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

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: Stage‐specific alternative splicing programs reveal SF3B1 as a key regulator in early mouse embryogenesis. (a) Pie chart depicting the relative proportions of alternative splicing event (ASE) types—mutually exclusive exons (MXE), skipped exon (SE), alternative 3'splice site (A3SS), alternative 5'splice site (A5SS), and retained intron (RI)—detected across mouse pre‐implantation stages: from 2‐cell to 4‐cell, 4‐cell to 8‐cell, and 8‐cell to Morula (FDR < 0.05). (b) Representative examples of the three major PSI (percent spliced‐in) trajectory clusters—Peak, Shift, and Other—identified by Mfuzz at mouse 4‐cell and 8‐cell stages. (d,e) Scatter plots of gene‐level changes across 4‐ to 8‐cell and 8‐cell to morula transitions. Gray dots indicate non‐significant genes; red dots mark up‐regulated genes, blue dots mark down‐regulated genes. The y‐axis displays log 2 (baseMean) expression level. Significance criteria: FDR <0.05 and absolute |(log 2 FC)| ≥ 2 (FC, fold change). (f) Heatmap represents expression for Cluster 5 dynamics of splicing factors (n = 140) that are sharply up‐regulated from the 4‐cell stage onward. Venn diagram depicting their overlap with the published 21 pluripotency‐associated splicing set; SF3B1 and Lsm2 (highlighted) are the two shared regulators. (g) A Line plots showing the mRNA profiles of SF3B1 various developmental stages were examined by RNA‐seq. (h) Quantitative PCR was performed to analyze the relative expression levels of Sf3b1 in embryos at different developmental stages. Five independent biological replicates were included in the experiment, with H2afz serving as the reference gene. In the boxplots, the center line represents the median, while the upper and lower edges of the boxes indicate the upper and lower quartiles, respectively. (i,j) Representative immunostaining images (i) and quantitative analysis (j) of mouse embryos at different stages stained with anti‐SF3B1 (Green) antibody (n ≥ 10 oocytes/embryos per group). Groups were compared by two‐tailed unpaired t ‐test. Nuclei were stained with DAPI (light blue). Scale bar: 20 µm.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Alternative Splicing, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Immunostaining, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: SF3B1 plays critical roles in preimplantation development. (a) Representative image represents embryonic development from the 2‐cell stage to the blastocyst stage. Mouse embryos at zygotic stage were injected with siRNA targeting SF3B1, while scrambled siRNA (siNC) served as the control. Scale bar: 100 µm. The total number of embryos in each group of treatment were as follows: siNC embryos (n = 100), siSF3B1 (n = 100). (b) Statistical evaluation of siSF3B1 injection on embryonic developmental rate. n = 3 biologically independent replicates. Data are mean ± SD. Two‐tailed Mann–Whitney U test, p = 0.05. (c,d) Same analyses as in panels (a,b), but performed with embryos injected with siRNA targeting SF3B6 instead of SF3B1. (e) Immunofluorescent staining images for SF3B1 in 4‐ and 8‐cell embryos after siNC or siSF3B1 microinjection (three independent experiments). Scale bar: 20 µm. Relative SF3B1 intensity was quantified with ImageJ and is expressed as mean ± SEM; groups were compared by two‐tailed unpaired t ‐test. (f) Quantitative PCR analysis of Sf3b1 expression at 4‐cell, 8‐cell, and morula stages. Transcript levels were normalized to endogenous H2afz and presented as mean ± SD of three biological replicates. (g) Immunofluorescent staining of 5‐ethynyl uridine (EU) showing RNA transcription in 2‐cell and 4‐cell embryos treated with siSF3B1.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Injection, Control, Two Tailed Test, MANN-WHITNEY, Staining, Microinjection, Real-time Polymerase Chain Reaction, Expressing

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: Disruption of SF3B1 leads to abnormal balance of pluripotency and totipotency gene expression in mouse 4‐cell and morula embryos. (a) Diagram illustrating the procedure of acquiring samples for total RNA‐seq analysis in embryos subjected to SF3B1‐KD and control conditions. (b) Bidimensional principal component (PC) analysis was conducted to assess the gene expression patterns in embryos undergoing preimplantation development under control and SF3B1‐KD conditions. (c) Venn diagrams comparing differentially expressed genes (DEGs) (FDR < 0.05 and |log 2 FC| ≥ 2) among different treatments. (d) Volcano plots of SF3B1‐KD versus control embryos at 4‐cell and morula: dots above the horizontal dashed line indicate DEGs (FDR ≤ 0.05, |log2FC| ≥ 2). FDR‐corrected with the Benjamini–Hochberg method (DESeq2). (e) Volcano plots showing the DEGs in morula‐stage embryos treated with plaB versus DMSO. Up‐regulated genes (red): n = 1224; down‐regulated genes (blue): n = 512. The highlighted gene Zfp352 (log 2 FC ≈ 8.48) is indicated. f GO enrichment analysis of differentially expressed genes in morula‐stage embryos upon SF3B1‐KD. Bars represent the –log 10 ( p‐ value) of the top enriched biological‐process terms; orange: up‐regulated genes, blue: down‐regulated genes. (g) Heat map showing the selected totipotency and pluripotency genes of RNA‐seq‐derived relative expression (log 2 ‐normalized) from MII oocyte to ICM stage. Adjacent bubble chart indicates genes altered in 4‐cell and morula SF3B1‐KD embryos. (h,i) qPCR quantification of pluripotency ( Nr5a2 , Myc , Oct4 , Nanog ) (h) and totipotency ( Zscan4d , Zscan4c , Zfp352 , Dux ) (i) transcript levels in SF3B1‐KD morula. Data are the mean ± SEM of three biologically independent replicates, normalized to endogenous H2afz .

