Journal: Advanced Science

Article Title: Elevator‐Like Hollow Channels in Porous Scaffolds Accelerate Vascularized Bone Regeneration via NETs‐Fibrin‐Mediated Macrophage Recruitment

doi: 10.1002/advs.202515693

Figure Lengend Snippet: Neutrophil activation and NETs formation mediated by fibrinogen. a) H&E staining of rat subcutaneous scaffolds 1 h postimplantation. b) Immunofluorescence costaining of fibrinogen and MPO. c–e) Serial paraffin sections were subjected to immunofluorescence costaining for: c) MPO and CitH3, d) MPO and NE, e) MPO and CD177. Nuclei were counterstained with Sytox Green. f) dHL‐60 cells were seeded onto PBS/hFibrinogen‐coated dishes. Morphological changes were observed by labeling cell membranes with Dil and nuclei with Sytox Green. g) Immunofluorescence costaining for MPO and CitH3 in dHL‐60 after 1 h, with nuclei counterstained with Sytox Green. h) 3D coculture scheme: Fibrinogen‐preadsorbed scaffolds were seeded with dHL‐60 cells for 1 h. i) Cell membranes were labeled with Dil, and the nuclei with Syto Green.

Article Snippet: dHL‐60 nuclei were stained with 20 n m Syto Green (KeyGEN Biotech, Nanjing, China) for 20 min.

Techniques: Activation Assay, Staining, Immunofluorescence, Labeling

Journal: Advanced Science

Article Title: Elevator‐Like Hollow Channels in Porous Scaffolds Accelerate Vascularized Bone Regeneration via NETs‐Fibrin‐Mediated Macrophage Recruitment

doi: 10.1002/advs.202515693

Figure Lengend Snippet: NETs drive macrophage infiltration into channel structures. a) MPO⁺ neutrophils and CD68⁺ macrophages in subcutaneously implanted scaffolds at 4 h, with nuclei counterstained using Sytox Green. b) H&E staining at 8 h postimplantation, along with immunofluorescence costaining of MPO and CD68, with Sytox Green nuclei counterstain. c) Left: Masson's trichrome staining at 24, 48 h; Right: CD68⁺ macrophages in serial sections. d) Gelation assay: A mixture of fibrinogen solution and dHL‐60 cell suspension transitioned from a liquid to a gel state within 3 h. e) Schematic showing the design of the 3D coculture experiment to assess the chemotactic effect of NETs on THP‐1 cells. f) THP‐1 cells (Dil, red) were seeded on the surface of NETs (Syto Green, green)/fibrin gel, and imaged at specified time points. g) Schematic of the Transwell assay for evaluating the chemotactic effect of NETs on THP‐1 cells. h) Migrated THP‐1 cells (crystal violet⁺) at 24 h. i) Quantification and statistical analysis of migrated THP‐1 cells ( n = 6 biological replicates). Data were analyzed using unpaired t ‐tests, with statistical significance defined as **** p < 0.0001.

Article Snippet: dHL‐60 nuclei were stained with 20 n m Syto Green (KeyGEN Biotech, Nanjing, China) for 20 min.

Techniques: Staining, Immunofluorescence, Suspension, Transwell Assay

Journal: PLOS Biology

Article Title: RNA-binding protein IMP1/ZBP1 directs local translation in microglial processes to regulate motility and phagocytosis during inflammation

