Journal: Frontiers in Pharmacology

Article Title: Establishment of a human nasal epithelium model of histamine-induced inflammation to assess the activity of fexofenadine as an inverse agonist and its link to clinical benefit

doi: 10.3389/fphar.2024.1393702

Figure Lengend Snippet: Reconstituted human nasal epithelium model (MucilAir™) to study the effect of HIS and FEX. CBF, cilia beating frequency; ELISA, enzyme-linked immunosorbent assay; LDH, lactate dehydrogenase; MCC, mucociliary clearance; RT-qPCR, quantitative reverse transcription–polymerase chain reaction; TEER, trans-epithelial electrical resistance.

Article Snippet: The basolateral medium was collected and measured using a Cytotoxicity Lactate Dehydrogenase (LDH) Assay Kit, following the manufacturer’s instructions (WST Dojindo, CK12-20).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor expression in synovial tissue of patients with rheumatoid arthritis. The representative results of HE staining (Figure 2A), immunofluorescence anti-CTGF antibody staining (Figure 2B; green), and anti-F4/80 antibody staining (Figure 2C; red) are shown using surgical samples from RA and OA patients. The samples were counterstained by DAPI (blue) for nuclear staining and merge images are shown (Figure 2D). A strong expression of CTGF and F4/80 was observed in the samples of RA compared to OA and the CTGF expression cells were not overlapped with F/40 expression cells indicating that CTGF is upregulated in synovial fibroblasts rather than macrophages.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Synergistic effects of connective tissue growth factor on M-CSF/sRANKL-mediated osteoclastic function. Figure 5A shows the results of the resorption of osteoclasts on calcium phosphate. Vacant regions indicated by arrows represent the areas where the osteoclasts actually absorbed. There was no vacant region in negative control cells (M-CSF alone). In contrast to negative control, significant vacant regions were observed in osteoclasts induced by M-CSF/sRANKL. CTGF further expanded the vacant areas in combination with M-CSF/sRANKL and anti-CTGF antibody neutralized this effect. Figure 5B shows the levels of expression of osteoclasts specific proteases (MMP-9 and cathepsin-K) measured by quantitative real time RT-PCR. Synergistic effect of CTGF was also observed for M-CSF/sRANKL-mediated osteoclastogenesis. Bars in Figure 5B indicate the SD.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Negative Control, Expressing, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor mediate ERK1/2 and focal adhesion kinase activation through integrin αVβ3 signal transduction. Figure 6A shows the immnoprecipitation and immunoblotting analysis. The cell extracts of osteoclasts stimulated with recombinant CTGF (10 or 50 ng/ml) at 60 minutes were precipitated using anti-integrin αVβ3 antibody and subsequently blotted with anti- phosphorylated ERK1/ERK2, conventional ERK1/ERK2, and integrin αVβ3 antibodies respectively. The phosphorylated ERK1/ERK2 was recruited with integrin αVβ3 by CTGF stimulation. Figure 6B shows the immunoblotting analysis using anti- phosphorylated FAK, conventional FAK, and β-actin antibodies in the osteoclasts extracts treated with CTGF (10 ng/ml) at 5, 15, 60, and 120 minutes in the presence or absence of anti-CTGF antibody (1 μg/ml). CTGF stimulation resulted in phosphorylation of FAK from 60 minutes and this effect was neutralized by anti-CTGF antibody suggesting activation of signal transduction pathways through integrin αVβ3.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Activation Assay, Transduction, Western Blot, Recombinant, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Altered IL-4 mRNA stability correlates with Th1 and Th2 bias and susceptibility to hypersensitivity pneumonitis in two inbred strains of mice.

doi: 10.4049/jimmunol.169.7.3700

Figure Lengend Snippet: FIGURE 2. Splenocytes and CD4 T cells from C57BL/6 mice produce less of the Th2-associated cytokine IL-4 relative to DBA/2 mice. A, Splenocytes were isolated from naive C57BL/6 and DBA/2 mice and cul- tured (2 106 cells/ml) in the presence of 2.5 g/ml Con A or PBS control for 24 h, after which cells and cell supernatants were harvested for analysis. IL-4 protein was measured directly from cell culture supernatants by ELISA, while real-time RT-PCR was used to quantitate relative IL-4 mRNA expression, as described in Materials and Methods. B, Same as A except splenocytes were activated with 5 ng/ml PMA and 500 ng/ml iono- mycin for 24 h. C, Purified CD4 T cells were isolated with positive selection from naive C57BL/6 and DBA/2 mice and then stimulated with 5 ng/ml PMA and 500 ng/ml ionomycin for 24 h. IL-4 protein and IL-4 mRNA was measured as described above. D, Same as C except CD4 T cells were activated with 1 g/ml plate-bound -CD3 and 1 g/ml -CD28 mAbs for 24 h. E, CD4 T cells from C57BL/6 and DBA/2 mice were isolated with positive selection and then stimulated for 24 h with 1 g/ml plate-bound -CD3 and 1 g/ml -CD28 mAbs. Total RNA was isolated at the end of the time period and relative expression of IL-2 was assessed with real-time RT-PCR. F, Same as in E except total RNA was analyzed for the expression of IFN- mRNA with real-time RT-PCR. Data are ex- pressed as mean SEM for three independent experiments (, p 0.05).

Article Snippet: Murine IL-4, IL-13, and IFN- were specifically detected in cell culture supernatants by use of ELISA DuoKits (IL-4 and IFN- ) and an ELISA Quantikine kit (IL-13), all purchased from R&D Systems.

