Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein binds to and enters the cell through STEAP2 (A) Comparison of the cell-binding capacity of SARS-CoV-2 wild type (WT) N protein and Omicron N protein expressed in either E. coli or mammalian cells. 1 × 10 5 A549 cells were used to mixed with 1 μg WT N or Omicron N proteins. One hour after protein addition, allophycocyanin (APC) conjugated anti-His antibody was used to detect the cell binding capacity of WT N protein or Omicron N protein. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). (B) Antibody blocking assay. Aliquots of 10 μg of SARS-CoV-2 N protein were pre-mixed with 0, 1, 3, 10, 30, and 100 μg of normal mouse IgG or anti-N monoclonal antibody (NP-mAb-40) and incubated at 4°C overnight. The antibody/N protein complex was used for the A549 cell surface binding assay. The blocking capacity of anti-N antibody was normalized to N protein only control. (C) Membrane fractions of A549 and HPAEpiC cells were extracted and incubated with N protein conjugated beads for 3 h binding at 4°C, and pull-downed for LC-MS-MS analysis (upper panels). A549 and HPAEpiC cells were suspended and treated with N protein for 1 h on ice. After incubation, cells were crosslinked with 3 mM DTSSP for 1.5 h. Then, cells were lysed in RIPA lysis buffer, and N protein complex in the lysate was immunoprecipitated for LC-MS-MS analysis (lower panels). Y axis denotes −logP values while the X axis shows log2 fold change values. Orange dots highlight the statistically significant proteins, with p value < 0.05 (-Log p > 1.3) and fold change>2, and the enriched plasma membrane protein was labeled on the plot. Identified proteins were further sorted by HuMemProtDB. (D) To knock-down (KD) STEAP2 expression, HPAEpiC cells were infected with lentivirus carrying STEAP2 shRNA followed by puromycin selection for 14 days. The STEAP2 mRNA expression levels were assessed by qRT-PCR, and the relative KD efficiency of shSTEAP2 was compared to shLacZ control (left-hand side panel). N protein binding capabilities to HPAEpiC STEAP2 KD cells and shLacZ control KD cells were assessed by flow cytometry analysis, and data were shown as mean fluorescence intensity (MFI). (right-hand side panel). (E) Western blot analysis of STEAP2 in wild type (WT) and knock-out (KO) A549 cells were shown. N protein binding to A549 STEAP2 KO cells was assessed by flow cytometry analysis and shown as mean fluorescence intensity (MFI). Ccr (crotonyl-CoAcarboxylase/reductase, a bacterial protein) binding was used as a control. (F) SARS-CoV-2 N protein enters alveolar cells. HPAEpiC cells were treated with 10 μg SARS-CoV-2 N protein overnight and then stained with anti-N antibody. The localization of N protein (Red) was checked by fluorescence microscope and cell morphology was observed by dimensional interference contrast (DIC). Nuclei of cells were stained by DAPI (blue). (G) N protein entering cells by endocytosis and N protein co-localization with STEAP2. HPAEpiC alveolar cells were seeded on 8 well slides. Cells were pretreated with endocytosis inhibitors HCQ, or Dynasore. Then the cells were treated with N protein overnight. After treatment, the cells were stained by specific antibodies to detected N protein (red), endosome marker (EEA1) (green), and STEAP2 (yellow). Cells were observed under fluorescent microscopy (Invitrogen tech.). Scale bar: 50 μm. All data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; t test. See also Figure S5 .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Comparison, Binding Assay, Flow Cytometry, Fluorescence, Antibody Blocking Assay, Incubation, Blocking Assay, Control, Membrane, Liquid Chromatography with Mass Spectroscopy, Lysis, Immunoprecipitation, Clinical Proteomics, Labeling, Knockdown, Expressing, Infection, shRNA, Selection, Quantitative RT-PCR, Protein Binding, Western Blot, Knock-Out, Staining, Microscopy, Marker

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein delivers nucleic acids into cells (A) N protein-RNA complex binding to the cell surface. Aliquots of 10 μg SARS-CoV-2 N protein were incubated with 1 μg of indicated RNAs for 1 h at 4°C, and added to A549 or HPAEpiC cultures. SARS-CoV-2 N protein only without RNA was used as a control. The samples were analyzed by flow cytometry and data are shown as mean fluorescence intensity (MFI). Data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; t test. (B) The observation of N protein-RNA enters into cells. HPAEpiC were seeded onto 8-well glass slides (40,000 cells/well). SARS-CoV-2 N protein 10 μg and 40 μg RNA-FAM (green) were mixed for 1 h at 4°C. cells were treated with SARS-CoV-2 N-RNA-FAM mixture for 1 h. The groups of non-treated cells and RNA-FAM only were as controls. After treatment, N protein was detected by anti-N antibody (Red). The localization of RNA-FAM was green. DAPI (blue) indicates cell nuclei. Scale bar: 15 μm. (C) Lattice light sheet microscopy time lapse imaging of N protein-RNA complex entering into HPAEpiC cells. SARS-CoV-2 N protein 10 μg was mixed with 40 μg RNA-FAM (fluorescein) for 1 h at 4°C and then treated with ice-cooled alveolar cells. The signals of RNA-FAM and Hochest 33,342 were monitored by lattice light sheet microscopy at different time points.

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Binding Assay, Incubation, Control, Flow Cytometry, Fluorescence, Microscopy, Imaging

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet: N protein-assisted nucleic acid dispersion and expression in the co-culture environment (A)The co-culture system consisted of A549 as recipient cells, and 293T pre-transfected with two plasmids, one expressing GFP and the other expressing SARS-CoV-2 N protein or the pcDNA3.1 empty vector. (B–E) After 24 h co-culture of the donor cells and recipient cells, cell pool was stained with cytokeratin 18 (an A549 marker) and SV40 large T antigen (a 293T marker). A549 cells in the cell pool were gated from cytokeratin 18 positive and large T antigen negative. A549 GFP positive percentage was further assessed by flow cytometry analysis. Effects of SARS-CoV-2 N variants (B), treatment with RANTES (C), the p38 inhibitor SB203580 (D), or anti-N neutralizing antibody (E) were accessed by adding these effectors to the medium. Experiments are performed in three to five biological replicates. ∗, p value <0.05 (paired two-tailed student’s t -test). (F) SARS-CoV-2 N protein promotes gene delivery by cell-free diffusion to neighboring cells. A549 cells were plated in the lower chamber, while 293T donor cells co-transfected with plasmids expressing EGFP and indicated N proteins in the upper chamber. After 3 days of co-culture, GFP positive A549 cells were observed and counted. See also Figures S10 and .

