Journal: STAR Protocols

Article Title: Protocol for visualization and quantitative analysis of murine lung immunological architecture using iBALT-UNMAP following bacterial challenge

doi: 10.1016/j.xpro.2026.104472

Figure Lengend Snippet: TSA multiplex staining Schematic workflow representing the steps required for TSA multiplex staining. In this example we use first a staining using GL7, follwed by CD3 for T cells, and finally B220 for B cells. We add the colors used for each secondary to guide each cycle. Incubation times, washing, and stripping steps are included.

Article Snippet: Immune “Surfaces” tables for B220, CD3, and GL7 with object IDs and centroid coordinates. c. A lineage or class label per nucleus if you already assigned B cell, T cell, and GL7 positive populations in Imaris.

Techniques: Multiplex Assay, Staining, Incubation, Stripping Membranes

Journal: eBioMedicine

Article Title: Migrasome-mediated clearance of excess PLK4 defines a targetable vulnerability

doi: 10.1016/j.ebiom.2026.106237

Figure Lengend Snippet: Targeting TSPAN6 suppresses tumour growth and metastasis. (A) Real-time cell proliferation assay of PLK4−OE cells treated with scramble or shTSPAN6 RNA. Data are presented as mean values ± SD. (B) Cystal violet staining of shTSPAN6 or control PLK4−OE cells after 14 days of culture. Colony number of each group was quantified on the right. Data are presented as the mean ± SD from three independent repeats. p values were calculated using unpaired two-tailed Student's t-tests, with p < 0.05 considered statistically significant. (C) Fluorescence staining for active apoptosis in shTSPAN6 or control PLK4−OE cells detected by GreenNuc Caspase-3 Assay Kit. The green fluorescence in nuclei indicate active apoptosis. Scale bar, 20 μm. (D) Western blot analysis of cleaved caspase3 in PLK4−OE cells treated with scramble or shTSPAN6 RNA. β-actin was used as the loading control. (E) Western blot analysis of PLK4 protein levels in primary tumour cells derived from 18 patients with triple-negative breast cancer (TNBC). β-Actin served as the loading control (left). Relative PLK4 protein levels were quantified as the ratio of sample to NC (sample/NC). Cases with sample/NC > 1 were classified as PLK4 high (red dots), whereas cases with sample/NC < 1 were classified as PLK4 low (blue dots) (right). (F) Growth curve of tumours in PDX-models treated with scramble or shTSPAN6 RNA. Tumour volumes are presented as mean values ± SD (n = 30 mice per group). p values were calculated by two-way ANOVA. p < 0.05 was considered statistically significant. (G, H) Immunohistochemical staining of Ki67 and cleaved-caspase3 in tumours from PDX-mice treated with scramble or shTSPAN6 RNA. Scale bar, 100 μm. (I) Immunofluorescent staining for PLK4, TSPAN6, and α-tubulin in frozen tumour sections from PLK4 high tumour mice treated with scramble or shTSPAN6 RNA. White arrowheads indicate PLK4 particles colocalized with TSPAN6 in the extracellular space. Yellow arrowheads indicate bipolar spindles in the control group and multipolar spindles in the shTSPAN6 RNA-treated group. Scale bar, 20 μm for each main image and 2 μm for the zoom in section. Regions marked by the arrowheads are shown at higher magnification in the right three columns. (J) Quantification of PLK4-containing particles in the extracellular space of frozen tumour sections from PLK4 high tumour mice treated with scramble or shTSPAN6 RNA. 20 fields of view from tumour sections of three mice per group were analysed. Data are presented as mean ± SD. p values were calculated using unpaired two-tailed Student's t-tests. p < 0.05 was considered statistically significant. (K) Proportion of bipolar spindles in mitotic tumour cells in frozen tumour sections from PLK4 high tumour mice treated with scramble or shTSPAN6 RNA. (L) Western blot analysis of intracellular PLK4 protein levels in PLK4 high tumours derived from PDX-mice treated with scramble or shTSPAN6 RNA. β-Actin was used as the loading control. Quantification is presented as mean ± SD from three independent biological replicates. p values were calculated using unpaired two-tailed Student's t-tests, with p < 0.05 considered statistically significant. (M) Liver and lung tissues were harvested from PDX-mice with or without detectable metastatic lesions. Arrowheads indicate representative metastatic nodules within the organs. (N) Metastatic incidence in the liver and lung tissues of PDX-mice treated with scramble or shTSPAN6 RNA. The number of mice analysed in each group is indicated above the corresponding bar. (O) Metastatic regions in liver and lung tissues visualised by H&E staining. Representative metastatic foci are indicated by arrowheads, with corresponding higher-magnification images shown on the right. Scale bar, 100 μm for each main image and 20 μm for the zoom in section. (P) Quantification of metastatic nodules in the lungs and livers of PDX-mice treated with scramble or shTSPAN6 RNA. Each dot represents an individual mouse bearing metastatic nodules in the indicated organ.