Techniques: Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Selection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Altered IL-4 mRNA stability correlates with Th1 and Th2 bias and susceptibility to hypersensitivity pneumonitis in two inbred strains of mice.

doi: 10.4049/jimmunol.169.7.3700

Figure Lengend Snippet: FIGURE 4. CD4 T cells from C57BL/6 mice do not polarize toward the Th2 immunophenotype to the same degree as CD4 T cells from DBA/2 mice. A, CD4 T cells from naive C57BL/6 and DBA/2 mice were polarized toward Th2 phenotype as described in Materials and Methods. After 7 days, cells were washed and then restimulated for 24 h (to assess protein release) or for 4 h (to assess mRNA expression) with 5 ng/ml PMA and 500 ng/ml ionomycin. IL-4 protein release (left panel) was measured with ELISA and IL-4 mRNA expression (right panel) was measured with real-time RT-PCR as described in Materials and Methods. B, CD4 T cells from naive C57BL/6 and DBA/2 mice were polarized toward Th1 immu- nophenotype as described in Materials and Methods. After 7 days, cells were washed and then restimulated for either 24 or 4 h with 5 ng/ml PMA and 500 ng/ml ionomycin. IFN- protein (left panel) and mRNA (right panel) was measured as described above. Data are expressed as mean SEM for three independent experiments (, p 0.05)

Article Snippet: Murine IL-4, IL-13, and IFN- were specifically detected in cell culture supernatants by use of ELISA DuoKits (IL-4 and IFN- ) and an ELISA Quantikine kit (IL-13), all purchased from R&D Systems.

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

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: L5-LDL from ST-elevation myocardial infarction patients induces IL-1β production via LOX-1 and NLRP3 inflammasome activation in macrophages.

doi: 10.1152/ajpheart.00509.2016

Figure Lengend Snippet: Fig. 4. Knockdown of lectin-type oxidized LDL receptor (LOX-1) reduced NF-B activation and L5-LDL-induced IL-1 and cleaved caspase-1. A: effects of copper oxidation on L5-LDL-induced IL-1 production in THP-1 macrophages. L5-LDL was oxidized with copper for 0, 6, and 16 h in vitro and then the copper-oxidized L5-LDL was used to treat THP-1 macrophages for 24 h. The levels of IL-1 in the medium were determined by ELISA. *P 0.05, compared with the 0 h. B: THP-1 cells were infected with lentivirus carrying specific shRNA for LOX-1 and CD36, and knockdown efficiency was evaluated by RT-qPCR. *P 0.05, compared with the shLuc control. C: macrophages derived from shLOX-1, shCD36, or shLuc knockdown cells were incubated with L5-LDL (20 g/ml) for 24 h. D: macrophages derived from shLOX-1 or shLuc knockdown cells were incubated with LPS (100 pg/ml) for 24 h. Levels of IL-1 in the medium were determined by ELISA. LPS was used as a positive control. *P 0.05, compared with the L5-LDL-treated shLuc cells. E: levels of cleaved caspase-1 in the medium of PBS or L5-LDL-treated shLuc and shLOX-1 knockdown cells were detected by Western blotting. Coomassie blue staining was used as loading control. Values are the means SD for 3 independent experiments.

Article Snippet: A Quantikine Human IL-1 Enzyme-Linked Immunosorbent Assay (ELISA) kit and the Quantikine High Sensitivity Human IL-1 ELISA were obtained from R&D Systems (Minneapolis, MN).

Techniques: Knockdown, Activation Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Infection, shRNA, Quantitative RT-PCR, Control, Derivative Assay, Incubation, Positive Control, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Lipocalin 2: A New Antimicrobial in Mast Cells

doi: 10.3390/ijms20102380

Figure Lengend Snippet: Sphingosine 1-phosphate (S1P) activates MCs and induces antimicrobial peptide (AMP) production of both LCN2 and Cathelicidin AMP (CAMP). hMCs and mMCs were stimulated with PBS (control) or 200 nM S1P for 4 (for q-PCR) or 24 (for ELISA) hr. ( a ) Human LCN2 and ( b ) CAMP expression measured by q-PCR. LCN2 secretion from ( c ) hMCs or ( d ) mMCs quantified by ELISA. ( e ) CAMP protein secretion from hMCs quantified by ELISA. *: p < 0.05, ****: p < 0.0001.

Article Snippet: A human LL-37 ELISA kit (Hycult Biotech, Wayne, PA, USA and a human S1P ELISA kit (MyBioSource, San Diego, CA, USA) were used to determine LL-37 (the human cathelicidin peptide) and S1P levels in hMCs or NHEKs in accordance with the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Lipocalin 2: A New Antimicrobial in Mast Cells

doi: 10.3390/ijms20102380

Figure Lengend Snippet: Both MCs and normal human epidermal keratinocytes (NHEKs) produce S1P against LTA or commensal bacterial supernatant stimulation ( a ) hMCs and ( b ) mMCs were stimulated with TSB (control for staphylococcus-bacterium), RCM (control for C. acnes ), or 200 µL/mL bacterial supernatant from S. epidermidis (S. epi) , S. aureus , or C. acnes for 24 h, and S1P release from ( a ) hMCs or ( b ) mMCs were quantified by ELISA. ( c ) After 10 μg/mL LTA stimulation, NHEK SPHK1 ( Sphingosine kinase 1) expression was measured at different time points by q-PCR. ( d ) NHEKs were stimulated with PBS (control), 10 μg/mL LTA, 200 µL/mL S. epidermidis 12228, or 1457 for 24 h and S1P concentration was measured by ELISA. ( e ) NHEK TLR2 was blocked by 1 μg/mL TLR2 antibody (TLR2Ab) or IgG isotype control for 1 h. After TLR2 blocking, NHEKs were stimulated with 10 μg/mL LTA for 24 hr and S1P concentration was measured by ELISA. *: p < 0.05, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: A human LL-37 ELISA kit (Hycult Biotech, Wayne, PA, USA and a human S1P ELISA kit (MyBioSource, San Diego, CA, USA) were used to determine LL-37 (the human cathelicidin peptide) and S1P levels in hMCs or NHEKs in accordance with the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Lipocalin 2: A New Antimicrobial in Mast Cells

doi: 10.3390/ijms20102380

Figure Lengend Snippet: S1P activates and enhances MC antimicrobial activity. hMCs or mMCs were conditioned with either PBS or 200nM S1P in an antibiotic-free culture medium for 48 hr and conditioned mediums (hMC-CM+PBS or +S1P, mMC-CM+PBS or +S1P) were collected for antimicrobial liquid assays. ( a – d ) Antimicrobial liquid assay with ( a , b ) mMC-CMs or ( c , d ) hMC-CMs. ( a , c ) S. aureus SA113 or ( b , d ) E. coli were incubated in vehicle control (antibiotic-free cell culture medium), mMC or hMC-CM+PBS, or, mMC or hMC-CM+S1P. ( a , c ) S. aureus SA113 or ( b , d ) E. coli growths were demonstrated by CFU/mL. **: p < 0.01, ****: p < 0.0001.

