Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: cfDNA promotes the polarization of macrophages towards the M1 phenotype by activating the TLR9-MyD88 and cGAS-STING pathways (A- D) CpG DNA treatment increased the protein levels of IL-1α, TNF-α, and IL-6. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h and then subjected to western blotting (A). IL-1α (B), TNF-α (C), and IL-6 (D) levels were normalized to the β-actin level in each sample, and the normalized values were used for statistical analysis, n = 4. (E-F) CpG DNA treatment significantly enhanced CD86 expression in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (E) and quantitative fluorescence graph (F) are shown, n = 6. Scale bar: 50 μm. (G-J) CpG DNA treatment significantly upregulated the expression of key proteins associated with the TLR9-MyD88 and cGAS-STING pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h and then subjected to western blotting, n = 8/4(G, I). STING, cGAS, TLR9, and MyD88 levels were normalized to the β-actin level in each sample. p-NF-κB, p-IκB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, IκB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis, n = 8/4(H, J). For (B-D, F, H, J), error bars represent mean ± standard deviation. P values were calculated using one-way analysis of variance (ANOVA) (B-D, F, H, J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: RBC-MBs promotes the polarization of macrophages towards the M2 phenotype by inhibiting the TLR9-MyD88 and cGAS-STING pathways (A-F) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of pro-inflammatory cytokines (A-B), key proteins in the TLR9-MyD88 (C-D) and cGAS-STING (E-F) pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, and then subjected to western blot analysis (A, C, E). IL-1α, TNF-α, IL-6, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample. p-NF-κB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis (B, D, F), n = 6. (G) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of CD86 in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (left panel) and quantitative fluorescence graph (right panel) are shown. Scale bar: 50 μm, n = 6. (H-J) RBC-MB treatment significantly inhibited hepatic IRI-induced polarization of macrophages toward the M1 phenotype (H) and activation of the TLR9-MyD88 and cGAS-STING pathways (I-J). MASLD rats underwent 45 min of hepatic ischemia followed by reperfusion. At 0, 6, 12, 18, and 24 h post-reperfusion, the rats were treated with either Con-MBs or RBC-MBs. Primary macrophages were isolated from rat livers, then co-stained with CD68 and CD163 followed by flow cytometric analysis (H), n = 3/4, or subjected to western blotting (I-J). CD206, iNOS, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample, and these normalized values were used for statistical analysis (J), n = 3. For (B, D, F, G right panel , H right panel , J), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (B, D, F, G right panel ) or one-way analysis of variance (ANOVA) (H right panel , J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Incubation, Concentration Assay, Western Blot, Fluorescence, Microscopy, Activation Assay, Isolation, Staining, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: RBC-MBs promotes the polarization of macrophages towards the M2 phenotype by inhibiting the TLR9-MyD88 and cGAS-STING pathways (A-F) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of pro-inflammatory cytokines (A-B), key proteins in the TLR9-MyD88 (C-D) and cGAS-STING (E-F) pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, and then subjected to western blot analysis (A, C, E). IL-1α, TNF-α, IL-6, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample. p-NF-κB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis (B, D, F), n = 6. (G) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of CD86 in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (left panel) and quantitative fluorescence graph (right panel) are shown. Scale bar: 50 μm, n = 6. (H-J) RBC-MB treatment significantly inhibited hepatic IRI-induced polarization of macrophages toward the M1 phenotype (H) and activation of the TLR9-MyD88 and cGAS-STING pathways (I-J). MASLD rats underwent 45 min of hepatic ischemia followed by reperfusion. At 0, 6, 12, 18, and 24 h post-reperfusion, the rats were treated with either Con-MBs or RBC-MBs. Primary macrophages were isolated from rat livers, then co-stained with CD68 and CD163 followed by flow cytometric analysis (H), n = 3/4, or subjected to western blotting (I-J). CD206, iNOS, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample, and these normalized values were used for statistical analysis (J), n = 3. For (B, D, F, G right panel , H right panel , J), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (B, D, F, G right panel ) or one-way analysis of variance (ANOVA) (H right panel , J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Incubation, Concentration Assay, Western Blot, Fluorescence, Microscopy, Activation Assay, Isolation, Staining, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: Synthetic RBC-MBs with high TLR9 expression (A-B) TLR9 protein levels on erythrocyte membranes were significantly decreased in both patients with MASLD (A), n = 9/7, and MASLD rats (B), n = 8/6. Erythrocytes were