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: 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

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: FAP + fibroblasts orchestrate tumor microenvironment remodeling in renal cell carcinoma with tumor thrombus

doi: 10.1038/s41467-025-64447-2

Figure Lengend Snippet: A UMAP plot of mesenchymal cells ( n = 13,965 cells) from our discovery dataset ( n = 22 patients; n = 22 AT, 22 PT, and 12 TT samples), colored by mesenchymal cell subsets. For the distribution of these cells by tissue region (AT, PT and TT), clinical subgroup (with and without TT), and patient identity, see also Supplementary Fig. . B Heatmap showing representative marker genes for each mesenchymal cell subset identified in the discovery dataset. C Systematic evaluation of alterations in the proportions of mesenchymal cell subsets in our discovery dataset, based on our comparison strategy (refer to Supplementary Fig. ). For more details, see the legend for Fig. . Full results are provided in Supplementary Data . D Schematic diagram of the scRNA-seq workflow used for the validation dataset, which applied a negative selection strategy to enrich for mesenchymal and epithelial cells. E ssGSEA analysis showing the enrichment scores of FAP + fibroblast (upper) and CYSLTR2 + fibroblast (lower) signatures—derived from our discovery dataset —in ATs (right; n = 18 and 54, respectively) and PTs (left; n = 99 and 431, respectively) from RCC patients with and without TT in the TCGA-KIRC bulk RNA-seq dataset. P -values were determined using the two-sided Wilcoxon rank-sum test. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. F Box plots showing the proportions of FAP + fibroblasts (upper) and CYSLTR2 + fibroblasts (lower) in PTs from patients with and without TT, based on scRNA-seq data from both our discovery dataset ( n = 8 and n = 5, respectively; PT samples with <50 mesenchymal cells were excluded) and the Yu et al . dataset ( n = 19, without TT only). Groups were defined as in Fig. , and P -values were calculated using the two-sided unpaired t -test. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. G Stacked bar plots showing the proportions of mesenchymal cell subsets in PTs from each patient in our validation dataset ( n = 6). We highlighted FAP + fibroblasts and CYSLTR2 + fibroblasts in this plot. Patient IDs were colored by TT status (blue: with TT; red: without TT). H Representative multiplex immunofluorescence images (left) and quantification (right) of FAP + fibroblasts—defined as DCN + FAP + double-positive cells—in tumor sections from RCC patients without ( n = 8) and with ( n = 8) TT. DCN (green) marks fibroblasts, and FAP (red) labels the specific fibroblast subset. FAP + fibroblasts were annotated and quantified using QuPath. Box plots show the distribution of the proportion of DCN + FAP + cells across groups. The box represents the interquartile range (IQR, 25th–75th percentile), with the horizontal line indicating the median. Whiskers denote minimum and maximum values. This analysis provides orthogonal validation for Fig. 3C. Scale bar = 200 µm. Scale bar inset = 50 µm. P -values were calculated using the two-sided Wilcoxon rank-sum test. I Violin plot showing the cell2location-inferred proportions of each mesenchymal cell subset within CN15, based on spatial mapping of 48 spatial transcriptomics samples using reference signatures estimated from our discovery dataset. J Representative multiplex immunofluorescence images (left) and quantification (right) of CN15-like regions—refined based on the spatial adjacency of FAP + fibroblasts (DCN + FAP + , green & red) and EMT-like cancer cells (PLOD2 + , white)—in tumor sections from RCC patients without ( n = 5) and with ( n = 5) TT. DCN + FAP + double-positive cells and PLOD2 + cells were annotated using QuPath. Their interface regions were manually delineated and quantified using Fiji software, followed by normalization to the total area of the tumor section. Box plots show the distribution of the interface area (% tissue area) of DCN + FAP + cells and PLOD2 + cells across groups. The box represents the interquartile range (IQR, 25th-75th percentile), with the horizontal line indicating the median. Whiskers denote minimum and maximum values. This analysis provides spatial validation for Figs. H and 3I. Scale bar = 200 μm; Scale bar inset = 50 μm. P -values were determined using the two-sided Wilcoxon rank-sum test. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. Source data are provided as a Source Data file.

Article Snippet: Cells were fixed in 4% formaldehyde for 10 min followed by permeabilization with 0.5% Triton X-100 for 5 min, then incubated for 30 min in PBS-T containing 5% goat serum, followed with the following antibodies: FAP (Cell Signaling Technology, 66562, 1:100); α-SMA (Invitrogen, 14-9760-82, 1:400); DCN (Proteintech, 14667-1-AP, 1:100) overnight at 4 °C.

