Journal: Nature Communications

Article Title: CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

doi: 10.1038/s41467-025-56703-2

Figure Lengend Snippet: a UMAP clustering of 13441 cardiac interstitial cells (left) and fibroblasts (right) isolated from C57BL/6 J wild-type (WT) mice at 7 and 14 days post-sham and post-MI surgery. Each dot represents a single cell. The expression of known marker genes annotated cell type. b UMAP and violin plots depict Postn , Acta2 , and Comp expression in fibroblasts. c Triple IF staining for periostin (green) and αSMA (red) in the peri-infarct zone of the hearts of C57BL/6 J WT mice 0, 7, 14, 21, and 28 days after MI surgery. Nuclei were stained blue. Scale bar = 100 μm. Quantification of the number of periostin + αSMA + F-Myo and periostin + αSMA - F-Act in each high-power field (HPF). n = 5 mice at each time point after MI. d Flow cytometry of fibroblasts isolated from hearts of C57BL/6 J WT and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. F-Myo and F-Act were marked with antibodies against periostin-AF488 and αSMA-AF647. Quantification of the pdgfrα + fibroblast ratio in live cells, periostin + fibroblasts ratio in pdgfrα + fibroblasts, periostin + αSMA + F-Myo ratio in pdgfrα + fibroblasts, and periostin + αSMA - F-Act ratio in pdgfrα + fibroblasts. n = 5 mice at each time point after MI. e Spatial distribution of the F-Act and F-Myo subpopulation in mouse heart sections from sham and 10 days post-MI conditions, visualized through the integration of scRNA-seq and stereo-seq methodologies. c, d Data are the mean ± SEM and p -values are displayed in the bar charts, one-way ANOVA followed by Tukey’s multiple comparisons tests. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this study included Periostin (Abcam, ab14041), αSMA-Alexa Fluor 647 (Novus, NBP2-34522), CD248-Alexa Fluor 647 (Santa Cruz, sc-377221), CD3-FITC (BioLegend, 317306 for human and 100204 for mouse), CD4-APC (BioLegend, 344614 for human and 100412 for mouse), CD8-PE (BioLegend, 344706 for human and 100708 for mouse), and goat anti-rabbit IgG (1:1000, Invitrogen, A11008 for Alexa Fluor 488).

Techniques: Isolation, Expressing, Marker, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

doi: 10.1038/s41467-025-56703-2

Figure Lengend Snippet: a Triple IF staining for periostin (green) and CD248 (red) in the peri-infarct zone of the hearts of C57BL/6J WT mice 0, 7, 14, 21, and 28 days after MI surgery. Nuclei were stained blue. Scale bar = 100 μm. Quantification of the periostin + CD248 + cell to DAPI ratio in each HPF. n = 5 mice at each time point after MI. Triple IF staining of periostin, αSMA, COMP (stained green), and CD248 (stained red) in the hearts of MI mouses ( b ) and a patient with ischemic cardiomyopathy ( c ). Nuclei were stained blue. Scale bar =100 μm. IF staining of mouse heart was repeated in heart sections from 5 independent mice, while IF of human heart was repeated 5 times in heart sections from one patient with ischemic cardiomyopathy, yielding similar results. d Flow cytometry of cells isolated from hearts of C57BL/6 J WT mouses and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. Quantification of the CD248 + cell ratio in live cells, pdgfrα + fibroblast ratio in CD248 + cells, CD248 + cell ratio in pdgfrα + fibroblasts, periostin + CD248 + cell ratio in pdgfrα + fibroblasts, CD248 + cell ratio in periostin + activated fibroblasts, and periostin + activated fibroblasts in CD248 + cells. n = 5 mice at each time point after MI. a, d Data are the mean ± SEM and p -values are displayed in the bar charts, one-way ANOVA followed by Tukey’s multiple comparisons tests. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this study included Periostin (Abcam, ab14041), αSMA-Alexa Fluor 647 (Novus, NBP2-34522), CD248-Alexa Fluor 647 (Santa Cruz, sc-377221), CD3-FITC (BioLegend, 317306 for human and 100204 for mouse), CD4-APC (BioLegend, 344614 for human and 100412 for mouse), CD8-PE (BioLegend, 344706 for human and 100708 for mouse), and goat anti-rabbit IgG (1:1000, Invitrogen, A11008 for Alexa Fluor 488).

