/kg, i.p.) was given on Day 5 and the LPS challenge (0.5 mg/kg, s.c.) was given after the final week of CORT. ( B ) Individual serum cytokine values normalized to the water (H2O), saline solution (SL), and SL condition values. Bars represent the mean ratios ± SEM for each cytokine. Significance was set at p < 0.05 compared to H2O SL SL (*), and within challenge groups (&: H2O DFP SL vs. H2O DFP LPS; and #: CORT DFP SL vs. CORT DFP LPS). The false discovery rate, as calculated via Storey et al. , was <0.1 for all significant measures. ( C ) Cytokine mRNA was measured in the frontal cortex ( n = 2–5/group) by qRT-PCR 6 h following exposure to LPS. Bars represent the mean ± SEM. Significance was set at p ≤ 0.05 for comparisons within challenge group (*: DFP vs. CORT DFP) or across challenge group (#: SL vs. LPS). Values for ( B , C ) are found in , respectively.

Article Snippet: Briefly, serum samples were analyzed using Q-PlexTM Mouse Cytokine Screen (16-plex), Quansys Imager, and Quansys reagents (Quansys Biosciences, Logan, Utah).

Techniques: Expressing, Saline, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of Tcf7l1 depletion on LCN2 expression in HFKs. HFKs were stimulated with calcium chloride (1.2 mM, 7 days) after siRNA transfection. ( a and b ) Intracellular ( a ) and soluble ( b ) LCN2 levels were evaluated by western blot and ELISA, respectively. ( c ) By immunofluorescence, the expression of involucrin and loricrin were detected in HFKs that were transfected with Tcf7l1 or control siRNA. ( d and e ) The expression of involucrin and loricrin was also detected in HFKs by western blot. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Representative images are shown. Scale bar=10 μ m. * P <0.05; ** P <0.01.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of Tcf7l1 overexpression on HFKs. After the transfection of Tcf7l1-inserted pcDNA vector, HFKs were stimulated with calcium chloride (1.2 mM, 7 days). ( a and b ) By qRT-PCR ( a ) and western blot ( b ), HFKs expressed more lipocalin 2 (LCN2) upon Tcf7l1 pcDNA transfection. ( c and d ) Both cell apoptosis ( c ) and proliferation ( d ) were determined in HFKs that were either transfected with Tcf7l1 pcDNA or not. Cell apoptosis and proliferation were also visualized by TUNEL and fluorescent Ki-67 staining, respectively. ( e ) qRT-PCR was performed for the mRNA levels of involucrin, loricrin and caspase-14 in HFKs. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Scale bar=10 μ m. In ( e ), * P <0.05, ** P <0.01, compared with nontreated groups accordingly; Δ P <0.05, compared with calcium alone-treated HFKs accordingly.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Over Expression, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, TUNEL Assay, Staining, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

doi: 10.1038/cddiscovery.2016.28

Figure Lengend Snippet: The effect of LCN2 inhibition on HFKs. ( a and b ) By western blot, involucrin and loricrin expression was determined in Tcf7l1-transfected HFKs that were then treated with LCN2 or control siRNA. ( c and d ) Similarly, involucrin and loricrin were assessed in E6/E7-transfected HFKs that were treated with neutralizing anti-LCN2 antibody or isotype control (2 μ g/ml, 2 days). ( e and f ) By immunofluorescence ( e ) and western blot ( f ), keratin 1 was detected in HFKs that were transfected with Tcf7l1 vector and then LCN2 or control siRNA. ( g ) HFKs were pre-transfected with Tcf7l1 vector, and then transfected with LCN2 siRNA. Cell apoptosis was quantitated by the CaspaTag caspase 3, 7 method. Data show mean±S.E.M. of triplicate cultures ( n =3). Statistical differences were tested with one-way ANOVA followed by a two-tailed t -test. Representative images are shown. Scale bar=10 μ m. * P <0.05; ** P <0.01.

