Journal: Advanced Science

Article Title: Endowing Flexibility of Alveolar Type II Cells to Recapitulate Development From Lung Bud to Alveoli

doi: 10.1002/advs.77773

Figure Lengend Snippet: Establishment and characterization of alveolar progenitor‐like organoids (APLOs), bud tip‐like organoids (BTLOs), and alveolar‐like organoids (ALOs). (A) Schematic diagram illustrating the differentiation protocol from APLCs to APLOs, BTLOs, and ALOs. Created in BioRender. Ma, H. (2026) https://BioRender.com/91lb473 (B) Representative immunofluorescence (IF) images showing expression of the bud tip marker SOX9 in P5‐APLOs and P15‐BTLOs. Scale bars, 50 µm. (C) Quantitative PCR (qPCR) analysis of AT2 and AT1 cell marker expression in APLC‐P5‐2D, P5‐APLOs, P5‐ALOs, and human lung tissue. Data are presented as mean ± SD from three independent experimental replicates using samples derived from a single donor (n = 3). Significance was evaluated by one‐way ANOVA with Tukey's multiple comparisons test; ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See also Supplementary Figure . (D) Representative transmission electron microscopy (TEM) images of APLOs and ALOs with lamellar body structures. Scale bars, 2 µm (left), 500 nm (upper right), 1 µm (lower right). (E) Immunofluorescence staining of the pan‐epithelial marker E‐cadherin and AT2 markers (proSFTPC, SFTPC, and HT2‐280) in APLOs, BTLOs, and ALOs. Scale bars, 50 µm. (F) Western blot analysis of bud tip markers (SOX9, SOX2), AT2 markers (proSFTPC, SFTPB dimer), and the lung lineage marker NKX2‐1 in APLOs, BTLOs, and ALOs.

Article Snippet: Primary antibodies were as follows: Pro‐surfactant protein C (abcam, ab90716, 1:800), E‐cadherin (Proteintech, 20874‐1‐AP, 1:500), SFTPC (ABclonal, A1835, 1:100), HT2‐280 (terracebiotech, TB‐27AHT2‐280, 1:250), NKX2‐1(Beyotime, AG3409, 1:200), Influenza A virus Nucleoprotein (GeneTex, GTX636247, 1:100).

Techniques: Organoids, Immunofluorescence, Expressing, Marker, Real-time Polymerase Chain Reaction, Analysis, Tissue, Derivative Assay, Transmission Assay, Electron Microscopy, Staining, Western Blot

Journal: Advanced Science

Article Title: Endowing Flexibility of Alveolar Type II Cells to Recapitulate Development From Lung Bud to Alveoli

doi: 10.1002/advs.77773

Figure Lengend Snippet: Influenza A virus (IAV) infection of APLOs, BTLOs and ALOs. (A) Schematic diagram outlining the method for IAV infection in APLOs, BTLOs and ALOs. To establish infection models, organoids were collected and broken to expose the luminal space. Then, the pieces of organoids were incubated with IAV at a multiplicity of infection (MOI) of 0.1 or 1.0 for 2 h before resuspended in Matrigel. Cells and supernatants were collected at 2,24,48 h after IAV infection for subsequent experiments. Created in BioRender. Ma, H. (2026) https://BioRender.com/jxmufa7 (B) Representative images for plaque assay using IAV‐infected cells at 24 hpi and 48 hpi. Dilution factors are shown above. (C) Plaque statistics of figure B. Data are presented as mean±SD (n = 3, three plaque assays at each time point).(D) Viral load of IAV in lysed APLOs, BTLOs and ALOs. Data are presented as mean±SD (n = 3, three qPCR assays at each time point). P5‐APLOs, P15‐BTLOs, P5‐ALOs and P15‐ALOs were used in the experiment, **** p < 0.0001. (E) Viral load of IAV in supernatant of APLOs, BTLOs and ALOs. Data are presented as mean±SD (n = 3, three qPCR assays at each time point). P5‐APLOs, P15‐BTLOs, P5‐ALOs and P15‐ALOs were used in the experiment, **** p < 0.0001. (F) Representative images of an infected ALOs at 48hpi. Viral components NP (green) are co‐stained with pro‐SFTPC (red). Scale bars, 25 µm. (G, I) Low‐magnification transmission electron microscopy (TEM) images of ALOs at 48 hpi, showing overall cellular architecture. Scale bars: 1 µm (G), 2 µm (I). (H, J) High‐magnification TEM views of infected AT2 cells within ALOs. Blue arrows (H) indicate individual IAV particles dispersed in the cytoplasm. Blue arrows (J) point to viral particles encapsulated within vesicular structures. Scale bars, 500 nm. (K) Volcano plot displaying differentially expressed genes in IAV‐infected ALOs compared to uninfected controls at 48 hpi. (L) Heatmap depicting dynamic transcriptional changes of key immune response genes including chemokines, interleukins, tumor necrosis factor (TNF), and interferon family members in APLOs, BTLOs, and ALOs at 0, 24, and 48 hpi. Expression values are normalized and shown as FPKM.

Article Snippet: Primary antibodies were as follows: Pro‐surfactant protein C (abcam, ab90716, 1:800), E‐cadherin (Proteintech, 20874‐1‐AP, 1:500), SFTPC (ABclonal, A1835, 1:100), HT2‐280 (terracebiotech, TB‐27AHT2‐280, 1:250), NKX2‐1(Beyotime, AG3409, 1:200), Influenza A virus Nucleoprotein (GeneTex, GTX636247, 1:100).

Techniques: Virus, Infection, Organoids, Incubation, Plaque Assay, Staining, Transmission Assay, Electron Microscopy, Expressing

Journal: Advanced Science

Article Title: Ultrafast Micrometric Laser Structuring of Polymer Surfaces for Highly Organized Skeletal Muscle Tissue Modeling

doi: 10.1002/advs.78129

Figure Lengend Snippet: Analysis of MHC and Desmin protein levels during C2C12 myoblast differentiation. Western blot analysis of C2C12 cells cultured on standard (Std) and LS surfaces at different stages of differentiation. Representative immunoblot for (a) MHC and Tubulin, and (c) Desmin and GAPDH. Densitometric analysis of (b) MHC and (d) Desmin protein levels. Data are presented as mean ± SD with individual data points ( n = 3). Statistical significance was assessed using Student's t ‐test ( * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Standard at each time point).

Article Snippet: Membranes were blocked with 5% non‐fat dry milk in Tris‐buffered saline containing 0.05% Tween (TBS‐Tween) and then incubated overnight at 4°C with primary antibodies against Myosin Heavy Chain (MHC; 1:1000, Bio‐Techne MAB4470, monoclonal, clone MF20), Desmin (1:3000, Abclonal A3736, monoclonal, clone ARC0235), GAPDH (1:40000, Sigma MAB374, monoclonal, clone 6C5) and Tubulin (1:4000, Sigma T6199, monoclonal, clone DM1A).

Techniques: Analysis, Western Blot, Cell Culture

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