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Dispersion, Expressing, Co-Culture Assay, Transfection, Plasmid Preparation, Staining, Marker, Flow Cytometry, Two Tailed Test, Diffusion-based Assay

Journal: iScience

Article Title: SARS-CoV-2 N protein mediates intercellular nucleic acid dispersion, a feature reduced in Omicron

doi: 10.1016/j.isci.2023.105995

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney 293T cells (American Type Culture Collection, CRL-3216), human cervical cancer HeLa cells (American Type Culture Collection, CCL-2) and mouse lung cancer LL2 cells (American Type Culture Collection, CRL-1642) were cultured in DMEM (Gibco, 11965-065) and human lung adenocarcinoma A549 cells (American Type Culture Collection, CCL-185), human colon adenocarcinoma HCT-8 cells (American Type Culture Collection, CCL-244) and mouse mammary gland epithelium 4T1 cells (American Type Culture Collection, CRL-2539) were cultured in RPMI 1640 Medium (Gibco, 22400-071), respectively.

Techniques: Bioprocessing, Recombinant, Magnetic Beads, Protease Inhibitor, Sequencing, Modification, SYBR Green Assay, shRNA

Journal: iScience

Article Title: A bacterial genotoxin reveals a p53-proteasome-LC3 regulatory axis that drives the suppression of autophagy in cells experiencing sublethal DNA damage

doi: 10.1016/j.isci.2025.112118

Figure Lengend Snippet:

Article Snippet: Escherichia coli BL21 (DE3) , New England Biolabs , Cat#C2527I.

Techniques: Virus, Recombinant, Cell Recovery, Modification, Gentle, Flow Cytometry, Reverse Transcription, Bicinchoninic Acid Protein Assay, RNA Extraction, Western Blot, Cloning, Mutagenesis, Plasmid Preparation, Software, Cell Culture, Microscopy, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies

doi: 10.1101/2024.11.17.623896

Figure Lengend Snippet: A) Characteristic log-log plots of MSD vs. lag time (τ). Slope α indicates diffusion type: Brownian (α=1), subdiffusive (α<1), superdiffusive (α>1). Plateau indicates confinement. B) Log-log plot of ensemble- and time-averaged MSD ( ate ) for particles longer than 10 frames (50 ms), corrected for static error and fitted to a power law with different lag time Δt values of 5, 10, 15, or 20 ms (τ = nΔt), the corresponding alpha values are given in the legend; C) image of U2OS with lower (fluorescent intensity = 16 r.u.) and higher GEM expression (fluorescent intensity = 147 r.u.). Sample size was 60 cells. D) Density probability function of collected D α , E) α,and F) D eff collected after fit with power-law relationship with σ/Δt = 0.5; G) Plot of D eff vs. fluorescence intensity in cells throughout the U2OS colony. The red line indicates the division between cells with “High GEM expression” (30 cells) and “Low GEM expression” (30 cells); H) Corresponding plot of α vs. fluorescence intensity.

Article Snippet: U2OS cells expressing pCMV-pfv-Sapphire-Ires-DsRed (Addgene #116934) or pCMV-pfv-paGFP were sorted by flow cytometry using a Cell Sorter SH800S, while U2OS cells expressing TRE-pfv-Sapphire were selected with 2 μg/mL puromycin.

Techniques: Diffusion-based Assay, Expressing, Fluorescence

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 1. UNBS1450 induces apoptotic cell death in U937 cells. (A) Molecular structure. (B) Analysis of UNBS1450-induced cell death was performed by Trypan Blue staining after 24, 48 and 72 h of treatment with UNBS1450 at 10, 15, 20 and 30 nM. (C) Cell cycle analysis after 24 h of incubation time with indicated concentrations. (D) Hoechst staining (upper panel) and quantification (lower panel) of the fraction of cells presenting fragmented nuclei. (E) Flow cytometry analysis after 24 h of incubation time at indicated concentrations. (F) Analysis of PBMCs viability after UNBS1450 treatment. PBMCs were seeded at 2 106, then after 24 h treated for 24 h with various concentrations (0–100 nM) of UNBS1450. Cells were then stained either by Trypan Blue (upper panel) or by Hoechst (panel below) to analyze either cell integrity or apoptosis induction. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Staining, Cell Cycle Assay, Incubation, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 2. Na+/K+-ATPase subunit a1 mRNA quantification. Na+/K+-ATPase subunit a1 mRNA content of untreated PBMCs and a wide panel of hematological cancer cell lines including K562, Jurkat and U937 cells was transcribed and then quantified by RT-PCR. The quantification of three independent experiments is expressed in brute 2^DCt values S.D.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 3. (A) Caspase activation. U937 cells were incubated in RPMI + 10% FCS UNBS1450 20 nM up to 24 h. Western blot analysis of UNBS1450-induced cleavage of pro-caspases-9, -8, -7 and -3. (B) Analysis of expression levels of anti- apoptotic proteins. UNBS1450-induced expression level alterations of XIAP, Bcl-2 and Mcl-1. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Activation Assay, Incubation, Western Blot, Expressing

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 4. UNBS1450 enables Bak/Bax activation. U937 cells were incubated for 24 h in RPMI + 10% FCS in presence or in absence of UNBS1450. Bak (A.) and Bax (B.) activation status were assessed by using primary antibodies specifically targeting activated forms of Bak (Ab-1; Calbiochem) and Bax (6A7; Santa Cruz). Counterstaining was done by Hoechst staining to assess apoptotic nuclei. The data shown here were representative for three independent experiments with similar results.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Activation Assay, Incubation, Staining