Article Snippet: For confocal analysis, rabbit anti-involucrin (Alexa Fluor 555-conjugated; Bioss), anti-loricrin (Cy5-conjugated) (Biorbyt LLC, San Francisco, CA, USA) or anti-cytokeratin 1 (Alexa Fluor 488-conjugated) (Abcam, Cambridge, MA, USA) antibodies were used by following the standard method.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Immunofluorescence, Plasmid Preparation, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: Coomassie brilliant blue staining of pull-down assays performed with 3 μg of recombinant IFI16 (A) or GST (B) in the presence or absence of biotin-labeled lipopolysaccharide (LPS) from E . coli O111:B4 (biotin-LPS-EB). (C) Saturation binding experiments performed with 2 μg/ml of IFI16 (red circles) and increasing amount of biotin-LPS-EB. Binding was detected by ELISA using HRP-conjugated streptavidin. Optical density (OD) of samples was measured at 450 nm. An excess of recombinant GST (blue circles) or BSA (green circles) and pre-treatment of biotin-LPS-EB with polymyxin B (PMB, empty circles) were used as negative controls. Data are expressed as mean values ± SD of three independent experiments. (D) Surface plasmon resonance (SPR) analysis of LPS-EB binding to immobilized IFI16. After immobilization of IFI16 on the CM5 sensor chip surface, increasing concentration of LPS-EB (3.125–100 nM) diluted in running buffer were injected over immobilized IFI16. Data are representative of three independent experiments. (E) Ex-vivo interaction analysis between increasing amount of recombinant IFI16 and formalin-fixed gram-negative ( E . coli and K . pneumonia ; pink circles and pink squares, respectively) or gram-positive ( S . aureus , S . epidermidis , S . pyogenes ; blue circles, squares and triangles, respectively) bacteria. Data are expressed as mean values ± SD of three independent experiments. (F) Lipid A structures of LPS derived from E . coli O111:B4 or F583 LPS (LPS-EB and LPS-F583, respectively; strong TLR4 agonists), P . gingivalis (LPS-PG; weak TLR4 agonist) and R . sphaeroides (LPS-RS, TLR4 antagonist). For LPS-PG, which harbors a mixture of di-, mono- and de-phosphorylated penta- or tetra-acylated lipid A moieties, a single isoform is represented for simplicity. PS-outer chain = polysaccharide outer chain. (G) Saturation binding experiments with increasing amount of recombinant IFI16 (8 to 12,288 ng/ml) and 10 μg/ml of LPS-EB (red line), LPS-F583 (green line), LPS-PG (blue line) or LPS-RS (purple line). Anti-IFI16 antibodies against the N-terminus of the protein and HRP-labelled anti-rabbit IgG were added as primary and secondary antibody, respectively, and binding was detected by ELISA at 450 nm. Data are expressed as mean values ± SD of three independent experiments.

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Staining, Recombinant, Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Concentration Assay, Injection, Ex Vivo, Bacteria, Derivative Assay

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: (A) Structures of di- or mono-phosphorylated lipid A from E . coli F583 (DPLA and MPLA, respectively) and detoxified LPS (detox-LPS) derived from E . coli O111:B4. PS-outer chain = polysaccharide outer chain. (B) Saturation binding experiments with increasing amount of recombinant IFI16 (from 8 to 12,288 ng/ml) and 10 μg/ml of MPLA (green line), DPLA (red line) or detox-LPS (purple line). Binding was detected by ELISA as described in the legend to . Data are expressed as mean values ± SD of three different experiments. (C) Competition ELISA assay for LPS-EB binding to IFI16 with increasing amount of LPS-EB, MPLA or detox-LPS as competitors. Briefly, microtiter plates were coated with 1 μg/ml LPS-EB, then 2 μg/ml of IFI16 were added to the wells in the presence of increasing concentration (5 to 25 μg/ml) of competitor. Binding was detected by ELISA as described in the legend to . Data are expressed as mean values ± SD of three independent experiments (*** P <0.001, ** P <0.01, Student’s t test). (D) Domain organization of the IFI16 protein. The numbers represent the amino acid positions based on NCBI Reference Sequence NP_005522. From the N- to the C-terminal (left to right), IFI16 comprises a pyrin domain involved in protein-protein interaction, and two hematopoietic interferon-inducible nuclear protein with 200-amino-acid repeats (HINA and HINB) domains, which are a hallmark of the absent in melanoma 2-like receptors (ALRs). S/T/P = serine/threonine/proline-rich repeats, which are regulated by alternative mRNA splicing. (E) Coomassie brilliant blue staining of pull-down assays performed with 3 μg of recombinant PYRIN, HINA, and HINB domains, in presence or absence of biotin-labeled LPS from E . coli O111:B4 (biotin-LPS-EB). (F) Saturation binding experiments performed by using increasing amount (8 to 4,096 ng/ml) of recombinant PYRIN, HINA or HINB domains (orange, purple and blue lines, respectively) and 10 μg/ml of LPS-EB, LPS-PG, MPLA or DPLA. Anti-IFI16 antibodies against the N- or C-terminus of the protein and HRP-labeled anti-rabbit IgG were added as primary and secondary antibody, respectively, and binding detected in ELISA at 450 nm. Data are expressed as mean values ± SD of three independent experiments.

