Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Tumor necrosis factor-like ligand 1A (TL1A) expression and secretion increase as asthma progresses. (A) Western blot analysis of TL1A protein expression in the lung tissues in the wild-type and ovalbumin (OVA)-induced model groups. (B) Changes in the cytokine concentrations of TL1A and immunoglobulin E (IgE) in serum over time following the final challenge in cases and controls. (C) Changes in the cytokine concentrations of TL1A, interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 13 (IL-13), and interferon gamma (IFN-γ) in bronchoalveolar lavage fluid (BAL) over time following the final challenge in cases and controls. (D) Schematic summarizing the timeline of the in vivo asthma modeling with different allergen exposure frequencies. (E) Comparison of total cell counts in the BAL in each group for different challenge numbers. (F) Comparison of IL-13 concentrations in BAL changed for different challenge numbers. (G) Representative plots showing changes in eosinophil counts for different challenge numbers. (H) Changes in IgE concentrations in blood samples for different challenge numbers. (I to K) Representative plots showing Masson staining, hematoxylin and eosin (HE) staining, and periodic acid–Schiff (PAS) staining of pulmonary airway tissue in mice for different challenge numbers. (L) Representative plots showing comparison of S100A4 + CD8 + effector memory T (Tem) cell counts for different challenge numbers. (M) Comparison of TL1A expression level in lung tissue samples from each group. (N and O) Comparison of TL1A concentrations in serum/BAL samples in each group. Data are presented as mean ± SD (error bars) from at least 3 independent experiments, with n = 6 mice per group per experiment (A and E to O). Time-course studies (B and C) used n = 6 mice per time point. * P < 0.05 and ** P < 0.01 compared with the respective control groups. Histological scoring and flow cytometry analysis were performed by investigators blinded to the experimental groups.

Article Snippet: The cells were subsequently washed with Flow Cytometry Staining Buffer (MultiSciences) and collected after centrifugation at 3,000 rpm for 5 min at 4 °C.

Techniques: Expressing, Western Blot, In Vivo, Comparison, Staining, Control, Flow Cytometry

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Identification and verification of the major tumor necrosis factor-like ligand 1A (TL1A)-expressing cell populations in the context of allergic lung inflammation. (A) Results of the immunohistochemical staining of TL1A in human lung tissue from the Human Protein Atlas (HPA) database. (B) Single-cell profiling via t-distributed stochastic neighbor embedding (t-SNE) following initial annotation using SingleR. (C) Cell clustering and selections made according to manual curation for the degree of relevance based on a marker gene. (D) Pie chart showing the TL1A expression distribution based on single-cell RNA sequencing (scRNA-seq). NK, natural killer. (E) Sample images of the colocalization of macrophages with TL1A in the lung tissue of asthmatic model mice. (F) Representative plots showing the number of TL1A + macrophages (CD45 + F4/80 + cells minus eosinophils). (G) Sample images of the colocalization of human macrophages with TL1A in the airways of patients with asthma. (H) Representative plots revealing the depletion of CD170 + CD11c + macrophages in lung tissue after treatment with clodronate (CLO) liposomes. (I to L) Changes in TL1A concentrations in serum/bronchoalveolar lavage fluid (BAL) after CLO liposome treatment. (M) Cell–cell communications between macrophages and other cell types were substantially altered after administration of an anti-TL1A antibody or Tnfsf15 depletion in the mouse model. DCs, dendritic cells; ENCs, endothelial cells; EPCs, epithelial cells; Grans, granulocytes; Monos, monocytes; Mφs, macrophages. Data are presented as mean ± SD. scRNA-seq analysis (B to D) was performed on lung cells pooled from 4 mice. Flow cytometry and imaging data (E and F) are representative of 3 independent experiments with n = 3 mice per group. For CLO experiments (H to L), n = 6 mice per group. All quantifications were performed in a blinded fashion.