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 5. Inhibition by UNBS1450 of TNFa-induced NF-kB activation. (A) K562 and (B) Jurkat cells were pretreated with UNBS1450 at various concentrations from 10 to 50 nM and incubated for 2 h, followed by TNFa addition (20 ng/ml) and an additional incubation period of 6 h. Results are represented as the ratio of the measured luminescence of the firefly luciferase vector divided by the measured luminescence of the Renilla plasmid. Untreated cells were used as a negative control, cells treated with TNFa only as a positive control. Results are presented as mean S.D. of 3 individual measurements performed in triplicates. (C) Effect of UNBS1450 on the binding affinity of NF-kB was assessed by an EMSA on the K562 and Jurkat cell lines. The data shown here were representative for three independent experiments with similar results. (D) For supershift/immunodepletion experiments, the nuclear extracts and labelled probes were incubated in the reaction mixture for 30 min on ice prior to a 30 min incubation with 2 mg of anti-p50 or anti-p65 antibodies. SS designates supershifted bands. (E) Jurkat cells were incubated with UNBS1450 (40 nM) for 2 h, followed by a TNFa (20 ng/ml) activation for the indicated time periods. Cytoplasmic and nuclear extracts were prepared, fractionated on a 10% SDS-page gel, transferred to a membrane and then tested for IkBa and p65. Protein loading and purity of nuclear/cytosolic extracts were verified by lamin B and a-tubulin Western blots. Data shown are representative for three independent experiments with similar results. K562 (F), and U937 (G) cells were incubated for 2 h in RPMI + 10% FCS in presence or in absence of various concentrations (10–50 nM) of UNBS1450 before being activated by TNFa during 22 h. After 24 h of incubation IL-8 concentrations in supernatants were measured. Untreated cells served as negative control whereas cells activated by TNFa only were used as a positive control. The data shown here were representative for three independent experiments with similar results.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Inhibition, Activation Assay, Incubation, Luciferase, Plasmid Preparation, Negative Control, Positive Control, Binding Assay, Immunodepletion, SDS Page, Membrane, Western Blot

Journal: Cell Reports Medicine

Article Title: Leucine restriction ameliorates Fusobacterium nucleatum- driven malignant progression and radioresistance in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2024.101753

Figure Lengend Snippet: Intracellular F. nucleatum promotes radioresistance in NPC cells by suppressing host apoptosis and DNA damage (A–G) Fn-infected and uninfected NPC cells were exposed to 2, 4, and 8 Gy irradiation, respectively. (A) Representative images of NPC cells. Fn (MOI = 10:1) or E. coli -infected NPC cells (MOI = 1:100). Scale bar: 150 μm. (B) Cellular viability with live/dead assay. Statistical results are presented in the below panels. Data are mean values of three biology repeats. Scale bar: 100 μm. (C) Representative photographs of colony formation assays. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (D) The apoptosis rates were determined by flow cytometry. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (E) LDH activity in supernatant was assessed by LDH Cytotoxicity Assay Kit; optical density (OD) values of 490 nm were present with histogram. (F) Representative images of the comet assay. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (G) Western blot analysis of γH2AX was performed. Statistical results are presented in the right panels. Data are mean values of three biology repeats. Data are shown as mean ± SD. p values were determined by independent sample t tests (C–E and G), ∗ p < 0.05, ∗∗ p < 0.001, and ∗∗∗ p < 0.001.

Article Snippet: E. coli DH5α , TIANGEN , Cat# CB101-02.

Techniques: Infection, Irradiation, Live Dead Assay, Flow Cytometry, Activity Assay, LDH Cytotoxicity Assay, Single Cell Gel Electrophoresis, Western Blot

Journal: Cell Reports Medicine

Article Title: Leucine restriction ameliorates Fusobacterium nucleatum- driven malignant progression and radioresistance in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2024.101753

Figure Lengend Snippet:

Article Snippet: E. coli DH5α , TIANGEN , Cat# CB101-02.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Viability Assay, ROS Assay, LDH Cytotoxicity Assay, ATP Assay, Sequencing, Virus, Synthesized, Software

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A A concentration range of Alexa Fluor 647-conjugated CD3xHER2 bsAbs varying in affinity and epitope recognition site on either arm, were added on top of collagen gels containing BT474 (HER2+) tumoroids. Localization of bsAbs to the tumoroids was analyzed after 24 h by confocal microscopy. Images show maximum projections. Bar = 100 μm. B Localization of three different bsAbs combining a CD3 high affinity arm with the indicated HER2 arms in the BT474 tumoroid. White arrows indicate penetration of bsAbs into the tumoroids. Blue, Hoechst; red, Alexa Fluor 647-conjugated bsAb. Images were obtained from a single z-section through the center of the tumoroid. Bar = 50 μm. C Cartoon showing the location on the HER2 receptor of the epitopes recognized by the different HER2 arms and whether the interactions are high or low affinity.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Concentration Assay, Confocal Microscopy

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing tumoroid (blue), T-cells (green) and loss of viability detected by PI (red) 6 days after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and bsAbs with either non-targeting CD3 arm or non-targeting HER2 arm. Bottom panel shows tumoroids treated with 10 μg/mL Cisplatin triggering near complete tumoroid killing versus tumoroids grown in absence of T-cells and bsAbs (negative control; NC) or T-cells only. Bar = 100 μm. B Quantification of image data as shown in A . Data were normalized to cisplatin condition. Graphs show mean and SEM of 3 independent experiments, each performed in triplicate (3 individual wells each containing 1 collagen embedded BT474 tumoroid). C Western blot showing loss of HER2 upon CRISPR-Cas9 mediated knockout in BT474 cells. Tubulin serves as a loading control. D Representative maximum projection images at 6 days after exposure of collagen embedded WT, shCTR, or sgHER2 BT474 tumoroids to a mixture of T-cells and 1 μg/mL CD3 wt xHER2 Herceptin bsAbs. Blue, tumor nuclei; green, T-cells; red, PI. bar = 100 μm. E Quantification of image data as shown in D . Data were normalized to cisplatin condition. Graph shows mean and SEM of 3 independent experiments, each performed in triplicate. P -value calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; *** P < 0.001.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Negative Control, Western Blot, CRISPR, Knock-Out, Control