doi: 10.1371/journal.pbio.3003463

Figure Lengend Snippet: (A) To identify the somatic domain and the peripheral structures in microglia, cells were stained with an anti-calreticulin (Calr) antibody (+Ab1) to visualize the “somatic” ER (soma, 1) and with phalloidin to visualize the actin-dense lamellipodia (2). The periphery was defined as the region where Calr was undetectable compared to a no-primary antibody negative control (−Ab1) which coincides with an intense phalloidin staining. Representative micrographs of the distribution of Calr and phalloidin are shown. Scale bar, 20 µm. (B) Newly synthesized proteins measured in the periphery of microglia treated with vehicle (PBS) or LPS for 24 h. Puromycilated proteins in the periphery of phalloidin-stained microglia are shown. Insets (1 and 2) show puromycin labeling at the edge of lamellae where filopodia emerge. Scale bar, 20 µm (insets, 5 µm) (B i ) . The box and whisker graph indicates the mean fluorescence intensity of puromycin in the periphery of microglia in 13 independent cultures ( n = 13) analyzed by two-tailed t test. ** p < 0.01 (B ii ). (C) Puromycin and SYTO-positive heatmaps are shown, as well puromycin and SYTO-positive foci in binarized images. Scale bar, 5 µm (C i ) . Box and whisker graphs represent the average SYTO-positive foci (C ii ) and SYTO-puromycin colocalization (C iii ) in the periphery of PBS- and LPS-treated microglia from 6 independent experiments ( n = 6) analyzed by two-tailed t tests. * p < 0.05; ** p < 0.01. (D) Active ribosomal protein Rsp6 was analyzed. Insets show the levels of pS6 in PeMPs from PBS (1)- and LPS (2)-treated cells. Scale bars 5 µm (D i ) . The box and whisker graph indicates the mean fluorescence intensity of pS6 in PeMPs in the periphery of microglia in 6 independent cultures ( n = 6) analyzed by two-tailed t test. * p < 0.05 (D ii ) . (E) pS6 s t aining in cortical microglia of fms-EGFP 1-month-old mice injected with saline or LPS. Insets show the levels of pS6 in GC-like PeMPs. Scale bars 20 µm (left panels), 10 µm (right panels) (E i ) . Violin plots represent the mean intensity of pS6 in 27–36 sampled GC-like PeMPs (E ii ) or primary processes (E iii ) from 3 to 4 mice ( n = 27–36; smaller dots. N = 3–4; bigger dots). Statistical analyses were performed by two-tailed t tests. ** p < 0.01. The data underlying this Figure can be found in .

Article Snippet: Cells were incubated for 20 min at room temperature with 500 nM SYTO RNA Select green fluorescent dye in PBS (Invitrogen).

Techniques: Staining, Negative Control, Synthesized, Labeling, Whisker Assay, Fluorescence, Two Tailed Test, Injection, Saline

Journal: PLOS Biology

Article Title: RNA-binding protein IMP1/ZBP1 directs local translation in microglial processes to regulate motility and phagocytosis during inflammation

doi: 10.1371/journal.pbio.3003463

Figure Lengend Snippet: Par3 mRNA in PeMPs in vitro . (A) Actb levels measured by FISH in cortical microglia (positive for P2Y12 and GFP) from fms-EGFP 1-month-old mice injected with saline or LPS. Linescans represent the mean distribution of the FISH signal ±SEM from the nucleus to the PeMPs in 50-58 individual cells per condition (A i ) . Violin plots represent the mean intensity of Actb in 26–31 sampled GC-like PeMPs (smaller dots) and 50–58 cell bodies (smaller dots) from 3 to 4 mice (larger dots). Two-tailed t tests. * p < 0.05; ** p < 0.01 (A ii ) . Two cells per condition are shown as examples in fluorescence micrographs (cells a and b). (1) and (2) indicate GC-like PeMPs and cell bodies represented in insets. Scale bars 10 µm (5 µm insets) (A iii ) . The bar graph shows the mean relative frequency distribution ±SEM of Actb intensity in PeMPs and the soma in cortical microglia from 3 to 4 mice ( N = 3–4). One-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; ** p < 0.01 (A iv ) . (B) New synthesis of β-actin in microglial peripheral structures was assessed with a 2-min puromycin pulse in PBS- and LPS-treated cells followed by proximity ligation assay (PLA) with antibodies against puromycin and β-actin. As a negative control, cells were preincubated with the protein synthesis inhibitor anisomycin. Micrographs show the PLA signal in microglia labeled with phalloidin. Arrowheads indicate PeMPs depicted in insets (4 rightmost panels). Scale bars 20 µm (insets 10 µm) (B i ) . The box and whisker graph represents the average PLA puncta within lamellae in phalloidin-stained microglia treated with PBS or LPS in 6 independent cultures ( n = 6). One-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; *** p < 0.001; n.s, not significant (B ii ) . The data underlying this Figure can be found in .