Techniques: Staining, Flow Cytometry, Isolation

Journal: Nature Communications

Article Title: CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

doi: 10.1038/s41467-025-56703-2

Figure Lengend Snippet: a Schematic diagram of the structure of the BBIR-T system. Created in BioRender. Haiting, C. (2025) https://BioRender.com/l46n352 . b Schematic images of dcAv. BBIR-28-137ζ and CD19. CAR-137ζ sequence. c Western blotting shows exogenous CD3ζ expression in HEK-293T cells transduced with lentivirus (LV)-dcAv.BBIR and LV-anti-CD19.CAR. The samples derive from the same experiment and that gels/blots were processed in parallel. Quantifying the relative protein expression of CD3ζ to GAPDH. n = 4 cell culture wells of each group. d Flow cytometry of human peripheral T cells transduced with LV-dcAv.BBIR and LV-anti-CD19.CAR. Quantifying GFP + cells ratio to total T cells and biotin + cells ratio to GFP + cells. n = 5 cell culture wells of each group. e Western blotting by BF shows CD248 expression in NIH-3T3 cells with or without Cd248 overexpression(OE) or knockout(KO). The samples were derived from the same experiment and that gels/blots were processed in parallel. Quantifying the relative protein expression of CD248 to GAPDH. n = 4 cell culture wells of each group. f Triple IF staining of periostin, αSMA, COMP (green), and biotinylated anti-CD248 F(ab’)2 (shorted as BF) (red) in the peri-infarct zone 14 days after MI surgery. Nuclei were stained blue. Scale bar =100 μm. Each experiment was repeated 5 times independently with similar results. c-e Data are the mean ± SEM and p -values are displayed in the bar charts, one-way ANOVA followed by Tukey’s multiple comparisons tests. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this study included Periostin (Abcam, ab14041), αSMA-Alexa Fluor 647 (Novus, NBP2-34522), CD248-Alexa Fluor 647 (Santa Cruz, sc-377221), CD3-FITC (BioLegend, 317306 for human and 100204 for mouse), CD4-APC (BioLegend, 344614 for human and 100412 for mouse), CD8-PE (BioLegend, 344706 for human and 100708 for mouse), and goat anti-rabbit IgG (1:1000, Invitrogen, A11008 for Alexa Fluor 488).

Techniques: Sequencing, Western Blot, Expressing, Transduction, Cell Culture, Flow Cytometry, Over Expression, Knock-Out, Derivative Assay, Staining

Journal: Cell Reports Medicine

Article Title: Pharmacological Inhibition of Acid Sphingomyelinase Prevents Uptake of SARS-CoV-2 by Epithelial Cells

doi: 10.1016/j.xcrm.2020.100142

Figure Lengend Snippet: VSV-SARS-CoV-2 Spike Particles Activate and Use the Acid Sphingomyelinase for Infection (A) Genetic downregulation of acid sphingomyelinase (ASM) with short hairpin RNA (shRNA) prevented infection of Caco-2 cells with pp-VSV-SARS-CoV-2 spike but had no impact on infection with particles that express the pp-VSV-G. Downregulation of neutral sphingomyelinase 2 (NSM2) did not affect the infection of Caco-2 cells with pp-VSV-SARS-CoV-2 spike. Control shRNA did not change the infection of Caco-2 cells by pp-VSV-SARS-CoV-2 spike. Cells were infected with pp-VSV-SARS-CoV-2 spike or pp-VSV-G, and cells positive for EGFP were counted in 2,000 cells per sample. Downregulation of ASM or NSM was confirmed by measuring the activity of the enzymes (right panels). (B) Vero cells were infected with pp-VSV-SARS-CoV-2 spike, pp-VSV, or pp-VSV-G for the indicated times or were left uninfected. Samples were pre-incubated with AT or with the solvent (0.9% NaCl) or were left untreated. In addition, recombinant (rec.) ACE2 protein or neutralizing anti-spike antibodies was added prior to and maintained during the infection. Cells were lysed in 250 mM sodium acetate (pH 5.0) and 0.2% NP40. ASM activity was determined by measuring the consumption of [ 14 C]sphingomyelin. Shown are the means ± SD of the percentage of infected cells or the activity of the ASM or NSM from 6 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests.