Techniques: Marker, Comparison, Biomarker Discovery, Selection, Derivative Assay, RNA Sequencing, Multiplex Assay, Immunofluorescence, Software

Journal: Nature Communications

Article Title: FAP + fibroblasts orchestrate tumor microenvironment remodeling in renal cell carcinoma with tumor thrombus

doi: 10.1038/s41467-025-64447-2

Figure Lengend Snippet: A ssGSEA score of FAP + fibroblast signature for the indicated clinical stage (Stage I–IV; n = 269, 57, 125 and 83 patients, respectively), T stage (T1–T4; n = 275, 69, 182 and 11 patients, respectively) and pathological grading (G1–G4; n = 14, 230, 207 and 78 patients, respectively) in the TCGA-KIRC dataset. P -values were determined by the two-sided Wilcoxon rank-sum test and adjusted for multiple comparisons using the Benjamini-Hochberg method. Box plots display median, upper and lower quartiles, with whiskers indicating maximum and minimum data points within 1.5 × interquartile range. B Kaplan–Meier analysis for overall survival in TCGA-KIRC dataset based on FAP + fibroblast (left; high, n = 241; low, n = 292) and CYSLTR2 + fibroblast (right; high, n = 199; low, n = 334) signature scores using optimal cutoff method. P -values were determined by the two-sided log-rank test. C Kaplan–Meier analysis for overall survival in the nivolumab arm (left; high, n = 132; low, n = 49) and everolimus arm (right; high, n = 63; low, n = 67) of Checkmate cohorts based on FAP + fibroblast signature score using optimal cutoff method. P -values were determined by the two-sided log-rank test. D Kaplan–Meier analysis for progression-free survival in the sunitinib arm (left; high, n = 204; low, n = 168) and avelumab + axitinib arm (right; high, n = 73; low, n = 281) of Javelin 101 cohort based on FAP + fibroblast signature score using optimal cutoff method. P -values were determined by the two-sided log-rank test. E Kaplan–Meier analysis for progression-free survival in the nivolumab arm (left; high, n = 38; low, n = 143) and everolimus arm (right; high, n = 73; low, n = 57) of Checkmate cohorts based on FAP + fibroblast signature score using optimal cutoff method. P -values were determined by the two-sided log-rank test. F Left: spatial mapping of cell niches in 2 representative samples ( n = 1778 and 4972 spots, respectively) to show the close proximity of fibroblast-enriched cell niches (CN6) and intra-tumoral vasculature (CN12). Right: spatial mapping of tumor EC 1 and pericyte 2, FAP + fibroblast in representative samples, showing only spots where the cell2location-inferred proportion of these cells exceeds 8% ( n = 561 and 1285 spots, respectively). Data are representative of n = 4 spatial transcriptomic slides. G Representative images of immunofluorescence staining of DCN (fibroblast, green), CSPG4 (pericyte, red), FAP ( FAP + fibroblast, yellow) and DAPI (blue) on a RCC section. Data shown are representative of three biologically independent replicates. Similar results were observed in all replicates.

Article Snippet: Cells were fixed in 4% formaldehyde for 10 min followed by permeabilization with 0.5% Triton X-100 for 5 min, then incubated for 30 min in PBS-T containing 5% goat serum, followed with the following antibodies: FAP (Cell Signaling Technology, 66562, 1:100); α-SMA (Invitrogen, 14-9760-82, 1:400); DCN (Proteintech, 14667-1-AP, 1:100) overnight at 4 °C.

Techniques: Immunofluorescence, Staining

Journal: PloS one

Article Title: Kinetic analysis of mouse brain proteome alterations following Chikungunya virus infection before and after appearance of clinical symptoms.

doi: 10.1371/journal.pone.0091397

Figure Lengend Snippet: Figure 5. Western blot validations of differentially regulated proteins identified by 2D-DIGE and/or iTRAQ analyses. (A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock (M), early (E), late paralytic (LP) and late tetanus-like (LT), separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye; green) and the specific immunoreactive proteins (FITC or Cy5 dye; red). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean 6 S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons. The significance of the differential protein expression are indicated *, p,0.05; **, p,0.01; ***, p,0.001. A.U., arbitrary units. ANXA2: annexin A2; ARRB1: b-arrestin; GABRA1: c-aminobutyric acid receptor subunit alpha-1; GRASP1: GRIP-associated protein; ITGAV: integrin aV; MYPT1: myosin phosphatase target subunit 1; N-Ras: N-Ras; RABEP1: rabaptin-5; SYNGR3: synaptogyrin-3. doi:10.1371/journal.pone.0091397.g005

Article Snippet: Blots were saturated with 5% nonfat dried milk in PBS containing 0.05% (v/v) Tween 20 (PBS-T-milk) for 1 h. Western blot (WB) analyses were carried out with rabbit mono- or polyclonal antibodies directed against b-arrestin (1:5000, ARRB1, no. 4674, Cell Signaling Technology, Danvers, MA), GRIP-associated protein (1:500, GRASP1, no. sc-135681, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), annexin A2 (1:100, ANXA2, no. sc-9061, Santa Cruz), integrin aV (1:100, ITGAV, no. 10179, Santa Cruz), myosin phosphatase target subunit 1 (1:100, MYPT1, no. sc25618, Santa Cruz), rabaptin-5 (1:1000, RABEP1, no. sc-15351, Santa Cruz), N-Ras (1:500, N-Ras, no. sc-519, Santa Cruz), synaptogyrin-3 (1:1000, SYNGR3, no. sc-68936, Santa Cruz), or with a goat polyclonal antibody directed against c-aminobutyric acid receptor subunit alpha-1 (1:100, GABAARa1 or GABRA1, no. sc-31045, Santa Cruz), diluted in PBS-T-milk and incubated overnight at 4uC.

Techniques: Western Blot, Multiplex sample analysis, Labeling, SDS Page, Fluorescence, Software, Expressing