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: The American Journal of Pathology

Article Title: MDM2

doi: 10.2353/ajpath.2006.051351

Figure Lengend Snippet: A: Representative RT-PCR analysis of total RNA from cultured human VSMCs (HASMC). RNA was amplified in the presence of oligonucleotide primers specific for MR (top panel) and 11β-HSD type 2 (bottom panel). Extracts from human kidney were used as a positive control (P). B: Representative immunoblotting studies demonstrating MR and 11β-HSD type 2 proteins in HASMC. Extracts from human kidney were used as a positive control (P). C and D: Immunocytochemical analysis demonstrated that immunopositive cells for MR appear brown as a result of diaminobenzidine colorimetric reaction in HASMC without ligand stimulation (C) or treated with 10 nmol/L aldosterone (D) (Original magnification, ×400).

Article Snippet: Cell Culture and Characterization A cultured human VSMC cell line, ie, HASMC (derived from human abdominal aorta) was commercially obtained from Kurabo Corp. (Osaka, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Positive Control, Western Blot

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

doi: 10.1165/rcmb.2013-0133OC

Figure Lengend Snippet: 6-Gingerol, 8-gingerol, and 6-shogaol potentiate isoproterenol-induced relaxation in airway smooth muscle (ASM). (A) In ASM strips (epithelium denuded) contracted with acetylcholine (ACh), isoproterenol elicited a concentration-dependent relaxation. Vehicle (0.2% dimethyl sulfoxide [DMSO], solid squares), 6-gingerol (100 μM, solid triangles), 8-gingerol (100 μM, open circles), or 6-shogaol (100 μM, open triangles) was added concurrently with the 300 pM concentration of isoproterenol. Each component of ginger significantly potentiated isoproterenol-induced relaxations and caused a leftward shift in the relaxation curve. Curves were fit with a four-parameter sigmoid to determine half-maximal effective concentrations (EC50) summarized in Table 1 (*P < 0.05, n = 5–6 strips per group). (B) In human ASM strips contracted with ACh (∼ EC50), 100 μM 6-gingerol, 8-gingerol, or 6-shogaol alone did not significantly relax tissue compared with vehicle (0.2% DMSO) within the first 7–14 minutes after addition. Moreover, the addition of 1 nM isoproterenol (ISO) to vehicle (open bars) did not produce significant relaxation compared with vehicle alone (solid bars). In tissues receiving the combination of either 6-gingerol plus ISO, 8-gingerol plus ISO, or 6-shogaol plus ISO, robust relaxation was observed that was greater than either ginger component or ISO alone (n = 5–9 strips from five patients; *P < 0.05, **P < 0.01, ***P < 0.001). N.S., not significant.

Article Snippet: Cell Culture Immortalized and primary human ASM cell lines were prepared as described previously ( 23 , 24 ) and grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 media (GIBCO, Grand Island, NY) with 10% fetal bovine serum and antibiotics.

Techniques: Concentration Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

doi: 10.1165/rcmb.2013-0133OC

Figure Lengend Snippet: Summary of Effects of Ginger Components of Isoproterenol Half-Maximal Effective Concentration Relaxation of an Established Acetylcholine Contraction

Article Snippet: Cell Culture Immortalized and primary human ASM cell lines were prepared as described previously ( 23 , 24 ) and grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 media (GIBCO, Grand Island, NY) with 10% fetal bovine serum and antibiotics.

Techniques: Concentration Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

doi: 10.1165/rcmb.2013-0133OC

Figure Lengend Snippet: 6-Gingerol and 8-gingerol do not effect ISO-induced heat shock protein (HSP) 20 phosphorylation. In primary human ASM cells, 20-minute treatment with ISO (1 μM) increased phosphorylation of HSP20 (Ser 16; p-HSP20) compared with vehicle controls (0.1% DMSO). The combination of ISO with rolipram (10 μM), 6-gingerol (100 μM), or 8-gingerol (100 μM) showed no difference in phosphorylation compared with ISO alone, but was significantly increased compared with vehicle controls. The combination of ISO and 6-shogaol (100 μM) showed significant attenuation of HSP20 phosphorylation compared with ISO alone; however, this combination remained significantly increased compared with vehicle (*P < 0.05 compared with vehicle, **P < 0.01 compared with vehicle; #P < 0.05 compared with ISO alone; n = 4).