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay, Sequencing, Staining, Labeling

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: (A) qRT-PCR analysis of IL-6, IL-8, TNF-α and IL-1β mRNA expression levels in 786-O or THP-1 cells stimulated for 24 h with IFI16 (25 μg/ml), IFI16ΔHINB (25 μg/ml), LPS from E . coli O111:B4 (LPS-EB, 10 ng/ml or 1 μg/ml), IFI16/LPS-EB complex (preincubated O/N at 4°C), IFI16ΔHINB/LPS-EB (preincubated O/N at 4°C), or left untreated (mock). Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SD (* P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA followed by Dunnett’s test). (B) Protein concentration of IL-6, IL-8 and TNF-α evaluated by ELISA in supernatants derived from 786-O or THP-1 cells stimulated for 24 h as described in A . Data are expressed as mean values ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA followed by Dunnett’s test).

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: (A) qRT-PCR analysis of IL-6, IL-8 and TNF-α mRNA expression levels in 786-O or THP-1 cells stimulated for 24 h with or without IFI16 (25 μg/ml), LPS from E . coli F583 (LPS-F583, 10 ng/ml) or LPS from R . sphaeroides (LPS-RS, 10 ng/ml), MPLA (10 ng/ml), DPLA (10 ng/ml) or in the presence of one of the following complexes: IFI16/LPS-F583, IFI16/LPS-RS, IFI16/MPLA or IFI16/DPLA. Values are normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. qRT-PCR data are presented as mean values of biological triplicates. Error bars indicate SD, and the P values refer to comparisons between IFI16 vs . IFI16/LPS or IFI16/lipid A complex-treated cells (* P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA followed by Dunnett’s test). (B) Protein concentration of IL-6, IL-8 and TNF-α evaluated by ELISA in supernatants derived from 786-O or THP-1 cells stimulated for 24 h as described in the legend to panel A . Data are expressed as mean values ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant; two-way ANOVA followed by Dunnett’s test). The P values are relative to comparisons between IFI16- and IFI16/LPS- or IFI16/lipid A-treated cells.

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: (A-C) Protein concentration of IL-6, IL-8 and TNF-α measured by ELISA in supernatants derived from 786-O or THP-1 cells transfected for 48 h with scramble control (siCTRL), or siRNAs against TLR4 (siTLR4, A ), MD2 (siMD2, B ) or MyD88 (siMyD88, C ). Cells were then stimulated for 24 h with IFI16 (25 μg/ml), LPS from E . coli O111:B4 (LPS-EB, 10 ng/ml) or IFI16/LPS-EB complex (preincubated O/N at 4°C), or left untreated (mock). Data are expressed as mean values ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant; unpaired Student’s t -test for comparison of silenced cells vs . their relative control counterpart). (D) Schematic representation of the probe containing the NF-κB binding site (highlighted in grey). (E) 786-O cells or THP-1 cells were stimulated with IFI16 (25 μg/ml), LPS from E . coli O111:B4 (LPS-EB, 10 ng/ml) or IFI16/LPS-EB complex (preincubated O/N at 4°C), or left untreated (mock). After 2 h, the cells were lysed and the nuclear fraction was analyzed for NF-κB binding activity using the Universal EZ-TFA transcription factor assay colorimetric kit and the probe described in D . Data are expressed as mean values ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA followed by Dunnett’s test).