Article Snippet: The expression of acid or neutral sphingomyelinase in Caco-2 cells was downregulated by transfection with commercial shRNA targeting acid sphingomyelinase (Santa Cruz Inc.; # sc-41650-SH) or neutral sphingomyelinase (Santa Cruz Inc.; # sc-62655-SH).

Techniques: Infection, shRNA, Control, Activity Assay, Incubation, Solvent, Recombinant

Journal: Cell Reports Medicine

Article Title: Pharmacological Inhibition of Acid Sphingomyelinase Prevents Uptake of SARS-CoV-2 by Epithelial Cells

doi: 10.1016/j.xcrm.2020.100142

Figure Lengend Snippet: VSV-SARS-CoV-2 Spike Particles Induce Ceramide Formation, Facilitating Infection (A) To determine ceramide levels, we organically extracted the samples and quantified ceramide levels by using the ceramide kinase method. We quantified C16 (C16-Cer)/C18 ceramide and C22/C24 ceramide. If indicated, cells were preincubated with 5, 10, 20, or 25 μM AT for 4 h before the addition of pp-VSV-SARS-CoV-2 spike. The rec. ACE2 protein or neutralizing anti-spike antibodies were added as above. In addition, cells were infected with pp-VSV or pp-VSV-G. Displayed are the means ± SD of the ceramide concentrations from each 5 independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests, as indicated or compared to the corresponding value without inhibitor. (B) Flow cytometry reveals the formation of ceramide in the outer leaflet of the cell membrane. Cells were treated with 25 μM AT, each 2 μg rec. ACE2 protein or neutralizing anti-spike antibodies and infected with pp-VSV-SARS-CoV-2 spike. Controls were infected with pp-VSV or pp-VSV-G. Shown are the mean fluorescence values (in a.u.) ± SD of 6 independent flow cytometry studies; ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests. (C) Reconstitution of ceramide in Vero cells (left panel) that had been treated with 10 or 25 μM AT or in Caco-2 cells (right panel) transfected with shRNA targeting ASM by the addition of C16-Cer (10 μM) or ASM (0.2 U/mL) during the infection with pp-VSV-SARS-CoV-2 spike restores viral infection of the cells. The rec. ACE2 protein or neutralizing anti-spike antibodies prevented the infection. Displayed are the means ± SD of the percentage of infected cells from 6 independent experiments. ∗∗∗p < 0.001; ANOVA, followed by post hoc Student’s t tests. See also Figure S3 .

Article Snippet: The expression of acid or neutral sphingomyelinase in Caco-2 cells was downregulated by transfection with commercial shRNA targeting acid sphingomyelinase (Santa Cruz Inc.; # sc-41650-SH) or neutral sphingomyelinase (Santa Cruz Inc.; # sc-62655-SH).

Techniques: Infection, Flow Cytometry, Membrane, Fluorescence, Transfection, shRNA

Journal: Cell Reports Medicine

Article Title: Pharmacological Inhibition of Acid Sphingomyelinase Prevents Uptake of SARS-CoV-2 by Epithelial Cells

doi: 10.1016/j.xcrm.2020.100142

Figure Lengend Snippet:

Article Snippet: The expression of acid or neutral sphingomyelinase in Caco-2 cells was downregulated by transfection with commercial shRNA targeting acid sphingomyelinase (Santa Cruz Inc.; # sc-41650-SH) or neutral sphingomyelinase (Santa Cruz Inc.; # sc-62655-SH).

