Journal: iScience

Article Title: LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates

doi: 10.1016/j.isci.2023.106880

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Article Snippet: Proteins were detected using the following antibodies: Monoclonal anti-GFP horseradish peroxidase-coupled (Miltenyi Biotech 130-091-833, 1/5,000), anti-ubiquitin P4D1 (Cell Signaling, 1/2,000).

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Expressing, Magnetic Beads, Reverse Transcription, Protease Inhibitor, Software

Journal: iScience

Article Title: circIFNGR2 regulating ankylosing spondylitis-associated inflammation through macrophage polarization

doi: 10.1016/j.isci.2023.107325

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Article Snippet: On day 21 from the first immunization, boost injection of 100 μL bovine type II collagen emulsified in incomplete Freund’s adjuvant (Chondrex, WA, USA) was conducted.

Techniques: Virus, Recombinant, Adjuvant, In Vivo, SYBR Green Assay, Transfection, Lysis, Blocking Assay, Western Blot, DNA Extraction, Protein Extraction, Enzyme-linked Immunosorbent Assay, Purification, RNA Immunoprecipitation, Luciferase, Software

Journal: Infection and Immunity

Article Title: Molecular and Functional Characterization of Fcγ Receptor IIIb-Ligand Interaction: Implications for Neutrophil-Mediated Immune Mechanisms in Malaria

doi: 10.1128/IAI.00924-17

Figure Lengend Snippet: Binding of FcγRIIIb alloforms onto IgG by ELISA and SPR. (A) ELISA. Biotin-labeled recombinant FcγRIIIb alloforms (HNA-1aa, -1bb, and -1bc) were added into microtiter wells coated with 2.5 μg IgG or BSA for 1 h at room temperature. After washings, bound FcγRIIIb protein was detected with HRP-conjugated streptavidin using TMB as the substrate. The color reaction was read on an ELISA reader at 450 nm. Data are presented as means ± SD from three independent experiments. (B) SPR. Different amounts of IgG fractions (50, 100, 200, 400, and 800 nmol/liter) were injected over three flow cells coated with different recombinant FcγRIIIb alloforms (HNA-1aa, -1bb, and -1bc). The binding response in real time was recorded as resonance units (response units) for 500 s. The dissociation constant (Kd) was analyzed using computer software (ProteOn Manager; Bio-Rad).

Article Snippet: Wells were washed twice, 100 μl of horseradish peroxidase-conjugated streptavidin (1:2,000 in PBS; GE Healthcare UK Limited, Buckinghamshire, UK) was added, and the mixture was incubated for 1 h at room temperature.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Recombinant, Injection, Software

Journal: Nature Communications

Article Title: Innate lymphocyte-induced CXCR3B-mediated melanocyte apoptosis is a potential initiator of T-cell autoreactivity in vitiligo