Article Snippet: Cells were incubated for 20 min at room temperature with 500 nM SYTO RNA Select green fluorescent dye in PBS (Invitrogen).

Techniques: In Vitro, Injection, Saline, Two Tailed Test, Fluorescence, Comparison, Proximity Ligation Assay, Negative Control, Labeling, Whisker Assay, Staining

Journal: PLOS Biology

Article Title: RNA-binding protein IMP1/ZBP1 directs local translation in microglial processes to regulate motility and phagocytosis during inflammation

doi: 10.1371/journal.pbio.3003463

Figure Lengend Snippet: (A) IMP1/ZBP1 relative distribution in microglia. Linescans represent the mean distribution ±SEM of the IMP1 fluorescence signal from the nucleus to the PeMPs in 40 individual cells per condition (A i ). Bar graphs show mean ±SEM IMP1/ZBP1 levels in LPS-treated cells relative to controls in PeMPs and the soma measured in 4 independent experiments ( n = 4). Two-tailed t tests. n.s, not significant (A ii ). Micrographs (binarized images) show the distribution of IMP1 in PBS- and LPS-treated cells. (1) and (2) indicate the PeMPs and the perinuclear regions, respectively, represented in insets. Scale bars, 20 µm (10 µm insets) (A iii ). The bar graph shows the mean relative frequency distribution ±SEM of IMP1 foci in the periphery and the soma of microglia after treatment with PBS or LPS for 30 min in 4 independent experiments ( n = 4). One-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; n.s, not significant (A iv ). (B) IMP1 levels measured by immunohistochemistry in cortical microglia (positive for P2Y12 and GFP) from fms-EGFP 1-month-old mice injected with saline or LPS. Linescans represent the mean distribution ±SEM of the fluorescent signal from the nucleus to the PeMPs in 55–72 individual cells per condition (B i ). (1) and (2) in micrographs indicate PeMPs (both GC-like and primary processes) and cell bodies, respectively, shown in insets. Scale bars 10 µm (5 µm insets) (A ii ). Violin plots represent the intensity of IMP1 in 28–36 sampled GC-like PeMPs (smaller dots), 55–72 primary PeMPs (smaller dots), and 55–72 cell bodies (smaller dots) from 3 to 4 mice (larger dots). Two-tailed t tests. ** p < 0.01; **** p < 0.0001; n.s, not significant (B iii ). The bar graph shows the mean relative frequency distribution ±SEM of IMP1/ZBP1 intensity in PeMPs and the soma in cortical microglia from 3 to 4 mice ( N = 3–4). One-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; **** p < 0.0001; n.s, not significant (B iv ). The data underlying this Figure can be found in .

Article Snippet: Cells were incubated for 20 min at room temperature with 500 nM SYTO RNA Select green fluorescent dye in PBS (Invitrogen).

Techniques: Fluorescence, Two Tailed Test, Comparison, Immunohistochemistry, Injection, Saline

Journal: PLOS Biology

Article Title: RNA-binding protein IMP1/ZBP1 directs local translation in microglial processes to regulate motility and phagocytosis during inflammation