Techniques: Produced, Virus, Recombinant, TUNEL Assay, In Situ, shRNA, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling.

doi: 10.1016/j.celrep.2023.113369

Figure Lengend Snippet: Figure 6. Netrin-1 interference suppresses metastasis and increases survival in murine PDAC models (A) Mice were pretreated with mAbNtn1 for 2 days and intrasplenically injected with 50,000 Ink4a.1 cells. Antibody treatment (10 mg/kg) continued every 2 days until liver harvest at day 14. Percentage tumor area in the liver was quantified as before (n = 5). (B) Kaplan-Meier graph showing survival of mice in- jected intrasplenically with 20,000 Ink4a.1 cells using mAbNtn1 (n = 16) as adjuvant treatment or vehicle (n = 19). Note that three mAbNtn1-treated mice have not recurred beyond day 200. (C) Immunohistochemistry of CK17-stained livers from C57Bl/6J mice injected via portal vein injection with KPC3 cells (left) and treated with IgG control or mAbNtn1, n = 10/group. Percentage of tumor metastasis area was quantified as before, n = 5 sec- tions/mouse. Note the significant reduction in mice treated with anti-Netrin-1 antibody (right). Scale bars, 200 mm. (D) Normalized Netrin-1 expression in KPC3 cells, normal liver, hepatic KPC3 metastases from control IgG-treated mice, and hepatic KPC3 metastases from mAbNtn1-treated mice as quantified by western blot. (E) Kaplan-Meier plot showing survival of KPC mice treated with IgG vehicle and mAbNtn1 antibodies. The vehicle group (n = 17) had MS = 18 days, while the mAbNtn1 group (n = 11) had MS 42 days, a doubling of lifespan. All experiments were completed at least in duplicate. Statistical analysis was completed using two-tailed Student’s t test (***p < 0.001, ****p < 0.0001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-netrin 1 antibody [EPR5428] Abcam ab126729 Human/Mouse ELF3 Antibody R&D Systems AF5787 RXRa (D6H10) Rabbit mAb Cell Signaling 3085S ELF3 Polyclonal Antibody Invitrogen PA5-89261 Normal Rabbit IgG Cell Signaling 2729P RARa/Retinoic Acid Receptor a Antibody (C-1) Santa Cruz Biotechnology sc-515796 Mouse (G3A1) mAb IgG1 Isotype Control Cell Signaling 5415S Anti-Desmin antibody [Y66] Abcam ab32362 Smooth muscle actin Polyclonal antibody ProteinTech 14395-1-AP Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 532 Invitrogen A-11009 Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 555 Invitrogen A-21428 Anti-CD9 antibody [EPR2949] Abcam ab92726 UNC5B (D9M7Z) Rabbit mAb Cell Signaling 13851 Purified Mouse Anti-Flotillin-1 BD Biosciences 610820 Cytokeratin 17 Monoclonal Antibody (CK17) Thermo Fisher 606–540 RARg1 (D3A4) XP Rabbit mAb Cell Signaling 8965S Anti-Retinoid X Receptor beta/RXRB antibody Abcam ab221115 RXRg Antibody (G-6) Santa Cruz Biotechnology sc-514134 Actin Antibody (H-6) Santa Cruz Biotechnology sc-376421 Alix (3A9) Mouse mAb Cell Signaling 2171S Alexa Fluor 647 anti-mouse/human CD324 (E-Cadherin) Antibody Biolegend 147307 Vimentin (D21H3) XP Rabbit mAb Cell Signaling 5741S alpha-Smooth Muscle Actin Antibody (1A4/asm-1) - Azide and BSA Free Novus Biologicals NBP2-34522 mAbNtn1 Patrick Mehlan N/A Bacterial and virus strains Edit-R Mouse Unc5b hEF1a All-in-one Lentiviral sgRNA Horizon Discovery VSGM11942-247910466 Edit-R Mouse Elf3 mCMV All-in-one Lentiviral sgRNA Horizon Discovery VSGM11942-248070215 Edit-R Mouse Ntn1 Lentiviral sgRNA Horizon Discovery VSGM10144-246774776 Biological samples BxPC-3 ATCC CRL-1687 Capan-2 ATCC HTB-80

Techniques: Injection, Adjuvant, Immunohistochemistry, Staining, Control, Expressing, Western Blot, Two Tailed Test