Article Snippet: A human LL-37 ELISA kit (Hycult Biotech, Wayne, PA, USA and a human S1P ELISA kit (MyBioSource, San Diego, CA, USA) were used to determine LL-37 (the human cathelicidin peptide) and S1P levels in hMCs or NHEKs in accordance with the manufacturer’s instructions.

Techniques: Activity Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Lipocalin 2: A New Antimicrobial in Mast Cells

doi: 10.3390/ijms20102380

Figure Lengend Snippet: LCN2 is important for MCs capacity to defend against E. coli . mMCs derived from WT, Camp -/- , and Lcn2 -/- mice were conditioned with PBS or 200nM S1P for 48 hr, and mMC-CMs were collected for antimicrobial liquid assays against E. coli . ( a ) Baseline antimicrobial activity of WT, Camp -/- or Lcn2 -/- derived mMC-CM against E. coli and ( b ) anti- E. coli inhibition activity of Camp -/- or Lcn2 -/- derived mMC+PBS or +S1P. Bacteria growth was measured by OD600. Data was shown by E. coli inhibition % calculated by the formula shown in materials and methods. ( c ) Camp expression measured by RT-qPCR among mMCs derived from WT, Lcn2 -/- and Camp -/- mice with PBS or 200nM S1P stimulation. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: A human LL-37 ELISA kit (Hycult Biotech, Wayne, PA, USA and a human S1P ELISA kit (MyBioSource, San Diego, CA, USA) were used to determine LL-37 (the human cathelicidin peptide) and S1P levels in hMCs or NHEKs in accordance with the manufacturer’s instructions.

Techniques: Derivative Assay, Activity Assay, Inhibition, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) AML cell lines (KG1a and NB4 cells) were treated with DMSO (Ctrl), DAC (1 and 5 μM) or AZA (1 and 5 μM) for 48 hours. Soluble levels of MICA, MICB, ULBP1, ULBP2 and ULBP3 ligands were quantified by sandwich ELISA. Each bar represents the mean ± SEM of at least four independent experiments. * p < 0.05 and * p < 0.001. ( B ) KG1a and NB4 cells were treated with 5 μM of DAC for 48 hours. Before and after treatment, cell surface expression of NKG2DL was analyzed by flow cytometry using monoclonal antibodies specific to each NKG2DL (MICA, MICB, ULBP1, ULBP2 and ULBP3), followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and shaded grey histograms show the NKG2DL expression. Dotted vertical lines indicate mean fluorescence intensity value in untreated samples.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Bioprocessing, Incubation, Control, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) NKL cells were co-cultured with cell-free supernatants (sn) obtained from KG1a and NB4 cells untreated (sn-DMSO) or treated with 1 μM DAC for 48 hours (sn-DAC). NKL cells grow in culture medium were considered as a control (Ctrl). NKG2D expression was analyzed by flow cytometry and represented as mean fluorescence intensity (MFI). Each bar represents the mean ± SEM of three independent experiments. * versus control and p < 0.01; # versus sn-DMSO and p < 0.05. ( B ) NKL cells were co-cultured with K562 cells at the indicated E:T ratio in a cell lysis assay, in the absence (Ctrl) or presence of cellular supernatant derived from KG1a (left panel) and NB4 (middle panel) cells previously treated with DMSO (sn-DMSO) or 1 μM DAC (sn-DAC) for 48 hours. Specificity of the NKG2D-NKG2DL interaction was corroborated using an anti-NKG2D blocking mAb and the effect of DAC was assayed to analyze the non-specific effects on the lytic capacity of NKL cells (right panel). Measurements were made in duplicate and the mean ± SEM of the two independent experiments are shown. * versus control and p < 0.05; * versus control and p < 0.01; # versus sn-DMSO and p < 0.05.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Cell Culture, Control, Expressing, Flow Cytometry, Fluorescence, Lysis, Derivative Assay, Blocking Assay

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with inhibitors specific to ADAM17 (10 μM GW280264X) and ADAM10 (50 μM of GI254023X) for 48 hours. Levels of soluble NKG2DL (sMICA/B and sULBPs1-3) were quantified by sandwich ELISA. Values are the mean ± SEM of at least three independent experiments. * p < 0.05 and * p < 0.01. ( B ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (1 μM) for 48 hours. After treatment, cell surface expression of ADAM17 was analyzed by flow cytometry using anti-human ADAM17 monoclonal antibody, followed by incubation with FITC-conjugated goat anti-mouse IgG. The white histograms represent the isotype control antibody and the shaded grey histograms show the ADAM17 expression. ( C ) ADAM17 activity in KG1a and NB4 cells was measured in whole-cell lysates after treatment with DMSO (Ctrl) or DAC (5 μM) for 48 hours. Data are expressed as relative fluorescence units (RLU) at Ex/Em=490/520 nm absorbance normalized with respect to micrograms of total protein (RLU/μg). Values are the mean ± SEM of three independent experiments. * p < 0.05 and * p < 0.01.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing, Flow Cytometry, Incubation, Control, Activity Assay, Fluorescence