Article Snippet: Cell Culture Immortalized and primary human ASM cell lines were prepared as described previously ( 23 , 24 ) and grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 media (GIBCO, Grand Island, NY) with 10% fetal bovine serum and antibiotics.

Techniques: Phospho-proteomics

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

doi: 10.1165/rcmb.2013-0133OC

Figure Lengend Snippet: 6-Gingerol, 8-gingerol, and 6-shogaol attenuate 17-kD PKC-potentiated inhibitory protein of type 1 protein phosphatase (CPI-17) phosphorylation. (A) In primary human ASM cells, 20-minute treatment with 10 μM ACh increased CPI-17 phosphorylation (Thr 38) compared with control (0.1% DMSO, P < 0.05, n = 5). The addition of Y-27632, 6-gingerol, 8-gingerol, or 6-shogaol (100 μM each), in combination with ACh, significantly reversed the ACh-induced increase in CPI-17 phosphorylation and approximated that of vehicle controls (P < 0.01 compared with Ach; n = 5). (B) Summary bar graph of five experiments.

Article Snippet: Cell Culture Immortalized and primary human ASM cell lines were prepared as described previously ( 23 , 24 ) and grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 media (GIBCO, Grand Island, NY) with 10% fetal bovine serum and antibiotics.

Techniques: Phospho-proteomics, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

doi: 10.1165/rcmb.2013-0133OC

Figure Lengend Snippet: 6-Shogaol, but not 6-gingerol or 8-gingerol, inhibits Ras homolog gene family member A (RhoA) activation. (A) In primary human ASM cells, treatment with bradykinin (10 μM, 5 min) increased RhoA activity measured by G-LISA compared with vehicle controls (0.2% DMSO). A 5-minute pretreatment with 6-shogaol (100 μM) significantly abrogated bradykinin-induced increases in RhoA activity, whereas neither 6-gingerol nor 8-gingerol had an effect (**P < 0.01, ***P < 0.001; n = 6). (B) Total RhoA protein was equivalent among all treatment groups, as determined by immunoblot.

Article Snippet: Cell Culture Immortalized and primary human ASM cell lines were prepared as described previously ( 23 , 24 ) and grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 media (GIBCO, Grand Island, NY) with 10% fetal bovine serum and antibiotics.

Techniques: Activation Assay, Activity Assay, Western Blot

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

doi: 10.1165/rcmb.2013-0133OC

Figure Lengend Snippet: 8-Gingerol attenuates ACh-induced increases in myosin light chain 20 (MLC20) phosphorylation. (A) In M3-overexpressing human ASM cells, 10-minute treatment with 100 μM ACh showed robust MLC20 phosphorylation (p-MLC20). In ACh-treated cells, concurrent treatment with 8-gingerol (100 μM) significantly attenuated the p-MLC20. The Rho kinase inhibitor, Y-27632 (10 μM), showed similar attenuation of the ACh-induced phosphorylation, and was used as a positive control. Samples were loaded in duplicate. (B) Summary bar graph of duplicate lanes in four separate experiments. Phosphorylated MLC20 was corrected for total MLC20 and expressed as a ratio (*P < 0.05 compared with Ach-only–treated cells; n = 4).

Article Snippet: Cell Culture Immortalized and primary human ASM cell lines were prepared as described previously ( 23 , 24 ) and grown in phenol red–free Dulbecco’s modified Eagle’s medium/F12 media (GIBCO, Grand Island, NY) with 10% fetal bovine serum and antibiotics.

Techniques: Phospho-proteomics, Positive Control