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing tumoroid (blue), T-cells (green) and loss of viability detected by PI (red) 6 days after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and bsAbs for one representative experiment of at least 4 biological replicates. Blue, tumor nuclei; green, T-cells; red, PI. Bar = 100 μm. B Quantification of the image data as shown in A . Tumoroids grown in absence of T-cells and bsAbs are shown as negative control (NC). Tumoroids treated with 10 μg/mL Cisplatin are shown as positive control triggering near complete tumoroid killing. Results are normalized to cisplatin condition. Graphs show mean and SEM of 5 (CD3 wt xHER2 variants) or 4 (CD3 Low xHER2 variants) independent experiments, each performed in triplicate (3 individual wells each containing 1 collagen embedded BT474 tumoroid). P -value calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to NC.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Negative Control, Positive Control

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing T-cell recruitment to the tumoroid and tumor killing with 12 h intervals from day 2 to day 5 after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and 0.1 μg/mL of the indicated bsAbs. Blue, tumor nuclei; green, T-cells; red, PI. Bar = 100 μm. B Quantification of time-lapse image data with 1-h intervals as shown in A . Green line represents the number of T-cells localized in the tumoroid. Red line represents the percentage of PI positive tumor cells, indicating loss of viability. Mean and SEM of three tumoroids is shown.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques:

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Cartoon illustrating experimental setup: BT474 tumoroids were printed below the higher surface of collagen gels with a diagonal upper surface. A mixture of T-cells and bsAbs was added on top of the lower surface. Maximum projection images are shown for bsAbs with a CD3 high affinity arm in combination with a high affinity HER2 arm either recognizing epitope 169 or epitope 153. BsAbs were used at 1 μg/mL. Time lapse starts upon initial contact of T-cells with tumoroids (indicated by red circle; 36 h after adding T-cells and bsAbs). Confocal image stacks were captured every hour. A time span of 40 h is shown with an 8-h interval, followed by an image displaying the final time point (day 5) where T-cell mediated tumoroid killing is near complete. Blue, tumor nuclei; green, T-cells; Red, PI. B , C 3D T-cell tracking analysis of time-lapse image data as shown in A . B MSD analysis of two typical T-cells in the vicinity of the tumoroid. The MSD was determined for time-lag from 1 to 25 h. Red color indicates the presence of CD3 wt xHER2 169 and blue color indicates CD3 wt xHER2 153 . Insets depict the 3D track of the two T-cells. The MSD for the two cells were characterized by D B = 0.x ± 0.x um 2 /h, V B = 0.x ± 0.x um/h, and D R = 0.x ± 0.x um 2 /h, V R = 0.x ± 0.x um/h, respectively. C MSD analysis of the total population of T-cells in the vicinity of the tumoroid over increasing time-lag from 1 to 30 h. In total 25 and 35 trajectories were analyzed for CD3 wt xHER2 169 and CD3 wt xHER2 153 , respectively. Insets depict median and SD for the parameters diffusion (D), velocity (v), and diffusive fraction (f D ) for the population analysis for the indicated bsAbs. Note that f D is lower for CD3 wt xHER2 169 bsAb. P -value calculated using multi comparison Dunn’s test following non-parametric Kruskal–Wallis test. * P < 0.05. D Confocal images of a single z-section through the center of a tumoroid exposed to T-cells and either CD3 wt xHER2 169 or CD3 wt xHER2 153 bsAbs. Initial contact of T-cells with the tumoroid (36 h after adding T-cells and bsAbs) and the same area 8 h later is indicated by the white circle. Red arrow indicates T-cell infiltration occurring only with the CD3 wt xHER2 169 bsAb. Blue, tumor nucleus; red, PI; green, T-cell. Bar = 100 μm.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Cell Tracking Assay, Diffusion-based Assay, Comparison

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Schematic illustration of the transwell assay. BT474 cells were seeded in the lower chamber of a transwell plate, with or without the addition of unlabeled T-cells and with or without CD3 wt xHER2 169 bsAbs. Cell Tracker CMFDA-labeled T-cells were added to the upper chamber. The number of green-fluorescent T-cells migrating to the lower chamber was analyzed after 48 h using confocal microscopy and flow cytometry. A condition using 100 ng/mL CXCL12 was used as positive control. B Bright field images (showing a mixture of unlabeled T-cells, infiltrating CMFDA-labeled T-cells, as well as tumor cells) and green fluorescence channel images (showing labeled T-cells) taken in the lower chamber at 48 h for the indicated conditions. Red dotted line outlines islands of tumor cells. Red arrow indicates a T-cell cluster. Bar = 100 μm. C Flow cytometry analysis of cell populations from the lower chamber at 48 h. Horizontal axis shows CMFDA signal (negative for unlabeled T-cells co-cultured with tumor cells in bottom chamber; positive for CMFDA-labeled T-cells recruited from the upper chamber); vertical axis shows CD3 levels. Flow cytometry analysis ( D ) and bar graph showing mean and SEM from 3 to 4 biological replicates ( E ), depicting cell counts in Q2 (CMFDA:CD3 double positive T-cells migrated from the upper to the lower chamber) for the indicated conditions. P -value calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ns, non-significant; ** P < 0.01; *** P < 0.001. F T-cell recruitment triggered by conditioned media (CM) from the indicated conditions. Graph shows cell counts of CMFDA-labeled infiltrated T-cells collected from the lower chamber. Mean and SEM from 2 to 4 biological replicates. P -value calculated using one - way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; * P < 0.05; ** P < 0.01. G Bright field images (showing a mixture of unlabeled T-cells, infiltrating CMFDA-labeled T-cells, as well as tumor cells) taken in the lower chamber at 48 h for the indicated conditions. Red dotted line outlines islands of tumor cells. Red arrows indicate T-cell clusters. Bar = 100 μm. H Bar graph showing mean and SEM from 3 biological replicates, depicting cell counts in Q2 (CMFDA:CD3 double positive T-cells migrated from the upper to the lower chamber) for the indicated conditions. P -value calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; ** P < 0.01.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Transwell Assay, Labeling, Confocal Microscopy, Flow Cytometry, Positive Control, Fluorescence, Cell Culture