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) KG1a and NB4 cells were treated with DMSO (Ctrl) or DAC (0.25, 0.5 and 1 μM) for 48 hours, and TIMP3 expression was analyzed by qRT-PCR. Each bar represents the relative expression of TIMP3 normalized with respect to the reference gene (GAPDH), using the 2 −ΔCt method. MICA transcription levels in the KG1a cell line untreated (DMSO, Ctrl) or treated with DAC at different concentrations were used as a positive control. Results are summarized as the mean ± SEM of five independent experiments. * p < 0.05 and * p < 0.01. ( B ) TIMP3 protein levels were evaluated by western blot in KG1a and NB4 cells after treatment with DMSO (Ctrl) or DAC (1 μM or 5 μM) for 48 hours. * p < 0.05. ( C ) The TIMP3 methylation pattern was quantified by pyrosequencing in AML cell lines (KG1a and NB4 cells) before and after treatment with 1 μM or 5 μM DAC. Pie charts show the average percentage of methylation for the CpGs analyzed in the TIMP3 gene. ( D ) TIMP3 expression was inhibited by transfection of KG1a cells previously treated with DAC (1 μM) with a TIMP3-specific siRNA or nonspecific scramble siRNA (200 nM). * p < 0.05 ( E ) Soluble NKG2DL were quantified by sandwich ELISA after TIMP3 inhibition. Values shown are the mean ± SEM of three independent experiments. * versus control and p < 0.05; * versus control and p < 0.01 # versus nonspecific scramble siRNA and p < 0.05.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Methylation, Transfection, Sandwich ELISA, Inhibition, Control

Journal: Oncotarget

Article Title: Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

doi: 10.18632/oncotarget.16657

Figure Lengend Snippet: ( A ) Soluble NKG2DL were quantified by sandwich ELISA in sera from twelve AML patients before and after Vidaza ® treatment. Lines represent the levels of each sNKG2DL (ng/mL) before and after treatment of each individual AML patient. ( B ) Expression of NKG2DL on the cell surface of blasts from five AML patients before and after Vidaza ® treatment (left panel). The right panel shows dot plots of NKG2DL expression on the cell surface of blats from a representative patient. Numbers included in the figure quadrants indicate the percentage of positive cells for each NKG2DL.

Article Snippet: After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL (rhMICA Ref: 1300-MA, rhMICB Ref: 1599-MB, rhULBP1 Ref: 1380-UL, rhULBP2 Ref: 1298-UL,and rhULBP3 Ref: 1517-UL all from R&D Systems) were added for 2 hours at RT.

Techniques: Sandwich ELISA, Expressing

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A expression is upregulated in airway epithelial cells in asthma. A‐B) Immunohistochemical (IHC) staining and quantification of OTUD6A in human lung tissues from healthy controls ( n = 2) and asthmatic patients ( n = 2). Scale bars: 100 µm. C‐D) Western blot analysis of OTUD6A expression in lung tissue from HDM‐CAM (C) and HDM‐AAM (D) mice. E‐F) Representative images of IHC for OTUD6A on mouse lung sections of HDM‐CAM and corresponding quantitative analysis ( n = 5). Scale bars: 100 µm. G‐H) Representative images of IHC for OTUD6A on mouse lung sections of HDM‐AAM and corresponding quantitative analysis ( n = 5). Scale bars; 100 µm. I‐J) Immunofluorescence staining for OTUD6A in the mouse lung tissue of HDM‐AAM and corresponding quantitative analysis ( n = 5). Scale bars: 50 µm. K) Western blot analysis of OTUD6A protein levels in BEAS‐2B cells stimulated with HDM (100 µg/mL) at indicated time points ( n = 5). L‐M) BEAS‐2B cells transfected with OTUD6A or control vector for 24 h. L) Western blot analysis of ZO‐1 and Occludin. M) RT‐qPCR analysis of IL‐25 , IL‐33 , and TSLP mRNA levels ( n = 5). N‐O) HBEpiC cells transfected with OTUD6A or control vector for 24 h. N) Western blot analysis of ZO‐1 and Occludin. O) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). P‐Q) HBEpiC cells transfected with siOTUD6A or control vector for 48 h and stimulated with HDM (100 µg/mL) for 6 h. P) Western blot analysis of OTUD6A, ZO‐1, and Occludin. Q) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by two‐tailed unpaired t‐test or one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Western Blot, Immunofluorescence, Staining, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A knockout alleviates asthma in HDM‐induced acute asthma model. A) Schematic diagram depicting the procedure of HDM‐AAM. B) AHR assessed via acetylcholine challenge ( n = 5). C) Serum IgE levels measured by ELISA ( n = 5). D) Representative H&E and PAS staining of lung tissue. Scale bars: 100 µm. E) Quantification of bronchial epithelial thickness ( n = 5). F) BALF eosinophil counts determined by Wright‐Giemsa staining ( n = 5). G‐J) IL‐5 and IL‐13 levels in BALF (G, H) and lung homogenates (I, J) measured by ELISA ( n = 5). K‐N) RT‐qPCR analysis of Il5 , Il13 , Muc5ac , and Muc5b mRNA levels in lung tissue ( n = 5). O) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF ( n = 5). P) Western blot and quantification of ZO‐1 and Occludin in lung tissues. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A knockout mitigates asthma in HDM‐induced chronic asthma model. A) Schematic diagram depicting the procedure of HDM‐CAM. B‐D) Rn, Ers, and Rrs assessed via methacholine challenge ( n = 5). E) Representative H&E staining of lung tissue. Scale bars: 100 µm. F) Serum IgE levels measured by ELISA ( n = 5). G‐H) Total cell counts (G) and protein concentration (H) in BALF ( n = 5). I‐L) IL‐5 and IL‐13 levels in BALF (I, J) and lung homogenates (K, L) measured by ELISA ( n = 5). M‐R) RT‐qPCR analysis of Il5 , Il13 , Muc5ac , Tslp , Il25 , and Il33 mRNA level in lung tissues ( n = 5). S‐U) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF ( n = 5). Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knock-Out, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: hResistin/PI3K mediates OTUD6A‐induced EMT process and the expression of epithelial‐derived alarmins. A‐G) BEAS‐2B cells transfected with siResistin for 48 h and then transfected with Flag‐OTUD6A for 24 h. A) RT‐qPCR analysis of TSLP , IL‐25 , IL‐33 mRNA level in BEAS‐2B cells ( n = 5). B) ELISA analysis of TSLP, IL‐25, and IL‐33 levels in cell supernatant ( n = 5). C) RT‐qPCR analysis of TGFB1 , ACTA2 , COL1A1 mRNA level in BEAS‐2B cells ( n = 5). D‐E) Western blot analysis of OTUD6A, hResistin, EMT, and PI3K/AKT markers. F) Immunofluorescence staining of E‐cadherin and N‐cadherin. Scale bars: 50 µm. G) Cell wound healing assay. Scale bars: 50 µm. H‐K) BEAS‐2B cells were pretreated with LY294002 (30 µ m ) for 30 min and transfected with Flag‐OTUD6A for 24 h. H) Western blot analysis of hResistin and EMT markers. I‐K) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Expressing, Derivative Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Wound Healing Assay

