Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: eNPM binds to TLR4 and the binding increases upon cytokines treatment in HaCaT and HFs. (A) Proximity ligation assay (PLA) of NPM and TLR4 interaction analysis in serum-starved HaCaT cells stimulated with a MIX of cytokines for 8 h (IL-17A, IL-22, TNF-α, and IFN-γ). Lower panels represent zoomed-in images of the original PLA images. Red dots indicate that even under physiological conditions, NPM interacts with TLR4. When a MIX of cytokines is administered, the rate of interaction increases. As a negative control, the IgG specific for each antibody (i.e., normal rabbit IgG used as the control of TLR4 antibody) and normal mouse IgG for NPM antibody (left panel) revealed the specificity of NPM and TLR4 interaction, since no red dots were present in the PLA. Scale bar 10 μm. (B) Bar graph of PLA interactions quantified by the mean number of red dots per cell ( n = 5; * p < 0.05). (C) PLA of NPM and TLR4 interaction analysis in serum-starved HFs derived from healthy subjects and stimulated with a MIX of cytokines for 8 h. Lower panels represent zoomed-in images of the original PLA images. Scale bar 10 μm. (D) Bar graph of PLA interactions quantified by the mean number of red dots per cell ( n = 6; * p < 0.05).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Binding Assay, Proximity Ligation Assay, Negative Control, Control, Derivative Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: rNPM promotes NF-κB translocation to the nucleus and induces inflammation in HaCaT and HFs via TLR4. (A) Representative image of immunofluorescence of NF-kB (red) with an α-p65rel NF-kB antibody, nuclei were counter-stained with DAPI (blue). NFκB translocated in the nucleus in HaCaT treated for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). Mouse IgG isotype control was used as a negative control. Left panels represent zoomed-in images of the original images. Scale bar 5 μm. (B) Representative image of immunofluorescence of NF-kB (red) with an α-p65rel NF-kB antibody, nuclei were counter-stained with DAPI (blue). NF-κB translocated in the nucleus in HFs treated for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). Mouse IgG isotype control was used as a negative control. Left panels represent zoomed-in images of the original images. Scale bar 5 μm. (C) HaCaT were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment ( n = 6; * p < 0.05; ** p < 0.01). (D) HFs were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment ( n = 6; * p < 0.05).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Translocation Assay, Immunofluorescence, Staining, Control, Negative Control, Quantitative RT-PCR

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: TRL4-based mechanism regulates NPM and miR-200c secretion in response to cytokine mix and miR-200c and NPM physically interact. HaCaT were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with cytokine MIX (IL-17A, IL-22, TNF-α, and IFN-γ). (A) Media were collected and analyzed by ELISA assay for the presence of eNPM. eNPM increased levels by cytokine MIX were significantly decreased by TLR4i ( n = 5; * p < 0.05; ** p < 0.01). (B) qRT-PCR of miR-200c from the supernatant show that miR-200c increase elicited by cytokine MIX treatment is significantly reduced by TLR4i ( n = 6; ** p < 0.01; *** p < 0.001). (C) HaCaT were treated with cytokine MIX for 8 h then supernatants were collected and treated either with RNase or 200 μM NPM inhibitor (NPMi) or 2 μg/ml RNAse together with 200 μM NPMi for 15 min at 37°C ( n = 6, * p < 0.05; * * p < 0.01).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Thoracic Disease

Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells

doi: 10.21037/jtd.2019.07.55

Figure Lengend Snippet: Primer sequences used for quantitative RT-PCR

Article Snippet: Pharmacological reagents treatment Unless otherwise indicated, all pharmacological reagents were obtained from Tocris Bioscience (Bristol, UK) or Cayman Chemical (Ann Arbor, MI, USA) and used at the following working concentrations: Toll-like receptor 4 (TLR4) inhibitor (TAK242, 2.5 µM; Cayman Chemical), phospholipase C (PLC) inhibitor ( {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}} U73122 , 10 µM; Tocris Bioscience), IP3/TRP channel inhibitor [2-aminoethoxydiphenyl borate (2-APB), 10 µM; Tocris], and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor [diphenyleneiodonium (DPI), 10 µM; Tocris].

Techniques: Sequencing

Journal: Molecular Medicine

Article Title: H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation

doi: 10.2119/molmed.2014.00189

Figure Lengend Snippet: H5N1 HA is detected by TLR4 expressed on respiratory epithelial cells, facilitating the activation of JAK3, which leads to the impairment of cAMP-dependent CFTR channels. (A) Isc and ΔIsc measurements in HA-challenged tracheal tissues of JAK3 +/+ mice with or without TLR4 inhibitor treatment (n = 5 per group, * p < 0.05 versus mice administered saline; # p < 0.05 versus mice pretreated with HA alone); (B) Immunofluorescent staining for phosphorylated JAK3 (p-JAK3) expression in 16HBE cells. Scale bars = 40 μm (C) Western blot for p-JAK3 in 16HBE cells. The mean optical densities of the bands for each group are presented (C, bottom) (* p < 0.05 versus saline control cells; # p < 0.05 versus cells incubated with HA alone for 60 min); c: control. (D) Quantitative RT-PCR analysis of TLR4 and CFTR gene expression in HA-treated calu-3 cells for the indicated periods compared with control.