doi: 10.1038/s41467-019-09963-8

Figure Lengend Snippet: CXCL10 activates melanocytic CXCR3B to induce apoptosis. a Effect of CXCL10 (5, 20, 100 pg/ml) on cell viability in unstimulated or IFNγ stimulated (50 ng/ml for 48 h) melanocytes from vitiligo patients, in presence or absence of CXCR3 antagonist AS612568 (0.02 μM, 0.2 μM or 2 μM) ( n = 5–8). Cell viability was monitored using lncuCyte® live cell fluorescence imaging system. b Illustrates live IncuCyte images of vitiligo melanocytes 24 h after exposure to CXCL10 in cells transfected with siC or siCXCR3. Melanocytes were tracked with CellTracker TM Red CMPTX dye and dead cells tracked with IncuCyte® Cytotox Green reagent. Co-localised yellow cells represent dead melanocytes. The effect of siCXCR3 (or its siC) on CXCL10 (100 pg/ml)-induced death of healthy ( n = 4–8) and vitiligo ( n = 4) melanocytes are shown in c . In d healthy and vitiligo melanocytes ( n = 6–12) were transfected with siCXCR3B (or its siC) and melanocyte death shown at 24 h following CXCL10, CXCL9 or CXCL11 (100 pg/ml) stimulation. In separate experiments, cell lysates was used to study the signalling pathway induced by chemokines, IFNγ (50 ng/ml) or Staurosporine (positive control, 1 μg/ml) at 24 or 48 h post stimulation, measuring the expression levels of phosphorylated and total p38 and total and cleaved poly(ADP-ribose) polymerase (PARP) by Western Blot analysis ( e ). HSP90 was used as an internal loading control. Representative blot of 3 separate experiments is shown. Total and cleaved caspase-3 activity ( f ) and proportion of apoptotic cells (counted as % Annexin V+DAPI+cells in FACS analysis) ( g ) from healthy ( n = 4) and vitiligo ( n = 4) melanocytes stimulated with 100 pg/ml CXCL10 for 24 h in presence or absence of QVd OPh (10 μM, caspase inhibitor) ( h ). Results are shown as individual dot plots with a line at mean ± SEM

Article Snippet: Finally, Incucyte ® green Cytotox Reagent (100 nM, Essen Bioscience, Michigan, USA) was added to all wells and melanocyte death monitored in real-time using IncuCyte ® Zoom live-cell imaging system (Essen Biosciences) which was inside a 37 °C humidified CO 2 incubator scanning the plate every 2 h. Multiple images were collected per well and quantification of dead melanocytes (yellow co-localised cells) was analysed using the integrated Zoom ® software.

Techniques: Fluorescence, Imaging, Transfection, Positive Control, Expressing, Western Blot, Activity Assay

Journal: Nature Communications

Article Title: Innate lymphocyte-induced CXCR3B-mediated melanocyte apoptosis is a potential initiator of T-cell autoreactivity in vitiligo

doi: 10.1038/s41467-019-09963-8

Figure Lengend Snippet: T cells enhance CXCL10-induced melanocyte death by induction of adaptive immunity. a CXCL10-induced death of vitiligo melanocytes in presence or absence of patients own autologous T cells. As above, IFNγ-pretreated melanocytes were exposed to CXCL10 in presence or absence of CXCR3 antagonist AS612568 (2 μM). The next day, media was replaced and 3 days later patient’s own CD3+ T cell were sorted and added to the melanocytes ( n = 3) prior to initiation of IncuCyte. b At the end of the experiment (~48 h), supernatant was collected, remaining cells trypsinised and cytospin sections prepared for immunofluorescence detection of co-stimulatory (CD40, CD80, HLA-DR) and adhesion (ICAM-1) molecules on melanocytes. c T cell-induced potentiation of melanocyte death in IFNγ-pretreated melanocytes (compared to untreated melanocytes) was associated with a parallel increase in the number of CD3+ T cells which was supported by increased expression of Ki67+cells in the same cytospin sections d . Results are shown as individual dot plots with a line at mean ± SEM. e T cell proliferation was quantified by flow analysis measuring the percentage of CD3+CSFE+ cells undergoing 0, 1, 2 or 3+divisions ( n = 3). Labelled cells at time zero was used as a negative reference, unstimulated cells left in culture for 72 h before labelling as a control and cells stimulated with PHA for 72 h (Phytohemagglutinin, 5 μg/ml) as a positive control

Article Snippet: Finally, Incucyte ® green Cytotox Reagent (100 nM, Essen Bioscience, Michigan, USA) was added to all wells and melanocyte death monitored in real-time using IncuCyte ® Zoom live-cell imaging system (Essen Biosciences) which was inside a 37 °C humidified CO 2 incubator scanning the plate every 2 h. Multiple images were collected per well and quantification of dead melanocytes (yellow co-localised cells) was analysed using the integrated Zoom ® software.

Techniques: Immunofluorescence, Expressing, Positive Control