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: Lung‐specific OTUD6A knockdown attenuates HDM‐induced asthma. A) Schematic diagram depicting the procedure of HDM‐induced chronic asthma model. B) Western blot analysis of OTUD6A and mRELMα in lung tissues. C) AHR assessed via acetylcholine challenge ( n = 5). D) H&E staining of lung tissues. Scale bars: 100 µm. E) Serum IgE levels measured by ELISA ( n = 5). F‐G) Total cell counts (F) and protein concentration (G) in BALF ( n = 5). H‐I) IL‐5 (H) and IL‐13 (I) levels in BALF measured by ELISA ( n = 5). J‐L) RT‐qPCR analysis of Il4, Il5 , and Muc5ac mRNA levels in lung tissues ( n = 5). M) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF (n = 5). N) Serum TGF‐β1 levels measured by ELISA ( n = 5). O) Masson's trichrome staining of lung sections. Scale bars: 100 µm. P) RT‐qPCR analysis of Tgfb1 , Acta2 , and Col1a1 mRNA levels in lung tissues ( n = 5). Q) Western blot analysis of Vimentin, TGFβ1, α‐SMA, and Fibronectin in the lung tissues. R) Western blot analysis of PI3K/AKT in the lung tissues. S) Western blot analysis of EMT markers in the lung tissues. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knockdown, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR

Journal: Frontiers in cell and developmental biology

Article Title: Pyrroline-5-Carboxylate Reductase 1 Directs the Cartilage Protective and Regenerative Potential of Murphy Roths Large Mouse Mesenchymal Stem Cells.

doi: 10.3389/fcell.2021.604756

Figure Lengend Snippet: FIGURE 3 | Pycr1 induces a glycolytic metabolism on MRL MSCs. (A,B) The metabolic activity of MRL MSC siCTL (MRL MSCs transfected with the siCTL siRNA) control (black line) and siPYCR1 (orange line) was evaluated by measuring the OCR (A) or the ECAR (B) with Seahorse analyzer. The knockdown of Pycr1 affects both the OCR and ECAR profiles, which translates to a preferentially oxidative metabolism. (C) The ratio between glycolytic rate and basal OCR confirms a significant decline in the glycolytic metabolism of MRL siPYCR1. (D) Similarly, we observed a significant decrease in lactate concentration in the extracellular media, as a result of the diminished glycolytic activity on MRL siPYCR1, compared with MRL siCTL. Results represent the mean ± SEM of three independent experiments with five different replicates each time. Statistics: Mann–Whitney unpaired t-test. ∗∗: p < 0.01. (E) The metabolic activity of BL6 MSC siCTL and BL6 MSC siPYCR1 was evaluated by measuring the OCR, ECAR and the ratio ECAR/OCR with Seahorse analyzer. MRL, Murphy Roths Large; MSCs, mesenchymal stem cell; siRNA, small interfering RNA.

Article Snippet: Briefly, MSCs were induced to differentiate using the protocol of micropellets, which consist in seeding the MSCs at 2.5 × 105 cells/well in 96-Well Polypropylene, centrifuged during 5 min at 400 g. The micropellets were cultured during 21 days in a medium containing DMEM (Invitrogen), 100 U/ml of penicillin/streptomycin, 10 μM of sodium-pyruvate, 1.7 μM of ascorbic acid-2-phosphate, insulin-transferrin-selenium (ITS; Sigma-Aldrich Corp., St. Louis, MO, United States), and 1 ng/ml of human Transforming Growth Factor β3 (hTGF-β3; R&D Systems, Minneapolis, MN, United States) prior to being recovered for RT-qPCR analysis.

Techniques: Activity Assay, Transfection, Control, Knockdown, Concentration Assay, MANN-WHITNEY, Small Interfering RNA

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Acyl-CoA synthetase 1 exacerbates lipotoxic inflammasome activation in primary macrophages

doi: 10.1002/JLB.3HI0219-045RR

Figure Lengend Snippet: (A, B) Peritoneal macrophages (pMACs) isolated from WT or TRIF KO mice (TRIF) were stimulated as indicated (LPS 100 ng, palmitate – 250 μM) for 16 h. Cell lysates (A) or mRNA (B) were prepared and ACSL1 protein and mRNA expression was assessed by Western blotting (A) and qRT-PCR (B), respectively. The bands are quantified and shown beneath the blot. Actin is shown as a loading control. (C, D) WT pMACs were stimulated as indicated for 16 h in the presence of an IFNAR-antibody (filled bars) or isotype control antibody (open bars). The expression of ACSL1 (C) and ISG15 (D) was quantified qRT-PCR and normalized to 36B4. (E) Protein was isolated from pMACs isolated from littermate WT or LysM-Cre X ACSL1flx/flx (KO) mice stimulated with control of palm-LPS for 16 h. ACSL1 protein expression was assessed by Western blotting. (F) WT (open bars) or mACSKO (filled bars) pMACs were treated with control (BSA-LPS) or palm (250 μM)-LPS (100 ng) for 20 h, and the release of IL-1β and TNF-α was determined by ELISA. (G) WT or mACSKO pMACs were treated with palm-LPS or stearate (stear; 150 μM)-LPS for 20 h and IL-1β release was quantified by ELISA. (F) pMACs from WT or mACSKO mice were stimulated with palm-LPS or treated with LPS 500 ng for 2 h followed by nigericin (nig; 1 μM) for 60 min or alum (500 μg/ml) for 6 h. IL-β release was quantified by ELISA. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. *, P < 0.05 for WT versus TRIFKO or mACSKO or control ab versus IFNAR ab; ns; nonsignificant