collected from patients with or without MASLD (A) and from rats fed a normal diet or a high-fat diet (B). Erythrocyte membranes were isolated and subjected to western blotting ( upper panels ). TLR9 levels were normalized to ATP1A levels in each sample, and the normalized values were used for statistical analysis ( lower panels ). (C) Lipopolysaccharide (LPS) treatment significantly increased TLR9 protein levels on erythrocyte membranes. Rats were intravenously injected with 12 mg kg -1 LPS for 6 h. Erythrocytes were collected, and the membranes were isolated and analyzed by western blotting ( upper panel ). TLR9 levels were normalized to ATP1A levels in each sample, and the normalized values were used for statistical analysis ( lower panel ), n = 6. (D) Schematic diagram illustrating the synthesis of RBC-MBs. (E) The ultrasound microbubbles appeared as a milky suspension. (F-G) Size distribution of Con-MBs (F) and RBC-MBs (G) in phosphate-buffered saline. (H-I) Average hydrodynamic diameters (H) and surface charges (ζ-potential) (I) of Con-MBs and RBC-MBs, as determined by dynamic light scattering, n = 6. (J) RBC-MBs were round, uniform, and well-dispersed. Erythrocyte membranes were labeled with DiL (red), and phospholipids were labeled with DiO (green). Scale bar: 30 μm. (K) RBC-MBs contained TLR9, CD47, and ATP1A proteins. Red blood cells, erythrocyte membranes, and RBC-MBs were subjected to western blotting. (L) RBC-MBs effectively adsorbed cfDNA in vitro . BRL-3A cells were treated with 250 μM sodium palmitate and 500 μM sodium oleate for 24 h, followed by hypoxia for 9 h and reoxygenation for 2 h. Subsequently, the cells were co-incubated with DiO-labeled (green) Con-MBs or RBC-MBs for 20 min, then immunostained with an anti-DNA antibody (red) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Scale bar: 20 μm. For (A-C, H-I), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (A, B, C, H, I). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Expressing, Isolation, Western Blot, Injection, Suspension, Saline, Labeling, In Vitro, Incubation, Fluorescence, Microscopy, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: cfDNA promotes the polarization of macrophages towards the M1 phenotype by activating the TLR9-MyD88 and cGAS-STING pathways (A- D) CpG DNA treatment increased the protein levels of IL-1α, TNF-α, and IL-6. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h and then subjected to western blotting (A). IL-1α (B), TNF-α (C), and IL-6 (D) levels were normalized to the β-actin level in each sample, and the normalized values were used for statistical analysis, n = 4. (E-F) CpG DNA treatment significantly enhanced CD86 expression in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (E) and quantitative fluorescence graph (F) are shown, n = 6. Scale bar: 50 μm. (G-J) CpG DNA treatment significantly upregulated the expression of key proteins associated with the TLR9-MyD88 and cGAS-STING pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h and then subjected to western blotting, n = 8/4(G, I). STING, cGAS, TLR9, and MyD88 levels were normalized to the β-actin level in each sample. p-NF-κB, p-IκB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, IκB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis, n = 8/4(H, J). For (B-D, F, H, J), error bars represent mean ± standard deviation. P values were calculated using one-way analysis of variance (ANOVA) (B-D, F, H, J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: RBC-MBs promotes the polarization of macrophages towards the M2 phenotype by inhibiting the TLR9-MyD88 and cGAS-STING pathways (A-F) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of pro-inflammatory cytokines (A-B), key proteins in the TLR9-MyD88 (C-D) and cGAS-STING (E-F) pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, and then subjected to western blot analysis (A, C, E). IL-1α, TNF-α, IL-6, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample. p-NF-κB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis (B, D, F), n = 6. (G) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of CD86 in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (left panel) and quantitative fluorescence graph (right panel) are shown. Scale bar: 50 μm, n = 6. (H-J) RBC-MB treatment significantly inhibited hepatic IRI-induced polarization of macrophages toward the M1 phenotype (H) and activation of the TLR9-MyD88 and cGAS-STING pathways (I-J). MASLD rats underwent 45 min of hepatic ischemia followed by reperfusion. At 0, 6, 12, 18, and 24 h post-reperfusion, the rats were treated with either Con-MBs or RBC-MBs. Primary macrophages were isolated from rat livers, then co-stained with CD68 and CD163 followed by flow cytometric analysis (H), n = 3/4, or subjected to western blotting (I-J). CD206, iNOS, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample, and these normalized values were used for statistical analysis (J), n = 3. For (B, D, F, G right panel , H right panel , J), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (B, D, F, G right panel ) or one-way analysis of variance (ANOVA) (H right panel , J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Incubation, Concentration Assay, Western Blot, Fluorescence, Microscopy, Activation Assay, Isolation, Staining, Standard Deviation