Journal: Nature Communications

Article Title: Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis

doi: 10.1038/s41467-018-04714-7

Figure Lengend Snippet: Mutations in the PsK domain compromise hMLKL-driven necroptosis in cells. a MLKL expression in parental U937, edited MLKL −/− U937, and MLKL −/− U937 cells reconstituted with a doxycycline-inducible wild-type hMLKL construct were assessed by western blot. Loading control: anti-actin reprobe. b Sensitivity of parental U937, edited MLKL −/− U937, and MLKL −/− U937 reconstituted with doxycycline-inducible wild-type hMLKL to apoptotic (TS) and necroptotic (TSI) stimuli was assessed by PI uptake and flow cytometry. Data represent mean ± SEM of three independent assays. c Sensitivity of MLKL −/− U937 cells reconstituted with doxycycline-inducible wild-type or mutant hMLKL to necroptotic death assessed at 6, 12, and 24 h post-TSI treatment by PI uptake and flow cytometry. Individual data points are plotted as circles. d Parental U937 and MLKL −/− U937 reconstituted with wild-type hMLKL or the E351K, E258K, T357E/S358E, E258K/E351K, or D107A/E111A mutants were assessed for oligomer formation by Blue Native PAGE post-TSI treatment at the indicated time points. Separation into cytoplasmic (C) and membrane (M) fraction was validated by SDS-PAGE western blots for VDAC1 (membrane) and GAPDH (cytoplasmic). All blots are representative of ≥2 independent experiments. e Sensitivity of MLKL −/− U937 cells reconstituted with doxycycline-inducible wild-type or mutant T357E/S358E, T357E/S358D, T357A/S358A, T357E/S358E/E351K, T357A, T357D, S358A, and S358E hMLKL to necroptotic death assessed at 6, 12, and 24 h post-TSI treatment by PI uptake and flow cytometry. Data in c and e represent mean±SEM of two independent assays of duplicate cell lines, except for T357E/S358D, which represents mean ± SD of two independent assays on a single line. Individual data points are plotted as circles. f Sensitivity of parental HT29, MLKL −/− HT29, and MLKL −/− HT29 cells reconstituted with doxycycline-inducible wild-type or mutant T357E/S358E, T357E/S358D hMLKL to necroptotic death at 48 h post-TSI treatment. Data represent mean±SEM of two independent assays on 4 (wild-type) or 2 (mutant) cell lines. Data for parental HT29 cells from a single experiment are shown for comparison. Individual data points are plotted as circles. g Streptavidin-binding peptide (SBP)-tagged hRIPK3 kinase domain immobilized on a streptavidin chip showed robust binding to wild-type, but not T357E/S358E, hMLKL (analyte applied over 0–1 μM) by surface plasmon resonance. Data are representative of four independent experiments

Article Snippet: The histiocytic lymphoma U937 (sourced from ATCC) and colorectal adenocarcinoma HT29 (a kind gift from Professor Mark Hampton and Dr Andreas Konigstorfer, University of Otago, New Zealand) cell lines were cultured in human tonicity RPMI medium and Dulbecco’s modified Eagle’s medium, respectively, supplemented with 8% v/v FCS, with puromycin (5 μg/mL) added for lines stably expressing MLKL constructs.

Techniques: Expressing, Construct, Western Blot, Control, Flow Cytometry, Mutagenesis, Blue Native PAGE, Membrane, SDS Page, Comparison, Binding Assay, SPR Assay

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s with Two Subsets Delineated by Expression of T-Bet (A) Representative contour plot showing gating strategy for splenic DCs in Tbx21 RFP-Cre mice. DCs defined as Lin(CD3,CD19,CD49b,Siglec-F) – Ly6C – CD64 – CD11c + MHCII + . (B) Frequency of T-bet + cDC2s across tissues. Each circle represents one mouse. In the peripheral and mesenteric LN (PLN and MLN), migratory DCs were defined as MHCII hi CD11c int and resident DCs as MHCII int CD11c hi . Error bars represent mean ± SEM. (C) Analysis of RFP + and YFP + splenic cDC2s from Tbx21 RFP-CreERT2 Rosa26 YFP mice, 3 days post tamoxifen gavage. (D) Percent RFP + and YFP + of cDC2 cells. Percent RFP + of YFP + cDC2s at indicated time points post tamoxifen gavage (right). Error bars represent mean ± SEM; n = 3–4 mice per time point. (E) t-SNE embedding of 4,464 DCs. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers (right panel). (F) Expression of canonical DC markers across the transcriptionally defined DC clusters from (E). (G) Proportion of T-bet (RFP + ) cells in each cell cluster identified in (D). (H) Violin plot showing expression of the cell-cycle signature across the DC clusters from (E). (I) Similarity of bulk T-bet – cDC2s, T-bet + cDC2, and cDC1 transcriptomes to the reference single-cell DC clusters (E). Colors represent the correlation coefficient between the cell population identified in the row label and the DC cluster identified by the column label. See also and .

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s, Related to Figure 1 A. Representative histogram showing expression of T-bet (RFP) in splenic cells from Tbx21 RFP-cre mice. (B). Expression of T-bet in CD11b + XCR1 + DCs from the intestinal lamina propria. Data representative of > 5 independent experiments, with at least 3 mice per experiment. (C). Expression of T-bet in splenic myeloid cells. Cells were defined as: (i) Ly-6C hi monocytes (Lin – Ly6C + Ly6G – CD11b + CX3CR1 + ); neutrophils (Lin – Ly6C + Ly6G + ); macrophages (Lin – CD64 + Ly6C – ). Lineages (Lin) were defined as: CD3e, CD90.2, CD19, CD49b and Siglec F. Each circle represents an individual mouse, error bars represent mean ± SEM. (D). Left: Gating strategy for single-cell sorting. DCs were defined as Lin(CD3, CD19, CD90) – Ly6C – CD64 – CD11c + MHCII + . Two populations were sampled: RFP + DCs and RFP – DCs (encompassing XCR1 + cDC1s, CD11b + RFP – and CD11b – XCR1 – DCs). Right: Post-sort purity of RFP + and RFP – cells. Contaminating population of Ly6C + cells identifiable on post-sort purity (lower panel). (E). Similarity of splenic CD11c + MHCII + cells to reference myeloid cells (ImmGen Consortium) Colors represent the Pearson correlation between the mean gene expression from the dendritic cell cluster in the rows and the bulk reference transcriptome in the columns. (F). Top 20 positive and negative gene loadings of PC1 for T-bet + cDC2 clusters after cell-cycle correction (left panel). Scatterplot of PC1 and PC2 for T-bet + cDC2 clusters after cell-cycle correction (right panel).