Article Snippet: Duoset ELISA kits (IL-1 β and TNF- α ) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Expressing, Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Acyl-CoA synthetase 1 exacerbates lipotoxic inflammasome activation in primary macrophages

doi: 10.1002/JLB.3HI0219-045RR

Figure Lengend Snippet: (A) WT (top panels) or mACSKO (bottom panels) pMACs were treated with the indicated stimuli for 16 h after which they were subjected to ORO staining to visualize crystals. Images were taken using confocal microscopy at 40× magnification. Nuclei were counterstained with DAPI (blue). (B) Quantification of ORO under all conditions is shown in A. (C) Electron microscopy of macrophages from WT and mACSKO mice demonstrating the ultrastructural appearance of intracellular crystals. (D) pMACs were treated with palm-LPS in the presence of the ACS inhibitor TC at 1 or 5 μM for 16 h after which the cells were stained with ORO, and the signal was quantified after extraction. (E) WT or mACSKO pMACs were treated with palm-LPS, and the indicated concentrations of TC and IL-1β release was quantified by ELISA. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate.*, P < 0.05 for WT versus mACSKO or veh versus TC

Article Snippet: Duoset ELISA kits (IL-1 β and TNF- α ) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Confocal Microscopy, Electron Microscopy, Extraction, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Frontline Science: Acyl-CoA synthetase 1 exacerbates lipotoxic inflammasome activation in primary macrophages

doi: 10.1002/JLB.3HI0219-045RR

Figure Lengend Snippet: (A) BODIPY-palmitate uptake was quantified by flow cytometry in WT and mACSKO pMACs at baseline and following LPS activation for 16 h. (B) WT pMACS were stimulated as indicated for 16 h after which cells were lysed and partitioned in to cytosolic (c) and mitochondrial (m) fractions. ACSL1 protein expression was assessed by Western blotting. Tubulin and VDAC were used as controls for fraction of cytosolic and mitochondrial compartments, respectively. Band quantification is shown beneath the blots. (C, D) pMACs from WT or mACSKO mice were stimulated as indicated for 16 h after which cells were lysed and C16 ceramide (C) or C16 acylcarnitine (D) were quantified by LC-MS/MS. (E) WT (black, gray lines) or mACSKO (red, blue lines) were treated with PBS (black, red lines) or LPS (gray, blue lines) for 16 h and cellular OCR was assessed using a Seahorse flux analyzer. The arrows indicate drug injections occurring during the run. (F) pMACs were treated with palm-LPS ± 50 μM etomoxir (eto) for 20 h, and IL-1β release was quantified by ELISA. (G) Macrophages from WT and mACSKO mice were stimulated with BSA-PBS (red line) or palm-LPS (black line) for 16 h and mitochondrial ROS was assessed by mitosox staining coupled with flow cytometry. As a control for mitochondrial ROS generation, WT cells were treated with rotenone for 6 h (rot). Representative histograms are displayed. Bar graphs report the mean ± SE for a minimum of 3 experiments, each performed in triplicate. *, P < 0.05 for WT versus mACSKO

Article Snippet: Duoset ELISA kits (IL-1 β and TNF- α ) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Activation Assay, Expressing, Western Blot, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Molecular & Cellular Proteomics

Article Title: Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages

doi: 10.1074/mcp.m112.024661

Figure Lengend Snippet: FIG. 3. The secretion of danger signal proteins, chemokines and interferon-induced proteins is induced on MSU stimulation from LPS-primed macrophages. A, Human macrophages were left unstimulated or primed with LPS for 21 h. After this macrophages were left untreated or activated with MSU for 3 h and the cell culture supernatants were collected. Exosomal fractions were enriched as depicted in Materials and Methods, and Western blotting analysis of danger signal proteins Annexin-1, Galectin-3 (Gal-3), and HSP90 was performed. B, Human macrophages were stimulated as in (A), and the secretion of chemokines CCL2, CCL3, CCL4, CCL5, and CXCL10 was quantified by Luminex assay. Representative data from one experiment is shown; n 2 independent experiments. C, Macrophages were left untreated or primed with LPS after which they were stimulated with MSU for 3 and 6 h. Total cellular RNA was isolated, and expression of chemokine genes was analyzed with quantitative RT-PCR as depicted in Materials and Methods. The data are reported as fold induction compared with controls. Mean with S.D. of two independent experiments is shown. D, Western blot analysis of IFIT-3 and MxA expression from total cellular lysates and cell culture supernatants of untreated and LPS-primed macrophages with and without MSU stimulation.

Article Snippet: Reaction was set up in PerfeCta qPCR FastMix (Quanta BioSciences) with predeveloped TaqMan Gene Expression Assay primers and probes (CCL2: Hs00234140_m1, CCL3: Hs00234142_m1, CCL4: Hs99999148_m1, CCL5: Hs00174575_m1, CXCL10: Hs00171042_m1, Applied Biosystems).

Techniques: Cell Culture, Western Blot, Luminex, Isolation, Expressing, Quantitative RT-PCR

Journal: Molecular & Cellular Proteomics

Article Title: Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages

doi: 10.1074/mcp.m112.024661

Figure Lengend Snippet: FIG. 4. Cathepsin activity is required for unconventional protein secretion on MSU stimulation. A, Macrophages were left untreated or LPS-primed after which they were activated with MSU for 3 h. Cathepsin B and D expression in total cellular lysates and secretion to supernatants was analyzed with Western blotting. B, Untreated and LPS-primed macrophages were activated with MSU for 3 h in the presence and absence of CA-074 Me. After this IL-1 secretion was measured from supernatants with ELISA. Representative data from one experiment is shown; n 4 independent experiments. C, Untreated and LPS-primed macrophages were activated with MSU for 3 h in the presence and absence of CA-074 Me. Secreted proteins were visualized by silver staining. D, Secretion of IL-1, IL-18, ASC, IFIT-3, Galectin-3 (Gal-3), and LAMP-1 was analyzed by Western blotting. E, The secretion of CCL2, CCL3 and CCL4 was quantified with Luminex assay. Representative data from one experiment is shown; n 4 independent experiments.