Journal: Materials Today Bio

Article Title: Hydrogel-integrated osteogenic microtissues promote repair of infected intervertebral defects through sequential immunomodulation

doi: 10.1016/j.mtbio.2026.103657

Figure Lengend Snippet: Osteogenic response supported by hydrogel under conditional inflammatory environment of RAW264.7 macrophages. A, B) Schematic illustrations of the inflammatory culture systems established using conditioned medium from LPS-stimulated RAW264.7 macrophages to evaluate osteogenic differentiation of rBMSCs (A) and osteogenic bone microtissues (BO) (B) . C, E) Representative ALP)staining and quantitative analysis of ALP activity on day 7. D, F) Representative ARS staining and quantitative analysis of mineral deposition on day 14. G) Immunofluorescence staining of OCN and OPN in BO after the different treatments. H) Western blot of RUNX2 and OCN expression. I,J) Quantification of RUNX2 and OCN. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Osteogenic protein expression in bone microtissues was assessed by immunofluorescence staining for OCN (ABclonal, A6205, 1:200) and OPN (ABclonal, A23658, 1:200) and by western blotting for RUNX2(Abcam, ab192256,1:1000), OCN (ABclonal, A6205, 1:1000), and GAPDH (ABclonal, AC001, 1:10,000).

Techniques: Staining, Analysis, Activity Assay, Immunofluorescence, Western Blot, Expressing

Journal: Materials Today Bio

Article Title: Human iPSC-EV-loaded nanofiber stent coatings accelerate vascular repair by enhancing EGFR/HIF-1α signaling and suppressing ROCK1-mediated remodeling

doi: 10.1016/j.mtbio.2026.103564

Figure Lengend Snippet: Characterization of human induced pluripotent stem cells (iPSCs) and iPSC-derived extracellular vesicles (EVs). (A) Phase-contrast microscopy image of the IBMS-iPSC-02-07 line, exhibiting typical stem cell morphology with tightly packed colonies, well-defined borders, and a high nuclear-to-cytoplasmic ratio. Scale bar: 50 μm. (B) Western blot analysis confirming the expression of key pluripotency transcription factors OCT4, SOX2, and NANOG. Tubulin served as the loading control. (C) Immunofluorescence staining demonstrating the nuclear localization of pluripotency markers OCT4 and SOX2 (red). The cytoskeleton was stained with F-actin (green) and nuclei were counterstained with DAPI (blue). Scale bar: 75 μm. (D) Validation of EV isolation purity by Western blot. Isolated EVs were enriched for the exosomal markers CD63 and HSP70 but were negative for the endoplasmic reticulum marker Calnexin compared to whole-cell lysates (Cell lysate), confirming the absence of cellular contamination. (E) Transmission electron microscopy (TEM) image revealing the characteristic cup-shaped morphology and lipid bilayer structure of the EVs. Scale bar: 200 nm. (F) Nanoparticle tracking analysis (NTA) determining the particle size distribution and concentration (2.45 × 10 11 particles/mL), showing a size range consistent with small EVs.

Article Snippet: Primary antibodies used for Western blotting and immunofluorescence were categorized as follows: Pluripotency markers: Nanog (1:1000, A3232, ABclonal), OCT4 (1:1000, ab19857, Abcam), and SOX2 (1:1000, ab97959, Abcam).

Techniques: Derivative Assay, Microscopy, Western Blot, Expressing, Control, Immunofluorescence, Staining, Biomarker Discovery, Isolation, Marker, Transmission Assay, Electron Microscopy, Concentration Assay