Article Snippet: The cells were subsequently washed with Flow Cytometry Staining Buffer (MultiSciences) and collected after centrifugation at 3,000 rpm for 5 min at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Staining, Single Cell, Marker, RNA Sequencing, Liposomes, Flow Cytometry, Imaging

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Myeloid-cell-specific Tnfsf15 -knockout resulted in the attenuation of allergic airway inflammation. (A) Myeloid-cell-specific Tnfsf15 -knockout strategy. (B) Genotyping was confirmed using tail DNA genomic polymerase chain reaction. (C) Immunomagnetic bead sorting was used to isolate F4/80 + cells, and the expression level of tumor necrosis factor-like ligand 1A (TL1A) was quantified using Western blotting. (D to G) Representative plots showing the proportions of 4 major types of immune cells (macrophages, CD8 + T cells, CD4 + T cells, and B cells) infiltrating the lung tissues in each group. (H) Representative hematoxylin and eosin (HE) staining among the different groups and quantification of the airway inflammation score. Black scale bar, 50 µm. (I) Representative periodic acid–Schiff (PAS) staining among the different groups and quantification of the airway mucus score. (J) Representative Masson’s trichrome staining among the different groups and quantification of the collagen volume fraction. (K and L) Lung eosinophil or neutrophil counts, as determined by flow cytometry. (M) Total cell counts in bronchoalveolar lavage fluid (BAL). (N) Concentrations of interleukin 4 (IL-4) in BAL. (O) Concentrations of interleukin 13 (IL-13) in BAL. Data are presented as mean ± SD. Experiments were repeated 3 times with n = 6 to 8 mice per group per experiment. Cell sorting and Western blot (C) used pooled cells from n = 4 mice. Flow cytometry analyses (D to G and K) and histological scoring (H to J) were performed by investigators blinded to genotype and treatment. * P < 0.05 and ** P < 0.01 compared with the respective groups.

Article Snippet: The cells were subsequently washed with Flow Cytometry Staining Buffer (MultiSciences) and collected after centrifugation at 3,000 rpm for 5 min at 4 °C.

Techniques: Knock-Out, Polymerase Chain Reaction, Expressing, Western Blot, Staining, Flow Cytometry, FACS

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Targeted therapy based on anti-tumor necrosis factor-like ligand 1A (anti-TL1A) interventions in vivo. (A) Representative micro-computed tomography (micro-CT) images in each group. (B) Experimental schematic for the anti-TL1A interventions in the acute asthma model. (C and D) Effects of the anti-TL1A intervention on C-C motif chemokine ligand 8 (CCL8) expression in serum and lung tissue in the different experimental groups. (E and F) Partitioning diagram for the subsets of T cells identified through single-cell sequencing based on marker genes. (G to I) Proportional distribution characteristics of the main T-cell subsets (naive T cells, T helper 2 [Th2] cells, and regulatory T [Treg] cells) in single-cell samples. (J) The percentages of eosinophils were analyzed by flow cytometry. (K) The percentages of Th2 cells were analyzed by flow cytometry. (L) The percentages of T helper 17 (Th17) cells were analyzed by flow cytometry. (M) The percentages of Treg cells were analyzed by flow cytometry. (N) Results of the CD8 + T-cell refinement analysis. (O) Proportions of S100A4 + GZMK + CD8 + effector memory T (Tem) cells in CD8 + T cells. GZMK, granzyme K. (P) Representative plots showing S100A4 intracellular staining of CD8 + Tem cells (CD62L low CD44 high ). Data are presented as mean ± SD. In vivo intervention studies used n = 6 mice per group, repeated twice. Single-cell RNA sequencing (scRNA-seq) analysis (E to I, N, and O) was performed on T cells isolated from n = 4 mice pooled for sequencing. * P < 0.05 and ** P < 0.01 compared with the respective groups; # P < 0.05, compared with the anti-TL1A 3-μg treatment group. Flow cytometry analyses (J to M and P) and micro-CT quantification were conducted by blinded investigators.

Article Snippet: The cells were subsequently washed with Flow Cytometry Staining Buffer (MultiSciences) and collected after centrifugation at 3,000 rpm for 5 min at 4 °C.

Techniques: In Vivo, Micro-CT, Expressing, Single Cell, Sequencing, Marker, Flow Cytometry, Staining, RNA Sequencing, Isolation

Journal: STAR Protocols

Article Title: Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

doi: 10.1016/j.xpro.2026.104471

Figure Lengend Snippet: Flow cytometry for MACS-isolated microglia purity (A and B) Total CD11b-positive cells from MACS columns; (C) Cell viability assessed by Zombie Red staining; (D) Infiltrating leukocytes, including neutrophils (Ly6G+) and T lymphocytes (CD3+); (E) Proportions of microglia (CD11b+CD45low) versus monocytes/border-associated macrophages (CD11b+CD45high); (F) Proportion of resting (homeostatic) microglia identified as CD11b+TMEM119+ cells.

Article Snippet: Note: We use commercial flow cytometry staining buffer from eBioscience (Cat# 00-4222-26), which is PBS-based formulation designed to prevent non-specific antibody binding and maintain cell stability during flow cytometry.

