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: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: Primer sequences for qRT-PCR.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: Sequencing, Amplification

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: B. Protein-protein interaction (PPI) network of key genes and their functional annotations. C-H. GSEA enrichment curves for key genes (CASP1, FABP4, BCL6, IL10, NR4A2, SHBG), displaying major enriched pathways in high- and low-expression groups.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: Functional Assay, Expressing

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: A-F. Molecular docking results of PFOA with SHBG, FABP4, BCL6, CASP1, IL10, and NR4A2.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: Docking Assay

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: H-N. Molecular dynamics simulation results for the SHBG-PFOA complex.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques:

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: B. Effect of PFOA on cell viability (CCK-8 assay). C. Kinetic analysis of SHBG and FABP4 interactions (SPR sensorgrams). D.Western Blot (WB) bands. E. Bar chart of Q-PCR results (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). F. Bar chart of WB grayscale analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: CCK-8 Assay, Western Blot

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: Primer sequences for qRT-PCR.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: Sequencing, Amplification

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: B. Protein-protein interaction (PPI) network of key genes and their functional annotations. C-H. GSEA enrichment curves for key genes (CASP1, FABP4, BCL6, IL10, NR4A2, SHBG), displaying major enriched pathways in high- and low-expression groups.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: Functional Assay, Expressing

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: A-F. Molecular docking results of PFOA with SHBG, FABP4, BCL6, CASP1, IL10, and NR4A2.

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: Docking Assay

Journal: PLOS One

Article Title: Environmental PFOA exposure and the risk of metabolic dysfunction-associated steatotic liver disease: An integrated computational toxicology and multi-omics study

doi: 10.1371/journal.pone.0357970

Figure Lengend Snippet: B. Effect of PFOA on cell viability (CCK-8 assay). C. Kinetic analysis of SHBG and FABP4 interactions (SPR sensorgrams). D.Western Blot (WB) bands. E. Bar chart of Q-PCR results (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). F. Bar chart of WB grayscale analysis (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: The following primary antibodies were used for Western blot analysis: Anti-BCL6 Rabbit Polyclonal Antibody (Catalog No. DF2903; Affinity, USA), Anti-IL10 Rabbit Polyclonal Antibody (Catalog No. DF6894, Affinity, USA), Anti-SHBG Rabbit Polyclonal Antibody (Catalog No. A7450, ABclonal, China), Anti-NR4A2 Rabbit Polyclonal Antibody (Catalog No. A22011, ABclonal, China), Anti-Cleaved Caspase-1 Rabbit Polyclonal Antibody (Catalog No. A27901 , ABclonal, China), Anti-FABP4 Rabbit Polyclonal Antibody (Catalog No. A25792, ABclonal, China), Anti-β-Actin Mouse Monoclonal Antibody (Catalog No. T0022, Affinity, USA), Sodium palmitate (PA) and sodium oleate (OA) were acquired as a pre-mixed solution (PA 6 mmol/L, OA 12 mmol/L, Catalog No. KC006) from Xi’an Kunchuang Technology Development Co., Ltd. (China).

Techniques: CCK-8 Assay, Western Blot