Journal: Materials Today Bio

Article Title: Human iPSC-EV-loaded nanofiber stent coatings accelerate vascular repair by enhancing EGFR/HIF-1α signaling and suppressing ROCK1-mediated remodeling

doi: 10.1016/j.mtbio.2026.103564

Figure Lengend Snippet: In vivo evaluation of re-endothelialization and inhibition of intimal hyperplasia following stent implantation. (A–E) SEM images of the luminal surface treated with EV-loaded (A, B) or DPBS-loaded (D, E) nanofibrous membranes. The EV-treated group exhibited a continuous, smooth, and intact endothelial layer (A, B), whereas the DPBS group displayed incomplete coverage with substantial endothelial disruption (D, E). (C, F) H&E-stained cross-sections revealed the extent of neointimal formation above the stent struts (black arrows). The EV group (C) demonstrated significantly reduced intimal thickening compared with the pronounced hyperplasia observed in the DPBS group (F). (G–H) Statistical analysis confirmed that EV treatment significantly increased endothelial coverage (G) and suppressed intimal hyperplasia thickness (H). (I–K) EV treatment significantly upregulated the expression of the angiogenic factor VEGF (I), the endothelial marker CD31 (J), and the proliferation marker PCNA (K), confirming accelerated vascular repair. (L–N) EV treatment prevented phenotypic switching and matrix accumulation, as evidenced by reduced levels of the contractile/hyperplasia marker α-SMA (L). Furthermore, the EV group showed decreased expression of matrix metalloproteinases MMP-2 and MMP-9 (M) and beneficial modulation of collagen composition, with increased Collagen I and the Collagen I/III ratio (N). (O–P) The EV group exhibited significantly downregulated levels of the profibrotic factor TGF-β1 (O) and the inflammatory cytokine IL-6 (P). N = 5 (G and H), and n = 3 (I-P). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.000.

Article Snippet: Proliferation and remodeling markers: PCNA (1:1000, ab29, Abcam), IL-6 (1:1000, A0286, ABclonal), TGF-β1 (1:1000, A2124, ABclonal), MMP-2 (1:1000, A6247, ABclonal), and MMP-9 (1:1000, A2095, ABclonal).

Techniques: In Vivo, Inhibition, Disruption, Staining, Expressing, Marker

Journal: Experimental and Therapeutic Medicine

Article Title: PDK1/AKT signaling is involved in the anti-tumor effect of gramine and cisplatin chemoresistance in ovarian cancer cells

doi: 10.3892/etm.2026.13273

Figure Lengend Snippet: PDK1 overexpression impaired the anti-proliferation effect of gramine on OC cells. (A) Western blotting assay determining the effect of gramine on PDK1 expressions. SK-OV-3 and OV-90 cells were incubated with gramine (50, 100 µM) for 24 h. * P<0.05, ** P<0.01, *** P<0.001 vs. the untreated control. (B) Western blotting assay assessing the transfection efficiency of PDK1 overexpression lentivirus. *** P<0.001 vs. the EV control. (C) Cell counting kit-8 assay detecting the effect of PDK1 overexpression on cell viability. (D) Clone formation assay assessing the effect of PDK1 overexpression on colony formation ability. (E) Western blotting assay examining the effect of PDK1 overexpression on Ki67 expression. * P<0.05, ** P<0.01 vs. the EV control or gramine-treated EV group (C-E). Data represent the mean ± SD of three independent experiments. EV, empty vector; OV, overexpression.

Article Snippet: The primary antibodies used were as follows: Ki67 (1:2,000; 28074-1-AP; Proteintech Group, Inc.), Bax (1:20,000; 50599-2-Ig; Proteintech Group, Inc.), Bcl-2 (1:5,000; 68103-1-Ig; Proteintech Group, Inc.), PARP1 (1:1,000; 13371-1-AP; Proteintech Group, Inc.), cleaved PAPR1 (1:5,000; 60555-1-Ig; Proteintech Group, Inc.), N-cadherin (1:20,000; 22018-1-AP; Proteintech Group, Inc.), E-cadherin (1:20,000; 20874-1-AP; Proteintech Group, Inc.), vimentin (1:20,000; 10366-1-AP; Proteintech Group, Inc.), GAPDH (1:50,000; 60004-1-Ig; Proteintech Group, Inc.), PDK1 (1:1,000; A0834; Abclonal Biotech Co., Ltd.), AKT (1:1,000; 4691; Cell Signaling Technology, Inc.) and p-AKT (Thr308) (1:1,000; 9275; Cell Signaling Technology, Inc.).