Article Snippet: Cultured 16HBE and calu-3 cells were treated with saline, JAK3 inhibitor VI (760 nmol/L), TLR4 inhibitor (candesartan, 5 μmol/L, 3B Scientific Corporation, Wuhan, China), forskolin (10 μmol/L) or glibenclamide (500 μmol/L) for 30 min prior to HA stimulation and then subjected to different experiments.

Techniques: Activation Assay, Saline, Staining, Expressing, Western Blot, Control, Incubation, Quantitative RT-PCR, Gene Expression

Journal: Molecular Medicine

Article Title: H5N1 Virus Hemagglutinin Inhibition of cAMP-Dependent CFTR via TLR4-Mediated Janus Tyrosine Kinase 3 Activation Exacerbates Lung Inflammation

doi: 10.2119/molmed.2014.00189

Figure Lengend Snippet: HA-induced JAK3 activation stimulates Gαimediated inhibition of AC, resulting in a decrease in the intracellular cAMP level 16HBE cells were pretreated with IBMX (1 mmol/L )for 15 min and then treated with forskolin (FSK, 10 μmol/L, 30 min) and either HA (40 μg/mL, 30 min) alone or HA (40 μg/mL, 30 min) in combination with IL-2 (100 UI/mL, 30 min) as indicated. In some instances, cells were pretreated with pertussis toxin (PTx, 100 ng/mL) for 16 h or a TLR4 inhibitor (TLR4inh, 5 μmol/L) or JAK3 inhibitor VI (JAK3inh, 760 nmol/L) for 30 min. The intracellular cAMP levels were examined in the cell lysates. * p < 0.05 versus forskolin-treated cells; # p < 0.05 versus HA-treated cells; and ## p < 0.05 versus PTx-treated cells.

Article Snippet: Cultured 16HBE and calu-3 cells were treated with saline, JAK3 inhibitor VI (760 nmol/L), TLR4 inhibitor (candesartan, 5 μmol/L, 3B Scientific Corporation, Wuhan, China), forskolin (10 μmol/L) or glibenclamide (500 μmol/L) for 30 min prior to HA stimulation and then subjected to different experiments.

Techniques: Activation Assay, Inhibition

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet: ( A ) Representative microscopy images of wild-type (WT) and Cd300a −/− bone marrow-derived dendritic cells (BMDCs) treated with pHrodo-labeled extracellular vesicles (EVs) to assess the localization of EVs (red) and early endosome antigen (EEA)-1 (green). Scale bar, 10 μm. Data are representative of two independent experiments. ( B ) Uptake of PKH-labeled tumor-derived EVs (TEVs) in WT ( n = 5) and Cd300a −/− BMDCs ( n = 5). ( C ) Representative microscopy images of WT and Cd300a −/− BMDCs treated with pHrodo-labeled exosomes to assess the localization of exosomes (green), TLR3 (red), and CD300a (blue). Scale bar, 10 μm. Data are representative of two independent experiments. ( D ) Quantitative RT-PCR analysis of Ifnb in WT and Cd300a −/− BMDCs treated with B16-derived exosomes in the presence of dimethyl sulfoxide (DMSO) (WT, n = 9; Cd300a −/− , n = 10), 100 nM TLR4 inhibitor ( n = 7 in each group), and 50 μM TLR3 inhibitor ( n = 6 in each group). ( E ) Quantitative RT-PCR analysis of Ifnb in WT, Cd300a −/− , ticam-1 −/− , and ticam-1 −/− ;Cd300a −/− mice-derived BMDCs treated with B16-derived EVs ( n = 5 in all group). ( F ) Representative immunoassay of WT and Cd300a −/− BMDCs left unstimulated (0 min) or stimulated for the indicated times with B16-derived exosomes, followed by immunoblot analysis of phosphorylated (p-) interferon regulatory factor 3 (IRF3) or total IRF3. Data are representative of two independent experiments. ( G and H ) Comparison of tumor growth and survival curves of B16 melanoma cells between ticam-1 −/− ( n = 6) and ticam-1 −/− ;Cd300a −/− ice ( n = 9) after inoculation of B16 melanoma. ( I and J ) Comparison of tumor growth and survival curves of B16 melanoma between MyD8 −/− ( n = 9) and MyD88 −/− ;Cd300a −/− mice ( n = 10) after inoculation of B16 melanoma. Data are given as means ± standard error of the means (SEMs). N.S.: not significant. *p < 0.05, **p < 0.01, and ***p < 0.001. p values were obtained by using the Student’s t -test ( B ), a two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test ( D, E, G, and I ), and the log-rank test ( H and J ). Data were pooled from two ( B, E, and H ) or three ( D, I, and J ) independent experiments. Figure 5—source data 1. Source data for .

Article Snippet: Chemical compound, drug , TLR4 inhibitor (TAK-242) , Merck , Cat. #: 614,316 , .

Techniques: Microscopy, Derivative Assay, Labeling, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

doi: 10.7554/eLife.61999

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , TLR4 inhibitor (TAK-242) , Merck , Cat. #: 614,316 , .

Techniques: Purification, Sequencing, Recombinant, Isolation, Software