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transfection, Control, Comparison, Binding Assay, Activity Assay, Transcription Factor Assay

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: (A) Western blot analysis of IFI16 in culture supernatants and total cell extracts of UVB-exposed (0 or 800 Jm -2 ) U2OS or U2OS-IFI16 -/- cells at 16 h after treatment. β-actin cellular expression was used for protein loading control. Data are representative of three independent experiments with similar results. (B) Protein concentration of IL-6, IL-8 and TNF-α evaluated by ELISA in supernatants derived from THP-1 cells stimulated for 24 h in the presence or absence of anti-TLR4 neutralizing antibodies (10 μg/ml) using conditioned medium from UVB-exposed (0 or 800 Jm -2 ) U2OS and U2OS-IFI16 -/- cells, or complete medium (mock), preincubated (O/N at 4°C), or not, with LPS from E . coli O111:B4 (LPS-EB, 10 ng/ml), or LPS from R . sphaeroides (LPS-RS, 10 ng/ml). Values were normalized to the initial protein concentration of the analyzed cytokines in the supernatants used for the treatment. Data are expressed as mean values ± SD of three independent experiments. The P values refer to comparison in each group with cells treated only with the medium without any addiction (white bar and black border; * P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA followed by Dunnett’s test).

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Comparison

Journal: PLoS Pathogens

Article Title: Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding

doi: 10.1371/journal.ppat.1008811

Figure Lengend Snippet: (A) qRT-PCR analysis of IL-6, IL-8, TNF-α and mRNA expression levels in 786-O or THP-1 cells stimulated for 24 h with IFI16 (25 μg/ml), LPS from E . coli O111:B4 (LPS-EB, 10 ng/ml), IFI16/LPS-EB complex, IFI16 + LPS-EB (not complexed), IFI16/LPS-EB complex + LPS-EB (10 ng/ml), LPS from Rhodobacter sphaeroides (LPS-RS, 10 ng/ml), IFI16/LPS-RS complex, IFI16 + LPS-RS (not complexed), IFI16/LPS-RS complex + LPS-RS (10 ng/ml), or left untreated (mock). Values were normalized to GAPDH mRNA and plotted as fold induction over mock-treated cells. The P values refer to comparisons with IFI16-treated cells (* P < 0.05, ** P < 0.01, *** P < 0.001; one-way ANOVA followed by Dunnett’s test). (B) Protein concentration of IL-6, IL-8 and TNF-α was measured by ELISA in supernatants derived from 786-O or THP-1 cells stimulated for 24 h as described in A . Data are expressed as mean values ± SD of three independent experiments. The P values refer to comparisons with IFI16-treated cells (* P < 0.05, ** P < 0.01, *** P < 0.001; one-way ANOVA followed by Dunnett’s test). (C) Surface plasmon resonance (SPR) analyses of IFI16, LPS-EB and IFI16/LPS-EB complex binding to immobilized TLR4/MD2 receptor. After immobilization of recombinant TLR4/MD2 on the CM5 sensor chip surface, increasing concentration of the different analytes—31.25–1000 nM for IFI16, 3.125–100 μM for LPS-EB, 31.25–1000 nM for IFI16/LPS-EB complex—diluted in running buffer were injected over immobilized TLR4/MD2. IFI16, LPS-EB, and IFI16/LPS-EB bind to TLR4/MD2 with an equilibrium dissociation constant (K D ) of 0.68 μM, 0.15 nM, and 4.01 μM, respectively. Data are representative of three independent experiments.