Techniques: Over Expression, Western Blot, Incubation, Control, Transfection, Cell Counting, Tube Formation Assay, Colony Assay, Expressing, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: PDK1/AKT signaling is involved in the anti-tumor effect of gramine and cisplatin chemoresistance in ovarian cancer cells

doi: 10.3892/etm.2026.13273

Figure Lengend Snippet: PDK1 overexpression impaired the pro-apoptotic effect of gramine on ovarian cancer cells. (A) TUNEL assay evaluating the effect of PDK1 overexpression on cell apoptosis. Scale bar, 200 µm. (B) Western blotting assay determining the effect of PDK1 overexpression on Bax and Bcl2 expressions. PDK1 overexpressing SK-OV-3 and OV-90 cells were treated with gramine (100 µM) for 24 h. * P<0.05, ** P<0.01, *** P<0.001 vs. the EV control or gramine-treated EV group. Data represent the mean ± SD of three independent experiments. EV, empty vector; OV, overexpression.

Article Snippet: The primary antibodies used were as follows: Ki67 (1:2,000; 28074-1-AP; Proteintech Group, Inc.), Bax (1:20,000; 50599-2-Ig; Proteintech Group, Inc.), Bcl-2 (1:5,000; 68103-1-Ig; Proteintech Group, Inc.), PARP1 (1:1,000; 13371-1-AP; Proteintech Group, Inc.), cleaved PAPR1 (1:5,000; 60555-1-Ig; Proteintech Group, Inc.), N-cadherin (1:20,000; 22018-1-AP; Proteintech Group, Inc.), E-cadherin (1:20,000; 20874-1-AP; Proteintech Group, Inc.), vimentin (1:20,000; 10366-1-AP; Proteintech Group, Inc.), GAPDH (1:50,000; 60004-1-Ig; Proteintech Group, Inc.), PDK1 (1:1,000; A0834; Abclonal Biotech Co., Ltd.), AKT (1:1,000; 4691; Cell Signaling Technology, Inc.) and p-AKT (Thr308) (1:1,000; 9275; Cell Signaling Technology, Inc.).

Techniques: Over Expression, TUNEL Assay, Western Blot, Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: PDK1/AKT signaling is involved in the anti-tumor effect of gramine and cisplatin chemoresistance in ovarian cancer cells

doi: 10.3892/etm.2026.13273

Figure Lengend Snippet: PDK1/AKT signaling pathway was involved in the anti-migratory and anti-invasive effects of gramine in ovarian cancer cells. (A) Wound healing and (B) transwell invasion assays detecting the effect of PDK1 overexpression on the abilities of cell migration and invasion respectively. Scale bar, 200 µm. (C) Western blotting assay determining the effect of PDK1 overexpression on N-cadherin, vimentin and p-AKT (Thr308) expressions. PDK1 overexpressing SK-OV-3 and OV-90 cells were incubated with gramine (50 µM) for 24 h. * P<0.05, ** P<0.01, *** P<0.001 vs. the EV control or gramine-treated EV group. Data represent the mean ± SD of three independent experiments. EV, empty vector; OV, overexpression.

Article Snippet: The primary antibodies used were as follows: Ki67 (1:2,000; 28074-1-AP; Proteintech Group, Inc.), Bax (1:20,000; 50599-2-Ig; Proteintech Group, Inc.), Bcl-2 (1:5,000; 68103-1-Ig; Proteintech Group, Inc.), PARP1 (1:1,000; 13371-1-AP; Proteintech Group, Inc.), cleaved PAPR1 (1:5,000; 60555-1-Ig; Proteintech Group, Inc.), N-cadherin (1:20,000; 22018-1-AP; Proteintech Group, Inc.), E-cadherin (1:20,000; 20874-1-AP; Proteintech Group, Inc.), vimentin (1:20,000; 10366-1-AP; Proteintech Group, Inc.), GAPDH (1:50,000; 60004-1-Ig; Proteintech Group, Inc.), PDK1 (1:1,000; A0834; Abclonal Biotech Co., Ltd.), AKT (1:1,000; 4691; Cell Signaling Technology, Inc.) and p-AKT (Thr308) (1:1,000; 9275; Cell Signaling Technology, Inc.).

Techniques: Over Expression, Migration, Western Blot, Incubation, Control, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: PDK1/AKT signaling is involved in the anti-tumor effect of gramine and cisplatin chemoresistance in ovarian cancer cells