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Disruption, Gene Expression, RNA Sequencing, Control, Derivative Assay, Expressing

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: SF3B1 directly shapes the alternative splicing landscape governing the cell cycle. (a) Proportions of five ASE categories‐MXE, SE, A3SS, A5SS, and RI‐significantly shifted in SF3B1‐KD versus control embryos at the 4‐cell and morula stages. (b) Counts of DEGs ( p < 0.05; |log 2 FC| > 1) corresponding to each AS event type. (c) Heatmap displaying the normalized expression patterns of transcripts A5SS and RI events at morula stage embryos. Values represent normalized relative mRNA abundance. (d) Venn diagrams showing the relationship of categorized as splicing active (SA) and splicing repressive (SR) between SF3B1‐KD and control embryos at the 4‐cell (left) and morula (right) stages. (e) A Venn diagram showing the overlap between ASEs and DEGs in siSF3B1 versus siNC (top). GO analysis of the 943 shared mRNAs is presented (bottom). (f) Sashimi plots depict read coverage and exon–exon junction usage for cdk11b . (g) Diagram of CDK11b protein architecture highlighting the kinase domain and basic region. Semiquantitative RT‐PCR ( n = 3 per group) mapped cdk11b ectopic splicing in SF3B1‐KD morula, and representative gels combined with pooled quantification of the specified fragment are showed accordingly. (h) Same as panel f, but for ccnb1 . (i,j) The ectopic splicing of ccnb1 by semiquantitative RT‐PCR. (k) Immunofluorescent staining of γH2AX showing DNA double‐strand breaks (DSBs) in 2‐cell and 4‐cell embryos treated with siSF3B1. Relative signal intensity was quantified with ImageJ and is expressed as mean ± SEM; groups were compared by two‐tailed unpaired t ‐test.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Alternative Splicing, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: The global landscape of SF3B1‐binding sites. (a) Venn diagram showing genomic distribution of the SF3B1‐binding reads in 4‐cell. (b) Venn diagram showing genomic distribution of the SF3B1‐binding reads in morula. (c) Enriched SF3B1‐binding motifs identified by LACE‐seq and their p values are shown. (d,e) The signal intensity distributions of 5’ splicing site of SF3B1 binding in morula (d) and 3’ splicing site (e). f GO enrichment analysis of SF3B1‐specific targets in morula. The represent results from two independent experiments.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Binding Assay

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: Characterization of SF3B1 IDR phase separation. (a) Domain structure and intrinsic disorder tendency of SF3B1. The top panel shows the domains of SF3B1, along with PONDR analysis. (b) Turbidity assays monitoring LLPS of SF3B1 IDR across different PEG8000 concentrations (n = 6). (c) Representative fluorescence (AF488‐labeled) and differential interference contrast (DIC) images demonstrating droplets of 10 µM SF3B1 IDR with varying concentrations of PEG8000. Scale bar: 10 µm. d‐e Quantitative analysis of SF3B1 IDR droplet average size (d) and droplet density per 100 µm 2 area (e) under increasing PEG8000 concentrations (0%, 1%, 5% and 10% w/v). n = 5 biologically independent samples. (f) Representative time‐lapse images showing fusion events of 10 µM SF3B1 IDR droplets in T50N100 buffer supplemented with 10% PEG8000. Insets show magnified views of tracked droplets highlighted in yellow circles. Scale bar, 10 µm. (g) Turbidity assays monitoring LLPS of SF3B1 IDR across varying concentrations in T50N100 buffer supplemented with 5% PEG8000 (n = 6). (h) Representative fluorescence (AF488‐labeled) and DIC images demonstrating SF3B1 IDR droplet formation at varying protein concentrations in T50N100 buffer supplemented with 5% PEG8000. Scale bar: 10 µm. (i) SF3B1 IDR condensate coverage (% Area) at varying protein concentrations (1.3–10.0 µM). n = 5 independent replicates. (j) Turbidity assays monitoring LLPS of 5 µM SF3B1 IDR in 50 mM Tris‐HCl buffer (supplemented with 5% PEG8000) at different NaCl concentrations. (n = 6). (k) Representative fluorescence (AF488‐labeled) and DIC images of 5 µM SF3B1 IDR in 50 mM Tris‐HCl buffer with 5% PEG8000, at different NaCl concentrations (50, 100, 200, 500 mM). Scale bar: 10 µm. (l) SF3B1 IDR condensate coverage (% Area) in 50 mM Tris‐HCl buffer (5% PEG) across different NaCl concentrations. n = 5 independent replicates. (m) Representative images depicting the effect of total RNA on SF3B1 IDR condensation. SF3B1 IDR was mixed with different concentrations of total RNA in T25N50 buffer (25 mM Tris‐HCl, 50 mM NaCl, pH 7.5) supplemented with 5% PEG8000. Scale bar = 10 µm. (n) SF3B1 IDR condensate coverage (% Area) in T25N50 buffer (5% PEG8000) across different RNA concentrations (7.8–250 ng/µL). All quantitative data are presented as mean ± SD. Turbidity assays (b, g, j) used n = 6 biologically independent samples; droplet/density quantifications (d, e, i, l, n) used n = 5 biologically independent samples. Statistical differences were determined by one‐way ANOVA with Tukey's multiple comparisons test. Significance levels: n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Fluorescence, Labeling