doi: 10.1371/journal.pbio.3003463

Figure Lengend Snippet: (A) IMP1/ZBP1 downregulation upon microglia transfection with an Imp1 -targeting siRNA. Representative western blot showing IMP1/ZBP1 and total protein levels following transfection with a control (ctrl KD) and an Imp1 -targeting siRNA ( Imp1 KD) (A i ) . The bar graph summarizes the quantification of western blots obtained from 3 independent cultures ( n = 3). Two-tailed t test; n.s, not significant (A ii ) . (B) Imp1 knockdown (KD) alters Actb mRNA localization toward the periphery of microglia. Linescans show the mean distribution ±SEM of the Actb FISH signal from the nucleus to the PeMPs in 38–48 individual cells per condition (B i ) . The bar graph represents the relative Actb intensity (compared to control-transfected cells exposed to PBS) in PeMPs from microglia treated with PBS or LPS and transfected with a control (ctrl KD) or an Imp1 -targeting (Imp1 KD) siRNA in 5 independent experiments ( n = 5). One-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; ** p < 0.01; n.s, not significant (B ii ) . (C) Relative distribution of Actb foci in binarized images. (1) and (2) indicate the PeMPs and the perinuclear regions, respectively, (C i ) represented in insets (C ii ) . Scale bars, 20 µm (C i ) and 10 µm (C ii ). The relative frequency distribution of Actb foci in the periphery and the soma of microglia transfected with a control (ctrl KD) siRNA or an Imp1 siRNA ( Imp1 KD) and exposed to PBS or LPS for 30 min in 5 independent experiments ( n = 5) is plotted in (C iii ) . Two-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; ** p < 0.01; n.s, not significant. (D) Imp1 KD blocks LPS-induced localized translation in microglia. Local β-actin synthesis was assessed with a 2-min puromycin pulse in PBS- and LPS-treated cells and transfected with control (ctrl KD) or Imp1 siRNAs ( Imp1 KD), followed by PLA with antibodies against puromycin and β-actin. As a negative control, cells were preincubated with the protein synthesis inhibitor anisomycin. Representative images of microglial PLA-labeled lamellae and stained with phalloidin are shown. Scale bar, 10 µm (insets, 5µm) (D i ) . The box and whisker graph represents the average of PLA puncta within lamellae in phalloidin-stained microglia cultured in 6 independent experiments ( n = 6) analyzed by two-way ANOVA followed by Holm–Šídák’s multiple comparison test. * p < 0.05; n.s, not significant (D ii ) . The data underlying this Figure can be found in .

Article Snippet: Cells were incubated for 20 min at room temperature with 500 nM SYTO RNA Select green fluorescent dye in PBS (Invitrogen).

Techniques: Transfection, Western Blot, Control, Two Tailed Test, Knockdown, Comparison, Negative Control, Labeling, Staining, Whisker Assay, Cell Culture

Journal: PLOS Biology

Article Title: RNA-binding protein IMP1/ZBP1 directs local translation in microglial processes to regulate motility and phagocytosis during inflammation

doi: 10.1371/journal.pbio.3003463

Figure Lengend Snippet: (A) Schematic representation of a transwell with 1-µm-diameter pore used for the isolation of PeMPs (A i ) . Cell material from the upper compartment was removed with a cotton swab and total RNA was isolated from PeMPs (lower panel) (A ii ) . Figure was created in BioRender. Baleriola, J. (2025) https://BioRender.com/urvkdx5 . (B) Significantly regulated RNAs in response to LPS in ctrl KD (B i ) and Imp1 KD cells (B ii ) . Volcano plots represent the upregulated (red dots) and downregulated (blue dots) transcripts obtained from 2 independent experiments. (C) Venn diagram showing the overlap between differentially localized transcripts upon LPS treatment in ctrl KD (magenta) or Imp1 KD (cyan) microglia and previously identified IMP1 targets (yellow). (D) Functional annotation with Metascape of the 198 transcripts whose regulation is lost by Imp1 knockdown. Only the top 20 categories from all significantly changed terms ( p < 0.05) are represented in the figure. (E) Heatmaps showing the relative levels (LPS vs. PBS) of transcripts belonging to the 3 most enriched categories in (C) in ctrl KD and Imp1 KD microglia. (F) Relative levels (LPS vs. PBS) of selected transcripts measured in 4–7 biological replicates by RT-qPCR. * p < 0.05; ** p < 0.01; n.s, not significant comparing LPS- vs. PBS-treated cells transfected with control or Imp1 siRNA. ## p < 0.01; ### p < 0.001; n.s, not significant comparing control vs. Imp1 siRNA in LPS-treated cells. The data underlying this Figure can be found in .

Article Snippet: Cells were incubated for 20 min at room temperature with 500 nM SYTO RNA Select green fluorescent dye in PBS (Invitrogen).

Techniques: Isolation, Functional Assay, Knockdown, Quantitative RT-PCR, Transfection, Control