doi: 10.3892/etm.2026.13273

Figure Lengend Snippet: Gramine downregulates PDK1/AKT linked to cisplatin sensitization of OC cells. (A) CCK-8 assay detecting the effect of cisplatin on cell viability. SK-OV-3 and SK-OV-3-R cells were incubated with cisplatin (5, 10, 20, 40, 80 µM) for 24 h. * P<0.05, ** P<0.01 vs. the untreated control. (B) CCK-8 assay detecting the effect of gramine and cisplatin on cell viability. SK-OV-3-R cells were incubated with gramine (50 µM) and cisplatin (5, 10, 20, 40, 80 µM) for 24 h. * P<0.05 vs. the DMSO group at the identical cisplatin concentration. (C) Clone formation assay assessing the effect of gramine and cisplatin on colony formation ability. (D) TUNEL assay evaluating the effect of gramine and cisplatin on cell apoptosis. Scale bar, 200 µm. (E) Wound healing and transwell invasion assays detecting the effect of gramine and cisplatin on the abilities of cell migration and invasion respectively. Scale bar, 200 µm. (F) Western blotting assay determining the effect of gramine and cisplatin on the expressions of PDK1 and p-AKT (Thr308). SK-OV-3-R cells were incubated with gramine (50 µM) and cisplatin (80 µM) for 24 h. * P<0.05, ** P<0.01, *** P<0.001 vs. the cisplatin group (C-F). Data represent the mean ± SD of three independent experiments. CCK-8, cell counting kit-8.

Article Snippet: The primary antibodies used were as follows: Ki67 (1:2,000; 28074-1-AP; Proteintech Group, Inc.), Bax (1:20,000; 50599-2-Ig; Proteintech Group, Inc.), Bcl-2 (1:5,000; 68103-1-Ig; Proteintech Group, Inc.), PARP1 (1:1,000; 13371-1-AP; Proteintech Group, Inc.), cleaved PAPR1 (1:5,000; 60555-1-Ig; Proteintech Group, Inc.), N-cadherin (1:20,000; 22018-1-AP; Proteintech Group, Inc.), E-cadherin (1:20,000; 20874-1-AP; Proteintech Group, Inc.), vimentin (1:20,000; 10366-1-AP; Proteintech Group, Inc.), GAPDH (1:50,000; 60004-1-Ig; Proteintech Group, Inc.), PDK1 (1:1,000; A0834; Abclonal Biotech Co., Ltd.), AKT (1:1,000; 4691; Cell Signaling Technology, Inc.) and p-AKT (Thr308) (1:1,000; 9275; Cell Signaling Technology, Inc.).

Techniques: CCK-8 Assay, Incubation, Control, Concentration Assay, Tube Formation Assay, Colony Assay, TUNEL Assay, Migration, Western Blot, Cell Counting

Journal: Materials Today Bio

Article Title: Human iPSC-EV-loaded nanofiber stent coatings accelerate vascular repair by enhancing EGFR/HIF-1α signaling and suppressing ROCK1-mediated remodeling

doi: 10.1016/j.mtbio.2026.103564

Figure Lengend Snippet: Characterization of human induced pluripotent stem cells (iPSCs) and iPSC-derived extracellular vesicles (EVs). (A) Phase-contrast microscopy image of the IBMS-iPSC-02-07 line, exhibiting typical stem cell morphology with tightly packed colonies, well-defined borders, and a high nuclear-to-cytoplasmic ratio. Scale bar: 50 μm. (B) Western blot analysis confirming the expression of key pluripotency transcription factors OCT4, SOX2, and NANOG. Tubulin served as the loading control. (C) Immunofluorescence staining demonstrating the nuclear localization of pluripotency markers OCT4 and SOX2 (red). The cytoskeleton was stained with F-actin (green) and nuclei were counterstained with DAPI (blue). Scale bar: 75 μm. (D) Validation of EV isolation purity by Western blot. Isolated EVs were enriched for the exosomal markers CD63 and HSP70 but were negative for the endoplasmic reticulum marker Calnexin compared to whole-cell lysates (Cell lysate), confirming the absence of cellular contamination. (E) Transmission electron microscopy (TEM) image revealing the characteristic cup-shaped morphology and lipid bilayer structure of the EVs. Scale bar: 200 nm. (F) Nanoparticle tracking analysis (NTA) determining the particle size distribution and concentration (2.45 × 10 11 particles/mL), showing a size range consistent with small EVs.

Article Snippet: Primary antibodies used for Western blotting and immunofluorescence were categorized as follows: Pluripotency markers: Nanog (1:1000, A3232, ABclonal), OCT4 (1:1000, ab19857, Abcam), and SOX2 (1:1000, ab97959, Abcam).

Techniques: Derivative Assay, Microscopy, Western Blot, Expressing, Control, Immunofluorescence, Staining, Biomarker Discovery, Isolation, Marker, Transmission Assay, Electron Microscopy, Concentration Assay