Article Snippet: Cytokines secreted in culture supernatants after treatments were analyzed using human IL-6 DuoSet ELISA and human IL-8 DuoSet ELISA (all from R&D Systems), human TNF-α Uncoated ELISA and human IL-1β Uncoated ELISA (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Derivative Assay, SPR Assay, Binding Assay, Recombinant, Concentration Assay, Injection

Journal: Journal of nanobiotechnology

Article Title: Regulating macrophage phenotypes with IL4I1-mimetic nanoparticles in IDD treatment.

doi: 10.1186/s12951-025-03241-0

Figure Lengend Snippet: Fig. 3 Selection and Expression of Core M-DEGs in the GEO Dataset. Note: (A) LASSO selection plot for M-DEGs; (B) SVM-RFE analysis results for M-DEGs; (C) Results from the Random Forest algorithm for M-DEGs; (D) Venn diagram showing the intersection of results from LASSO, SVM-RFE, and Random For est algorithms; (E) Expression levels of IL4I1 in NP samples from each group (n = 6) in the merged dataset; (F) Pseudo-time gene expression curve of IL4I1 in MΦ subtypes from scRNA-seq data, with time on the x-axis and gene expression level on the y-axis; ** P < 0.01 compared between the two groups

Article Snippet: In brief, 1 ml of 2% (w/v) PLGA in ethyl acetate solution was mixed with 10 μg of recombinant human IL4I1 (R&D Systems, 5684-AO) dissolved in 100 μl of sterile DNase/RNase.

Techniques: Selection, Expressing, Gene Expression

Journal: Journal of nanobiotechnology

Article Title: Regulating macrophage phenotypes with IL4I1-mimetic nanoparticles in IDD treatment.

doi: 10.1186/s12951-025-03241-0

Figure Lengend Snippet: Fig. 4 Influence of IL4I1 on M1-M2 Polarization of MΦs. Note: (A) Schematic representation of IL4I1 treatment on MΦ induced M1 or M2 polarization; (B) RT-qPCR analysis of expression of M1 and M2 MΦ marker genes in different groups of MΦ; (C) Representative immunofluorescence images of MΦ in differ ent groups (scale bar = 25 μm), quantification of fluorescence intensities of CD80 and CD86, CD163 and CD206 co-localization; (D) Levels of IL-1β, TNF-α, TGF-β, and IL-10 in the supernatant of cultured MΦ in each group; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, all experiments were repeated thrice

Article Snippet: In brief, 1 ml of 2% (w/v) PLGA in ethyl acetate solution was mixed with 10 μg of recombinant human IL4I1 (R&D Systems, 5684-AO) dissolved in 100 μl of sterile DNase/RNase.

Techniques: Quantitative RT-PCR, Expressing, Marker, Immunofluorescence, Fluorescence, Cell Culture

Journal: Journal of nanobiotechnology

Article Title: Regulating macrophage phenotypes with IL4I1-mimetic nanoparticles in IDD treatment.

doi: 10.1186/s12951-025-03241-0

Figure Lengend Snippet: Fig. 5 Preparation, characterization, and In Vitro Uptake of IL4I1-NPs and IL4I1-MNPs. Note: (A) Schematic depiction of the preparation process for IL4I1-NPs and IL4I1-MNPs; (B) Representative images of IL4I1-NPs and IL4I1-MNPs using TEM (scale bar = 200 nm); (C-D) DLS analysis of the size and zeta potential of IL4I1-NPs and IL4I1-MNPs; (E) SDS-PAGE protein analysis of cell lysates, MΦCM, IL4I1-NPs, and IL4I1-MNPs; (F) Loading capacity of IL4I1 in IL4I1-NPs and IL4I1-MNPs; (G) The loading capacity and encapsulation efficiency of IL4I1 within IL4I1-NPs and IL4I1-MNPs, respectively, with monitoring of encapsulation rates within 72 h at 4 °C; (H) In vitro cumulative release curve of IL4I1 from IL4I1-NPs and IL4I1-MNPs in PBS at 37 °C; (I) Particle size detec tion of IL4I1-MNPs in deionized water, 1×PBS, and 50% FBS over three days; (J) Schematic diagram of the preparation process for DiR-NPs and DiR-MNPs; (K) Flow cytometry analysis of fluorescence intensity of DiR in MΦ after incubation with DiR-NPs and DiR-MNPs for 1, 2, and 4 h; (L) Representative images (scale bar = 15 μm) and quantitative analysis of fluorescence intensity of MΦ uptake of DiR-NPs and DiR-MNPs; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, all experiments were conducted in triplicate

Article Snippet: In brief, 1 ml of 2% (w/v) PLGA in ethyl acetate solution was mixed with 10 μg of recombinant human IL4I1 (R&D Systems, 5684-AO) dissolved in 100 μl of sterile DNase/RNase.