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, FACS, Gene Expression

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen, Related to Figure 5 (A). Gating strategy for the identification of DC progenitors in the bone marrow (BM) (B). Palantir pseudo-time analysis of differentiation potential and branch probabilities from the Siglec-H + pre-DC state to T-bet + cDC2 and T-bet – cDC2 terminal states. (C). Plots showing Palantir differentiation potential (y axis) along Palantir pseudo-time (x axis) for Siglec-H + DC and T-bet + cDC2s (top) or Siglec-H + DC and T-bet – cDC2 clusters (bottom) (D). Plots showing the top two diffusion component embeddings for Siglec-H + DC and T-bet + cDC2 clusters (top) or Siglec-H + DC and Tbet – cDC2 clusters (bottom). Black arrow indicates Siglec-H + DC cluster cells adjacent to cells from the proliferative T-bet + cDC2 clusters 6 and 8. (E). Top panel: plots showing probability of each cell being within 20 nearest neighbors of randomly sampled shortest paths from the Siglec-H + DC to the indicated end points. Middle panel: plots showing the proportion of cells belonging to Siglec-H + DC, T-bet + cDC2, or T-bet – cDC2 from 20 nearest neighbors of randomly sampled shortest paths. Bottom: plots showing diffusion distance step sizes for each step along the indicated shortest paths (bottom panel). Colors illustrate cluster membership. (F). Graph showing AUC (x axis) for genes differentially expressed between Siglec-H + DC cluster (cluster 11) and all other cDC2 clusters. EMD on the y axis. Dashed lines represents μ EMD ± 3σ EMD . (G). Gating strategy for FACS-isolation of MHCII + ILC3s: Lin = CD3, CD19, CD49b, Siglec-F. (H). Heatmap reports scaled expression of 3550 differentially expressed genes (log 2 FC > 1, FDR < 0.01) between ILC3s and Rorγt fm cDC2s. Selected genes listed to the right. (I). Representative flow cytometric analysis of phenotypes of splenic progeny from Tbx21 RFP-cre CD45.2 + Ly6C − CD64 – MHCII + CD11c + Siglec-H + pre-DCs adoptively transferred into sub-lethally irradiated CD45.1 recipient mice 7 days earlier (data from one experiment with n = 3). J. Sort purified T-bet + or T-bet – cDC2 were cultured for 24hrs in the presence of LPS, CpG, TNF-α or IFN−γ. Representative overlay histogram showing the expression of RFP(T-bet) at 24hrs. Data representative of 2 (TNF-α) or 4 (all other cytokines/TLR agonists) independent experiments, n = 2-3.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Diffusion-based Assay, Isolation, Expressing, Irradiation, Purification, Cell Culture

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Human DC Heterogeneity, Related to Figure 7 (A). Violin plots showing expression distribution of mouse DC subset marker genes across human peripheral blood DC and monocyte clusters identified in Villani et al. (2017) . (B). Representative flow cytometric analysis of mouse peripheral blood cDC2s showing absence of T-bet (RFP) + cDC2s. (C). Gating strategy for FACS-isolation of human spleen DCs for scRNA-seq. DCs were defined as live, LIN(CD3,CD56,CD19) − CD14 – CD11C + HLA-DR + . (D). Representative flow cytometry analysis of human spleen cDC2s gated as Lin(CD3,CD56,CD19) – CD14 – CD11c + HLA-DR + CD123 – XCR1 – CLEC4A + cells. Left panel: cell surface expression of CD1c and CLEC10A by cDC2s. Right panel: overlay of CLEC10A + and CLEC10A – cDC2s distinguished by differential expression of CLEC4A and FcεR1a. Summary bar graphs show frequency of CD1C + CLEC10A + and CD1C + CLEC10A – cDC2s as a percentage of cDC2s (n = 4 individuals). (E). t -SNE embedding of 9,315 FACS-isolated CD45 + immune cells from two melanoma tumors. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers and correlations with bulk RNA-seq data (right panel). Each dot represents an individual cell. (F). Pearson correlations between cluster centroids in (F) and bulk RNA-seq data from purified immune populations ( Jeffrey et al., 2006 , Novershtern et al., 2011 ) (G). t-SNE map of 2,122 myeloid cells identified in (F). Colors indicate patient sample (left) or unsupervised clustering by Phenograph (right panel). Each dot represents an individual cell. (H). Heatmap of normalized, log transformed and MAGIC imputed expression of top 20 differentially expressed genes, defined by the highest earth mover’s distance (EMD), per Phenograph cluster in E. The colored bar at the top of the heatmap shows assignment of cells to clusters labeled in F, right panel. (I). t-SNE map of human melanoma myeloid cells (H) colored by imputed expression of labeled genes.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Quantitative Proteomics, RNA Sequencing, Purification, Transformation Assay, Labeling

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Recombinant, Staining, Multiplex Assay, Cell Isolation, Gene Expression, Software