Techniques: Flow Cytometry, Isolation, Staining

Journal: bioRxiv

Article Title: NFATC2 in pancreatic cancer-associated fibroblasts predicts treatment response and facilitates ERBB-targeted therapies

doi: 10.64898/2026.04.04.716465

Figure Lengend Snippet: (A) Western blot showing ectopic NFATC2 expression in pancreatic CAFs (pCAF) generated using lentiviral transduction. GAPDH served as loading control. WT, wild-type pCAFs; NFATC2+, NFATC2-expressing pCAFs. Uncropped blots are shown in Supplementary Figure 3A. (B) Representative fluorescence microscopy images of PANC1-pCAF co-cultures. PANC1 cells are labeled with mCherry (red) and pCAF cells with EGFP (green). WT and NFATC2+ pCAF co-cultures are shown on days 1-3 under vehicle or FOLFIRINOX treatment. Scale bar, 10μm. (C) Quantification of PANC1 cell numbers using CellProfiler from fluorescence images showing that NFATC2+ CAFs significantly reduce cancer cell proliferation, particularly under FOLFIRINOX treatment. Data are presented as mean ± SEM (n=3 independent experiments). Upper panel: p<0.0001. Lower panel: p=0.0092, two-way ANOVA. (D) Flow cytometry analysis of PANC1-pCAF co-culture systems with WT pCAFs (left) and NFATC2+ pCAF (right). Scatter plots display mCherry fluorescence (x-axis) versus eGFP fluorescence (y-axis), PANC1 cells (mCherry+, eGFP-) seen in the lower right quadrant. (E) Annexin V-FITC apoptosis assay by flow cytometry. Density plots showing the fraction of apoptotic cells (Q2, top-right quadrant) relative to live cancer cells (Q3, bottom-right quadrant) following co-culture with WT or NFATC2+ pCAFs ± FOLFIRINOX. In FOLFIRINOX-treated cells, the apoptotic fraction increased from 19.1% in WT pCAF co-cultures to 33.0% in NFATC2+ pCAF co-cultures, indicating enhanced chemotherapy-induced apoptosis in cancer cells co-cultured with NFATC2+ pCAFs.

Article Snippet: Cell pellets were resuspended in 1 mL flow cytometry buffer (Invitrogen, #00-4222-26) and passed through 40 μm cell strainers (Corning, #CLS431750) to remove cell aggregates and debris.

Techniques: Western Blot, Expressing, Generated, Transduction, Control, Fluorescence, Microscopy, Labeling, Flow Cytometry, Co-Culture Assay, Apoptosis Assay, Cell Culture

Journal: bioRxiv

Article Title: NFATC2 in pancreatic cancer-associated fibroblasts predicts treatment response and facilitates ERBB-targeted therapies

doi: 10.64898/2026.04.04.716465

Figure Lengend Snippet: (A) Representative fluorescence microscopy images of PANC1-pCAF co-culture system under different treatment conditions. Cancer cells (mCherry+, red) and CAFs (eGFP+, green) were treated with vehicle control, standard chemotherapy regimens (FOLFIRINOX or Gemcitabine plus Paclitaxel) alone, or chemotherapy combined with ERBB-targeted inhibitors including Trastuzumab, Erlotinib and Neratinib. Scale bar, 10μm. (B) Quantification of PANC1 cancer cell numbers from fluorescence microscopy analysis. Data represent mean ± SEM from three independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, t-test. (C) Normalized cell numbers of PANC1 from fluorescence microscopy analysis based on vehicle control from each group. Data represent mean ± SEM from three independent experiments. Same statistical significance marks used as (B). (D) Flow cytometry analysis of the co-culture system after three-day treatment. Density plots display mCherry fluorescence (x-axis) versus eGFP fluorescence (y-axis), revealing distinct cell populations: PANC1 cells (mCherry+, eGFP-, lower right quadrant), CAFs (mCherry-, eGFP+, upper left quadrant), and cellular debris (lower left quadrant). Quantification of PANC1 cell proportions demonstrating a marked reduction in the cancer cell population following chemotherapy combined with ERBB-targeted inhibitors, particularly Trastuzumab and Neratinib, in the presence of NFATC2+ pCAFs.

Article Snippet: Cell pellets were resuspended in 1 mL flow cytometry buffer (Invitrogen, #00-4222-26) and passed through 40 μm cell strainers (Corning, #CLS431750) to remove cell aggregates and debris.

Techniques: Fluorescence, Microscopy, Co-Culture Assay, Control, Flow Cytometry