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: SF3B1 undergoes LLPS in a concentration dependent manner. (a) Immunofluorescence staining of endogenous SF3B1 (green) in HEK293T and HeLa cells. Nuclei were counterstained with DAPI (blue). Scale bar: 10 µm. (b) Representative confocal image of HEK293T and Hela cells transfected with different forms of recombinant GFP‐SF3B1 constructs, including GFP, GFP‐SF3B1 WT , GFP‐SF3B1 IDR, and GFP‐SF3B1 C‐term . Nuclear area visualized by Hoechst staining. Scale bar: 5 µm. (c) Representative confocal images of HEK293T cells expressing GFP, GFP‐SF3B1 WT , GFP‐SF3B1 IDR, and GFP‐SF3B1 C‐term at 10, 30, and 50 arbitrary units (a.u.). Scale bar: 5 µm. Representative images were selected based on relevant statistical analyses from three independent experiments with similar results. (d) Quantitative phase diagram depicting the cellular fluorescence intensity of SF3B1 domains observed. Each dot represents the SF3B1 concentration measured in an individual cell. Blue dots indicate positive phase separation, while red dots indicate negative phase separation. (e) Representative images of the FRAP experiment in HEK293T cells transfected with GFP‐SF3B1 WT or GFP‐SF3B1 C‐term . Images depict the Pre‐Bleach (before photobleaching), Bleach (during photobleaching), and Post‐Bleach (30s and 60s after photobleaching, relative to 0s at bleaching). The red circle highlights the organelle targeted for bleaching. Scale bar: 5 µm. (f) The quantification of FRAP data for GFP‐SF3B1 WT or GFP‐SF3B1 C‐term puncta. Time 0 refers to the time point of the photobleaching pulse. Data are presented as the means ± SEMs (n = 6), n = individual SF3B1 nuclear condensate.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Concentration Assay, Immunofluorescence, Staining, Transfection, Recombinant, Construct, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Phase Separation of SF3B1 Serves as a Critical Post‐Transcriptional Regulator During Early Mouse Embryogenesis

doi: 10.1002/advs.77605

Figure Lengend Snippet: LLPS of SF3B1 is essential for early mouse embryonic development. (a) Representative immunostaining images indicate NSN, SN, and GVBD oocyte stained with anti‐SF3B1 (Green) antibody (n ≥ 5 oocytes per group). Nuclei were stained with DAPI (light blue). Scale bar: 20 µm. (b,c) Confocal images of embryos microinjected at the 1‐cell stage with GFP‐tagged mRNAs encoding GFP–SF3B1 IDR (b) or GFP‐SF3B1 ΔIDR− FUS −IDR (c), and imaged at zygote and 2‐cell stages. Scale bar: 20 µm. (d) Representative image represents embryonic development from the 2‐cell stage to the blastocyst stage. Mouse embryos at zygotic stage were injected with siRNA targeting SF3B1 combined with GFP‐SF3B1 FL , GFP‐SF3B1 IDR or GFP‐SF3B1 ΔIDR‐FUS‐IDR . Scale bar: 100 µm. n = 3 biologically independent replicates. (g) Model of SF3B1 mediated alternative splicing in mouse early embryo development.

Article Snippet: Membranes were blocked with TBST buffer supplemented with 5% non‐fat milk (BBI,A600669) for 2 h at room temperature, followed by overnight incubation at 4°C with primary antibodies: mouse monoclonal GFP antibody (Beyotime, AG281, 1:2000), rabbit polyclonal SF3B1 antibody (ABclonal, A15801, 1:1000), and GAPDH as a loading control (YEASEN, 30202ES60, 1:5000).

Techniques: Immunostaining, Staining, Injection, Alternative Splicing