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet: ( A ) Ex vivo primary murine splenic B cells were treated with 100 μM CK-689 or CK-666 for 1 hr. SPT was then carried out as in , using Cy3-labeled Fab fragments of antibodies to CD19. Single-particle trajectories from a representative cell are plotted using a color-coded temporal scale (left panels). Scale bars: 5 µm. Diffusion coefficients (center panels) and the diameter of maximum displacement (confinement diameter, right panels) over the 10 s period of observation were calculated for each track and cumulative frequency curves are shown. The dots on the curves indicate the median values. ****p<0.0001; Kolmogorov-Smirnov test. ( B–E ) Primary murine B cells were pre-treated with 100 μM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Ig κ surrogate Ag. Cells were fixed at the indicated time points and stained with an antibody that recognizes the surrogate Ag and with an antibody that recognizes phosphorylated CD19. Representative cells are shown ( B ). Scale bars: 2 µm. For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. Beeswarm plots in which each dot represents one cell are plotted with the median (red line) and interquartile ranges (red box) for >125 cells per time point from a representative experiment ( C ). For each cell in ( C ), the fraction of total pCD19 fluorescence that overlaps with BCR-Ag microclusters was quantified by calculating the Manders’ coefficient ( D ). For each cell in ( C ), the total fluorescence intensity of pCD19 that was within BCR-Ag microclusters in cells was quantified ( E ). ****p<0.0001; ***p<0.001; ns, not significant; Mann-Whitney U test.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Ex Vivo, Labeling, Single Particle, Diffusion-based Assay, Expressing, Staining, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet: ( A ) Primary murine B cells were treated with CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the anti-Ig κ surrogate Ag. The cells were fixed at indicated times and stained for surrogate Ag (anti-Ig κ ) and pCD19. For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. For each condition, the median pCD19 fluorescence intensity was determined for >15 cells per experiment. For each experiment, the median pCD19 fluorescence intensity for CK-666-treated cells was expressed as a percent of the median value for CK-689-treated cells (=100%). This ratio is plotted for four independent experiments. ( B ) Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then stimulated with 10 µg/ml soluble anti-Ig κ for the indicated times. pCD19 and total CD79a (loading control) immunoblots are shown (left panels) and the pCD19/total CD79a ratios are graphed (right panels). Dotted red line corresponds to the pCD19/total CD79a ratio value for unstimulated CK-689-treated B cells. Representative data from one of three experiments. See for full blots. ( C ) The co-localization of pSyk with BCR-Ag clusters is not dependent on Arp2/3 complex activity. The co-localization of pSyk and Ag in the cells that were analyzed in was quantified using Manders’ coefficient.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Expressing, Staining, Fluorescence, Control, Western Blot, Activity Assay

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet: Images of blots that were cropped for presentation in , , and are shown. The portions of the blots that were shown in the indicated figures are outlined by a red dashed box. Molecular weight markers are shown in kDa. ( A ) Full blots for and . Primary murine B cells were pre-treated with CK-689 (lanes 1–5) or CK-666 (lanes 6–10) for 1 hr then stimulated with COS-7 APCs expressing anti-Ig κ (left) or with soluble anti-Ig κ (right) for 0, 3, 5, 15 or 30 min. The upper blots were probed with anti-pCD79 antibodies and the lower blots with anti-CD79a antibodies. ( B ) Full blots for , an additional independent experiment carried out as in ( A ). ( C ) Full blots for . Primary murine splenic B cells were treated with DMSO (lane 1), CK-689 (lane 2), CK-666 (lane 3) for 1 hr, or stimulated with anti-Ig κ antibodies for 5 min (lane 4). The blots were probed with anti-pAkt plus anti-pERK antibodies (upper blot) or with anti-ERK plus anti-Akt antibodies (lower blot). ( D ) Full blots for . Primary murine B cells were pre-treated with CK-689 (lanes 1–5) or CK-666 (lanes 6–10) for 1 hr then stimulated with soluble anti-IgΚ for 0, 3, 5, 15 or 30 min. The left blot was probed with anti-pCD19 antibodies and the right blot with anti-CD79a antibodies as a loading control.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Molecular Weight, Expressing, Control

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet:

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Sequencing, Immunofluorescence, Western Blot, Single-particle Tracking, Flow Cytometry, Labeling, Cell Isolation, Electroporation, Recombinant, Software

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Schematic view of TBEV viral proteins. b. Comprehensive interactome analysis was conducted to study interactions between TBEV and host proteins. c. Viral baits expressed in HEK293T cells and analyzed by immunoblot assay using anti-Flag antibody. d, e. Enrichment analysis of TBEV-interacted host proteins (d) and the proteins (e) significantly deregulated by TBEV infection. f. UpSet plot showing the overlap of flavivirus-host protein–protein interactions from other studies.( , ) Each bar shows the interactions shared by only the marked studies at the bottom.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Western Blot, Infection, Protein-Protein interactions

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. An integrated protein-protein interaction network showed high-confidence TBEV-host interactions. Viral proteins are represented as rhombus nodes. Edge intensity reflects the MIST value associated with the interaction. Solid thick edges represent the most significant interactions, while light edges correspond to low MIST (Molecular Interaction Search Tool) value (0.5 < MIST < 0.99). b-f. Validation of virus-host interactions using co-immunoprecipitation. Flag-tagged empty vector (EV) or viral proteins were overexpressed in HEK293T cells, and CHUK co-precipitated with prM ( b ), SYVN1 co-precipitated with NS2B ( c ), SPCS1 co-precipitated with NS4A ( d ), endogenous Ephrin B1(EFNB1) co-precipitated with NS4B ( e ), along with PAF1 and LEO1 co-precipitated with NS5( f ), were detected using flag beads and immunoblot assay.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Biomarker Discovery, Virus, Immunoprecipitation, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. The schematic illustration outlines the network diffusion analysis process. The proteome results were initially integrated with the virus-host interactome, and subsequently mapped to the global human protein-protein interactome to identify affected host pathways. b. Subnetworks derived from network diffusion predict the correlation of TBEV NS5 with spliceosome (| Log 2 FC|>0.1, P value < 0.05). c. Subnetworks resulting from network diffusion depict the correlation of TBEV prM with factors involved in autophagy (|Log 2 FC|>0.1, P value < 0.05). d, e. Additional subnetworks derived from network diffusion predict the correlation of TBEV NS5 with DNA damage response ( d ) and cell cycle ( e ) (|Log 2 FC|>0.1, P value < 0.05). Black edges denote connections present in ReactomeFI. The thickness of the line reflects the combined score provided by STRING. The thickness of directed edges is proportional to the random-walk transition probability.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Diffusion-based Assay, Virus, Derivative Assay