Techniques: In Vitro, Zeta Potential Analyzer, SDS Page, Encapsulation, Flow Cytometry, Fluorescence, Incubation

Journal: Journal of nanobiotechnology

Article Title: Regulating macrophage phenotypes with IL4I1-mimetic nanoparticles in IDD treatment.

doi: 10.1186/s12951-025-03241-0

Figure Lengend Snippet: Fig. 6 Impact of IL4I1-MNPs on MΦ Polarization in IDD Mice In Vivo. Note: (A) RT-qPCR analysis of expression of M1 and M2 MΦ marker genes in IVD MΦs of mice in different groups (n = 6); (B) Percentage of M1 MΦs (CD80+CD86+) in IVD MΦs of mice in different groups (n = 6); (C) Percentage of M2 MΦs (CD163+CD206+) in IVD MΦs of mice in different groups (n = 6); (D) ELISA detection of IL-1β, TNF-α, TGF-β, and IL-10 levels in IVD of mice in different groups (n = 6); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: In brief, 1 ml of 2% (w/v) PLGA in ethyl acetate solution was mixed with 10 μg of recombinant human IL4I1 (R&D Systems, 5684-AO) dissolved in 100 μl of sterile DNase/RNase.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Marker, Enzyme-linked Immunosorbent Assay

Journal: Journal of nanobiotechnology

Article Title: Regulating macrophage phenotypes with IL4I1-mimetic nanoparticles in IDD treatment.

doi: 10.1186/s12951-025-03241-0

Figure Lengend Snippet: Fig. 8 Exploration of the Role of FGR in Regulating MΦ Polarization Balance Mediated by IL4I1-MNPs. Note: (A) Illustration of FGR-OE transfection; (B) RT- qPCR analysis of FGR expression in different groups of MΦ; (C) Schematic representation of MΦ polarization induction post-NC-OE or FGR-OE transfection, followed by treatment with PBS or IL4I1-MNPs; (D) RT-qPCR analysis of expression of M1 and M2 MΦ marker genes in different groups of MΦ; (E) Repre sentative immunofluorescence images of MΦ in different groups (scale bar = 25 μm), quantification of CD80 and CD86, CD163 and CD206 co-localization fluorescence intensity; (F) Levels of IL-1β, TNF-α, TGF-β, and IL-10 in the supernatant of cultured MΦ in different groups; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, all experiments were repeated three times

Article Snippet: In brief, 1 ml of 2% (w/v) PLGA in ethyl acetate solution was mixed with 10 μg of recombinant human IL4I1 (R&D Systems, 5684-AO) dissolved in 100 μl of sterile DNase/RNase.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Marker, Immunofluorescence, Fluorescence, Cell Culture

Journal: Journal of nanobiotechnology

Article Title: Regulating macrophage phenotypes with IL4I1-mimetic nanoparticles in IDD treatment.

doi: 10.1186/s12951-025-03241-0

Figure Lengend Snippet: Fig. 9 Effect of CHG@IL4I1-MNPs on MΦ Polarization in IDD Mice. Note: (A) RT-qPCR analysis of the expression of M1 and M2 MΦ marker genes in IVD MΦs of mice (n = 6); (B) Percentage of M1 MΦs in IVD MΦs of mice (n = 6); (C) Percentage of M2 MΦs in IVD MΦs of mice (n = 6); (D) ELISA detection of IL- 1β, TNF-α, TGF-β, and IL-10 levels in the IVD of mice (n = 6); n.s. P > 0.05 for comparison between groups, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: In brief, 1 ml of 2% (w/v) PLGA in ethyl acetate solution was mixed with 10 μg of recombinant human IL4I1 (R&D Systems, 5684-AO) dissolved in 100 μl of sterile DNase/RNase.