Article Snippet: Reaction was set up in PerfeCta qPCR FastMix (Quanta BioSciences) with predeveloped TaqMan Gene Expression Assay primers and probes (CCL2: Hs00234140_m1, CCL3: Hs00234142_m1, CCL4: Hs99999148_m1, CCL5: Hs00174575_m1, CXCL10: Hs00171042_m1, Applied Biosystems).

Techniques: Activity Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Silver Staining, Luminex

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cancer-Associated Fibroblasts Facilitate Squamous Cell Carcinoma Lung Metastasis in Mice by Providing TGFβ-Mediated Cancer Stem Cell Niche

doi: 10.3389/fcell.2021.668164

Figure Lengend Snippet: Cancer-associated fibroblasts increased self-renewal and invasion of CSCs in a TGFβ-dependent manner via direct contact and paracrine effects. (A) Representative images of GFP + A223 CSCs and RFP + CAFs in spheroid co-culture. (B) Quantification of sphere number in each of the indicated A223 CSC treatment [vehicle (DMSO) or TGFβ inhibitor (“TGFβi”)] ± fibroblast co-culture conditions. (C,D) Representative images and quantification of A223 CSC spheres cultured with the indicated conditioned media (CM) and vehicle (DMSO) or TGFβi. (E) Transwell invasion assay: A223 CSCs were seeded in the top Matrigel-coated chamber, and CAFs or NAFs were cultured in the bottom well. After 48 h, invaded CSCs attached to the underside of the upper chamber were quantified. (F) TGFβ1 concentration in each of the indicated CM was determined by ELISA. Either three or four technical replicates were conducted for each cell type. (G) The CM of CAFs or unconditioned control media was applied to recipient A223 cells in a 24 h sphere initiation assay. RNA harvested from A223 recipient cells was evaluated by RT-qPCR for the expression of TGFβ-target genes Spp1 , Junb , and Vegfa normalized to the expression of Gapdh . n s P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: RT-qPCR was performed using 40 ng RNA, Brilliant II QRT-PCR 1-Step Master Mix (Agilent, Santa Clara, CA, United States) and TaqMan gene expression assays for Junb (Mm04243546_s1), Spp1 (Mm00436767_m1), Vegfa (Mm00437306_m1), and Gapdh (Mm99999915_g1) (ThermoFisher, Rockford, IL, United States).

Techniques: Co-Culture Assay, Cell Culture, Transwell Invasion Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 1. The Recombinant Soluble CD89 Receptor Interacts Directly with Bacteria (A and B) Dose-dependent binding of soluble recombinant CD89 (sCD89) to fixed S.p (A) and E. coli (B). Binding to albumin (Alb) was used as a control. (C) Comparison of sCD89 binding to various types of fixed bacteria. (D) Interaction of sCD89 with live (green bar) versus fixed (black bars) 106 E. coli or 106 S. p. (E and F) S.p (E) and E. coli (F) binding to BMMs grown from CD89 transgenic mice (CD89Tg) or from littermates, visualized by confocal laser-scanning mi- croscopy. Right: quantification of binding (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (G and H) S.p (G) and E. coli (H) binding to BMMs isolated from CD89Tg mice or from littermates in the presence or absence of the anti-CD89 blocking antibody MIP8a F(ab’)2 (10 mg/mL) or of sCD89 (500 mg/mL), analyzed by flow cytometry. MFI, mean fluorescence intensity. Data are presented as mean ± SEM; n = 5. *p < 0.05, **p < 0.01; t test. See also Figure S1.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Recombinant, Bacteria, Binding Assay, Control, Comparison, Transgenic Assay, Isolation, Blocking Assay, Cytometry

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 2. Bacterium-CD89 Interaction on Mouse Cells Induces Activating ITAM Signaling, Leading to Inflammatory Cytokine Production, Bacterial Phagocytosis, and Killing (A and B) IL-6, TNF-a, and IL-1 production in the supernatant of BMMs obtained from CD89Tg and CD89R209L transgenic mice and littermate controls. Cells were incubated for 16 h in the presence of S.p (A) and E. coli (B) and cytokines in the supernatants were measured by ELISA. All data are presented as mean ± SEM; n = 3. *p < 0.05, **p < 0.01; t test. (C) Confocal analysis of E. coli-pHrodo phagocytosis by BMMs obtained from CD89Tg mice compared with littermates in the presence or absence of MIP8a F(ab)’2 or sCD89 in a dose-dependent manner (100–800 mg/mL). Left: representative images. Right: quantification. Data are presented as mean ± SEM; n = 3. ***p < 0.001, t test. (D) ROS production over 30 min by littermate, CD89Tg, and CD89R209L transgenic BMMs stimulated by live S.p (left) or E. coli (right), measured by confocal microscopy. All data are presented as mean ± SEM; n = 15. **p < 0.01, t test. (E) Quantification of bacterial survival after 2 h of incubation with BMMs from CD89Tg, CD89R209L Tg, and littermate mice. Data are presented as mean ± SEM; n = 3. ***p < 0.001, t test.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Transgenic Assay, Incubation, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 3. IgA-Deficient CVID Phagocytes Mediate Phagocytosis, ROS Production, and Bacterial Killing through CD89 Interaction (A) Representative plots of CD89 expression on blood monocytes isolated from healthy donors (HDs) (left) and CVID patients (right) using a phycoerythrin (PE)-conjugated anti-CD89 antibody and its isotype control. (B) Binding of S.p or E. coli to blood monocytes from HDs (purple symbols) or from CVID patients (red symbols) in the presence of monomeric IgA (500 mg/mL) or of MIP8a F(ab’)2 (10 mg/mL). All data are presented as mean ± SEM; n = 4. ***p < 0.001, t test. (C) Phagocytosis of E. coli-pHrodo by human blood monocytes and/or macrophages isolated from HDs or from CVID patients. Left: representative images. Scale bars, 200 mm. Right: quantification (n = 3). All data are presented as mean ± SEM. ns, not significant. (D) IL-6, TNF-a, and IL-1 production in the supernatant of monocytes obtained from CVID patients. Cells were incubated for 16 h in the presence of E. coli or S.p and in the presence or absence of MIP8a F(ab)’2 (500 mg/mL), and cytokines in the supernatants were measured by ELISA. All data are presented as mean ± SEM; n = 3. *p < 0.05, **p < 0.01, ****p < 0.0001; t test.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Expressing, Isolation, Control, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 4. Role of CD89-Bacterium Interaction under Physiological Conditions (A) Competitive ELISA assays between sCD89 and S.p (blue line) or E. coli (red line) and ns-IgA. (B) Competitive ELISA assays between sCD89 and S.p (blue line) or E. coli (red line) and pd-IgA. (C) Bacterial phagocytosis by BMMs obtained from CD89Tg mice (left) compared with littermates (right). Bacteria were allowed to be phagocytosed by BMMs from the indicated mice in the presence or absence of ns-IgA at physiological concentration (2 mg/mL) or MIP8a F(ab)’2 (500 mg/mL). Cells were washed and analyzed by flow cytometry. Data are presented as mean ± SEM; n = 3. *p < 0.05, ***p < 0.001; t test. (D) S.p (left) or E. coli (right) phagocytosis by BMDCs obtained from CD89Tg mice compared with littermates. Bacteria were allowed to be phagocytosed by BMDCs from the indicated mice in the presence or absence of ns-IgA at physiological concentration (2 mg/mL). Cells were washed and analyzed by flow cytometry. Data are presented as mean ± SEM; n = 3. *p < 0.05, ***p < 0.0001; t test. (E) Representative images of E. coli (blue) and CD11c (red) staining by BMDCs derived from CD89Tg or wild-type (WT) mice captured by imaging flow cytometry (scale bars, 5 mm) and the percentages of the bacterial phagocytosis score. See also Figures S1C and S5C.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Competitive ELISA, Bacteria, Concentration Assay, Cytometry, Staining, Derivative Assay, Imaging