Article Snippet: Subsequently, cells were incubated in the dark with GreenNucTM Caspase-3 Substrate (1:200 dilution, Beyotime, #C1168) for 30 min to label positive apoptosis signal, followed by Hoechst 33342 (1:100 dilution) staining for 20 min to visualise nuclei.

Techniques: Proliferation Assay, Staining, Control, Two Tailed Test, Fluorescence, Caspase-3 Assay, Western Blot, Derivative Assay, Immunohistochemical staining

Journal: bioRxiv

Article Title: De novo masking domains that gate TNF-family ligand assembly and activity

doi: 10.64898/2026.04.20.719557

Figure Lengend Snippet: A single masked TNFα or 4-1BBL module was fused to a one-armed, affinity-attenuated trastuzumab variant to generate an antibody format that undergoes activation-dependent homotrimerization. Homotrimerization was first validated using the masked TNFα construct (b–d), and functional consequences were then evaluated using an analogous masked 4-1BBL fusion in assays with HER2-positive SK-BR-3 cells and Jurkat-Lucia h4-1BB reporter cells (e, f). Protein concentrations are reported per molecular species (monomeric or trimeric antibody, as indicated). Where indicated, trimeric species were re-isolated by SEC before downstream assays. a, Proposed mechanism for delivery of membrane-type TNFLs. Before activation, the construct is predominantly monomeric with reduced apparent target engagement and masked TNFL function. Protease-triggered activation promotes assembly into a trimeric state with increased functional valency and the capacity to drive receptor crosslinking, thereby enhancing signaling. b, Design of antibody formats used to test the concept: OATmab_mTNFα, OATmab, and Tmab. All constructs are based on an affinity-attenuated trastuzumab variant and contain a C-terminal peptide tag on the light chain for site-selective drug conjugation . In OATmab_mTNFα, TNFα (orange) within the masked TNFα module was fused to the C-terminus of one heavy chain. One-armed formats were assembled using knob-into-hole Fc heterodimerization mutations. c, Oligomeric states of OATmab and OATmab_mTNFα assessed by analytical SEC before (black) and after (gray) TEV activation. The arrow indicates the TEV-dependent shift in the main peak consistent with homotrimer formation. d, Binding of monomeric and trimeric antibody species to HER2 (left) and TNFR2 (right) measured by ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. e, Binding of monomeric and trimeric antibody species to HER2-positive SK-BR-3 cells measured by cell-based ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. f, Functional activity of monomeric and trimeric antibody species measured in Jurkat-Lucia h4-1BB reporter cells cultured alone (dashed lines) or co-cultured with SK-BR-3 cells (solid lines). Data are shown as mean ± s.d. from n = 3 technical replicates.

Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).

Techniques: Variant Assay, Activation Assay, Construct, Functional Assay, Isolation, Membrane, Drug discovery, Conjugation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Activity Assay, Cell Culture

Journal: bioRxiv

Article Title: De novo masking domains that gate TNF-family ligand assembly and activity

doi: 10.64898/2026.04.20.719557

Figure Lengend Snippet: The antibody formats shown in were conjugated with MMAE, and cell binding and cytotoxicity were evaluated in HER2-positive SK-BR-3 cells. Protein concentrations are reported per molecular species (monomeric or trimeric ADC, as indicated). Where indicated, trimeric species were re-isolated by SEC before downstream assays. a, Proposed mechanism for the switchable ADC concept. Before activation, the construct is predominantly monomeric with reduced apparent binding and low payload density. Protease-triggered activation promotes assembly into a trimeric state with increased functional valency and complex-level payload density, which may enhance target-cell killing. b, Binding of monomeric and trimeric ADC species to HER2-positive SK-BR-3 cells measured by cell-based ELISA. Data are shown as mean ± s.d. from n = 3 technical replicates. c, Cytotoxic activity of ADCs in HER2-positive SK-BR-3 cells. Cell viability (%) was calculated as 100 × S/V, where S is the sample signal and V is the 100% viability control (cells plus medium). Data are shown as mean ± s.d. from n = 3 technical replicates.

Article Snippet: The biological activity of antibody-fused masked 4-1BBL was evaluated using Jurkat-Lucia h4-1BB reporter cells in the presence or absence of HER2-positive SK-BR-3 cells (ATCC).

Techniques: Binding Assay, Isolation, Activation Assay, Construct, Functional Assay, In-Cell ELISA, Activity Assay, Control