Techniques: Quantitative RT-PCR, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Communications Biology

Article Title: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 low resting monocytes

doi: 10.1038/s42003-022-03057-w

Figure Lengend Snippet: a , b Cytokine profile arrays of supernatant collected from cord blood CD34 + cells infected with SCR - and TET2- shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: * P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: * P < 0.05; *** P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2 -depleted ( TET2 shRNA, gray bars) and control ( SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: * P < 0.05; *** P < 0.001; **** P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 ( n = 5), M07e ( n = 5), UT-7 ( n = 4) and TF-1 ( n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: * P < 0.05; ** P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2 -deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2 +/+ n = 4, wt/exc or Tet2 +/− n = 5 and exc/exc or Tet2 −/− n = 5); ANO knock-down model (wt/wt or Tet2 + /+ n = 5, wt/LacZ or Tet2 +/− n = 5 and LacZ/LacZ or Tet2 −/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 + collected supernatants were analyzed using human cytokine antibody array (panel A; R&D Systems).

Techniques: Infection, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Western Blot, Software, Clinical Proteomics, Knock-Out, Knockdown, Comparison

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir. Data represent the average of three independent experiments ± SD. (C) POC for percentage of infection of the Huh7.5 drug screen performed at 0.5 μM. 33 drugs had >60% reduction in infection with >80% cell viability. (D) Distribution of 23 validated antivirals by drug target class. (E) Dose-response analysis of the candidates with a selectivity index (SI) > 3 identified in the screen. Data represent the average of three independent experiments ± SD.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: High Throughput Screening Assay, Infection, Microscopy

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: Cell-type-specific dependencies of entry inhibitors (A) Calu-3 human lung epithelial cells were infected with SARS-CoV-2 (MOI = 0.5) and processed for microscopy 48 hpi. (B) Dose-response analysis of Calu-3 cells treated with quinolines or remdesivir. Data represent the average of four independent experiments ± SD. (C) IC 50 , CC50, and SI for Vero, Huh7.5, and Calu-3 cells treated with a panel of quinolines or remdesivir. Data represent the average of four independent experiments ± SD. (D) Dose-response analysis of Calu-3 cells treated with the cathepsin inhibitor Z-FA-FMK. Data represent the average of four independent experiments ± SD. (E) Dose response analysis of Calu-3, Vero, and Huh7.5 cells treated with camostat. Data represent the average of ≥2 independent experiments ± SD. (F) IC 50 , CC50, and SI for camostat across cell types. (G) Immunoblot of Vero, Huh7.5, and Calu-3 cells probed for Ace2 and tubulin as a loading control. Representative blot is shown. (H) qRT-PCR of Ace2 or TMPRSS2 comparing Huh7.5 and Calu-3 cells. Data represent the mean ± SEM for ≥2 independent experiments.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Infection, Microscopy, Western Blot, Quantitative RT-PCR

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: Validation of antiviral activity of nine candidates in Calu-3 cells (A) Dose-response analysis of Huh7.5 candidate antivirals in Calu-3 cells with a SI > 3. Data represent the average of ≥4 independent experiments ± SD. (B) qRT-PCR analysis of nine candidate antivirals in Calu-3 cells. Data represent the mean ± SEM for ≥2 independent experiments.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Activity Assay, Quantitative RT-PCR

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: Cyclosporine is antiviral against SARS-CoV-2 independent of calcineurin (A) Dose-response analysis of Huh7.5 cells treated with a panel of cyclosporins and related drugs. Data represent the average of ≥2 independent experiments ± SD. (B) Dose-response analysis of Calu-3 cells treated with a panel of cyclosporins and related drugs. Data represent the average of five independent experiments ± SD. (C) Dose-response analysis of Calu3 cells treated with cyclophilin-selective drugs NIM811 and alisporivir. Data represent the average of five independent experiments ± SD. (D) Table of IC 50 s, CC50s, and SI of Huh7.5 cells and Calu-3 cells treated with the indicated drugs. (E) qRT-PCR analysis of viral replication in Huh7.5 and Calu-3 cells treated with the indicated drugs. (F) qRT-PCR analysis of viral replication in iAT2 cells treated with the indicated drugs. (G) qRT-PCR of NHBE cells treated with the indicated drug. For (E)–(G), data represent the mean ± SEM for ≥2 independent experiments.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Quantitative RT-PCR

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet:

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Recombinant, SYBR Green Assay, Software