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Vero cells were either mock-infected or infected with TBEV at various MOI (0.1, 1.0 and 10), cells were collected at 48 hours post infection (hpi) and their distribution was analyzed by flow cytometry. b. The experiments in ( a ) were repeated for three times, and the proportions of cells at different cell cycle phases were shown in column graph. c. Vero cells were mock-infected or infected with TBEV at the MOI of 1.0, cells were collected at 12, 18, 24, 36 and 48 hpi, and the percentages of cells in G0/G1 ( up ), S ( middle ) and G2/M ( down ) phase were analyzed by flow cytometry. d-i . Vero cells were synchronized to G0/G1 ( d ), S ( f ) and G2/M ( h ) phases via no-serum starvation, adding 0.85mM Thymi or 25 ng/mL Noco for 20 h. Subsequently, the cells were either mock-infected or infected with TBEV at an MOI of 10, cells were collected and analyzed using flow cytometry. The experiments were repeated for three times, and the proportion of cells in were shown in column graphs. j-l. Vero cells were synchronized to different stages and subsequently infected with TBEV, the mRNA level of TBEV envelope (E) gene was analyzed at 48 hpi by probe qPCR ( j ). The protein level of TBEV NS1 was analyzed at 48 hpi by immunoblot ( k, l ).

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Cells were transfected with the plasmids of Flag-tagged empty vector (EV) and ten TBEV viral proteins, the cell distribution was analyzed by flow cytometry. b. The Flag-EV, 0.5 and 1.0 μg Flag-NS5 were transfected into A549 cells, the cell distribution was analyzed by flow cytometry. The experiment was repeated for three times, and the percentage of cells were shown in column graph. c. Cells were transfected with Flag-EV or Flag NS5 together with HA-P300, the interaction between NS5 and P300 was analyzed by co-immunoprecipitation analysis. d. The colocalization of P300 (green) and NS5 (red) were examined by immunofluorescence analysis. Scale bar: 10 μm. e-g . Cells transfected with NS5 were collected after 48 h, the expression of CDK4, CDK6 and P16 was analyzed by immunoblot ( e ) and qPCR analysis ( f ). The relative expression of CDK4, CDK6 and P16 was shown in column graph ( g ). h. A549 cells transfected with HA-EV, HA P300 and P300 Hm were further transfected with NS5, the distribution of cells was analyzed by flow cytometry. The experiments were repeated for three times, and the proportions of cells were shown in column graph, the expression of indicated proteins was assessed by immunoblot assay. i. Cells transfected with P300 siRNA and NC siRNA (siNC) were further transfected with NS5, the distribution of cells was analyzed by flow cytometry, and the proportions of cells and the relative expression of P300 mRNA were shown in column graph. j. Cells transfected with HA-EV, HA P300 and P300 Hm were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. k. Cells transfected with P300 siRNA and NC siRNA were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. l. Outline of G0/G1 cell cycle arrest regulated by TBEV NS5.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Immunoprecipitation, Immunofluorescence, Expressing, Western Blot, Infection

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Cells were either mock-infected or infected with TBEV, and then collected at 12, 24, 36 and 48 hpi, the expression levels of cell cycle regulators were detected by immunoblot using indicated antibodies. M: mock, I: infection. b. Representative images of brain organoid generation from iPSCs. Scale bar, 500 µm. c. Microscopic images of brain organoids, either mock-infected or infected with TBEV at 0- and 3-days post-infection (dpi). Scale bar, 200 µm. d. Microscopic images depicting 42-day-old iPSC-derived organoids, featuring TUJ1 (neurons), SOX2 (progenitors), Olig2 (oligodendrocytes), and NeuN (neuronal nuclei) markers. Scale bar, 500 µm. e, f . Brain organoids were either mock-infected or infected with TBEV at the MOI of 2.0, the expression of CDK4 ( e ) and P16 ( f ) in brain organoids were examined using immunofluorescence analysis. CDK4 and P16 were visualized in red, TBEV NS1was visualized in green, and the nuclei were stained in blue. Scale bar: 50 μm.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Expressing, Western Blot, Derivative Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a, b . A549 cells were either mock-infected or infected with TBEV, the cells were then treated with indicated concentration of C646 ( a ) and CPI-637 ( b ), the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. c-f . Cells mock-infected or infected with TBEV at the MOI of 10 were treated with 10 μM C646 and CPI-637, the expression of TBEV NS1 protein was detected by immunoblot ( c, d ) and immunofluorescence analysis ( e ), and the cell cycle distribution was analyzed by flow cytometry ( f ). Scale bar: 50 μm. g. Cells transfected with HA-EV, HA P300 and P300Hm were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. h. Cells transfected with P300 siRNA and NC siRNA were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. i. A549 cells were treated with the indicated concentrations of chrysin for 2 h, the cells were then infected with TBEV, the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. j-l. Cells were treated with 10 μM chrysin for 2 h, the cells were then mock-infected or infected with TBEV at the MOI of 10, the expression of TBEV NS1 protein was detected by immunoblot ( j ), and the cell distribution was analyzed by flow cytometry ( k ). The percentage of cells ( l ) upon chrysin treatment was shown in column graphs. Scale bar: 50 μm.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Virus-protein interaction network and predicted drug targets. Potential drug-protein interactions were predicted by NeDREx using the TrustRank algorithm, integrating data from various biomedical databases and experimentally validated targets. Nodes represent proteins and edges indicate their interactions. b-d. Cells were pretreated with indicated inhibitors targeting RIOK1/BRD9 ( b ), XIAP ( c ) and KAT6A ( d ) before TBEV infection for 48 h. The IC 50 value was determined using probe qPCR. Cell viability was assessed using cell counting (CCK-8) assay in the absence of viral infection. The percentage of viral titer compared with DMSO treatment (red) and cell viability (black) depicted. Error bars represent the mean ± SEM of three biological replicates.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Virus, Infection, Cell Counting, CCK-8 Assay