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 5. CD89-Bacterium Interaction Protects against Infection-Related Mortality in Mice (A) Survival of CD89Tg mice (red line) and littermates (black line) after intranasal inoculation (at time 0) with S. pneumonia (n = 25). Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. *p < 0.05. (B) Decreased lung contents of S.p in CD89 transgenic compared with littermate mice. All data are presented as mean ± SEM; n = 8. ***p < 0.001, t test. (C) H&E staining of lung sections from representative CD89Tg and littermate animals after intranasal infection. Scale bars, 200 mm. (D) Alveolitis invasion score of monomorphic inflammatory cells. All data are presented as mean ± SEM; n = 6. ***p < 0.001, t test. (E) mRNA expression of cytokines (IL-1, TNF-a, and IL-6) was assessed by qRT-PCR of 5 independent lung tissue RNA samples collected 6 and 48 h after intranasal infection. mRNA levels were normalized to b-actin mRNA levels. All data are presented as mean ± SEM; n = 6. *p < 0.05, t test. (F) Increased survival of CD89Tg mice (red line, n = 26) compared with littermates (black line, n = 22) after CLP. Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. **p < 0.01. (G–I) 48 h after CLP, peritoneal fluid was evaluated for total bacteria (G), E. coli (H), and Enterococcus (I) in CD89Tg mice and littermates. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; t test. (J) IL-1, TNF-a, and IL-6 levels in peritoneal lavage, assessed by ELISA 6 and 48 h after CLP. All data are presented as mean ± SEM; n = 3. *p < 0.05, t test. See also Figures S6A–S6C.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Infection, Transgenic Assay, Staining, Expressing, Quantitative RT-PCR, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

doi: 10.1016/j.celrep.2019.03.062

Figure Lengend Snippet: Figure 7. CD89 Protection against Sepsis Is Independent of CRP and IgA Antibodies during the Early Phase of Infection (A) Increased survival of CD89TgCRP-KO animals after intranasal infection with S.p compared with CRP-KO mice (n = 12 per group). CD89Tg mice and their littermates were used as controls. Kaplan-Meier curves and log rank test were used to compare mortality rates. All data are presented as mean ± SEM. (B) Decreased lung counts of S.p in CD89TgCRP-KO mice at 48 h compared with CRP-KO mice (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (C) Expression of cytokine mRNA (IL-1, TNF-a, and IL-6) was assessed by qPCR of independent lung tissue RNA samples collected 6 and 24 h after intranasal infection. Cytokine mRNA levels were normalized to b-actin mRNA levels, as indicated in Figure 5E (n = 4). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (D) Increased survival of CD89TgCRP-KO (n = 10) compared with CRP-KO mice (n = 10) after CLP. Kaplan-Meier curves and log rank test were used to compare mortality rates. CD89Tg mice and their littermates were used as controls. All data are presented as mean ± SEM. (E) Peritoneal fluid counts of bacteria 48 h after CLP (n = 4). All data are presented as mean ± SEM. **p < 0.01, t test. (F) IL-1, TNF-a, and IL-6 levels in peritoneal lavage, assessed by ELISA 6 and 48 h after CLP (n = 4). All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (G) Phagocytosis of S.p (left) and E. coli (right) after incubation with BMMs isolated from CD89TgCRP-KO or CRP-KO mice. (H) Phagocytosis of S.p (left) and E. coli (right) after incubation with BMMs isolated from CD89TgCRP-KO or CRP-KO mice in the presence of MIP8a anti-CD89 F(ab’)2. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01; t test. (I) Measurement of mouse IgA antibodies against the indicated bacteria 48 or 168 h after S.p infection (left) or CLP (right) in CD89Tg or CD89TgCRP-KO mice. Data are presented as mean ± SEM. See also Figure S7.

Article Snippet: The Escherichia coli (E. coli-K12, Strain SMG 123 (PTA-7555)), Staphylococcus aureus subsp. aureus Rosenbach (S. aureus, ATCC 25923), Streptococcus pyogenes Rosenbach (S. pyogenes, ATCC 19615) and Escherichia coli-K12 WzxE (Coli genetic stock center, Yale university) were used for sCD89-bacteria interaction assays shown in Figure 1C.

Techniques: Infection, Expressing, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Isolation