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$Time-dependent evolution of myelin debris phagocytosis by macrophages and endogenous <t>FGF4</t> expression following spinal cord injury. A Representative immunofluorescence images of transverse spinal cord sections at the lesion epicenter from sham-operated mice and at 1, 3, 7, 10, 14, and 42 days post-injury (dpi). Sections were stained for macrophages (F4/80, green), myelin debris (MBP; red), and nuclei (DAPI, blue). Scale bars: 500 μm and 100 μm (magnified insets). B Quantitative analysis of MBP-positive area fraction (%) ( n = 3 animals). C Quantitative analysis of the number of phagocytic F4/80⁺ macrophages per field ( n = 3 animals). D Representative immunofluorescence images showing the localization of macrophages (F4/80, green) and FGF4 (red) in spinal cord sections from sham and injured mice at 1, 3, 7, 10, and 14 dpi. Nuclei are stained with DAPI (blue). Scale bars: 500 μm and 100 μm (magnified insets). E Quantitative analysis of the mean fluorescence intensity of FGF4 ( n = 4 animals). F Pearson’s correlation analysis between the fluorescence signals of F4/80 and FGF4 across the examined time points ( n = 4 animals). G Representative Western blot images of FGF4 protein expression in spinal cord tissue lysates from sham and injured mice at the indicated time points. β-actin served as the loading control. H Densitometric quantification of relative FGF4 protein levels normalized to β-actin ( n = 4 animals). * p < 0.05, ** p < 0.01, n.s. = not significant$
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1) Product Images from "FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair"

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-026-03743-0

$Time-dependent evolution of myelin debris phagocytosis by macrophages and endogenous FGF4 expression following spinal cord injury. A Representative immunofluorescence images of transverse spinal cord sections at the lesion epicenter from sham-operated mice and at 1, 3, 7, 10, 14, and 42 days post-injury (dpi). Sections were stained for macrophages (F4/80, green), myelin debris (MBP; red), and nuclei (DAPI, blue). Scale bars: 500 μm and 100 μm (magnified insets). B Quantitative analysis of MBP-positive area fraction (%) ( n = 3 animals). C Quantitative analysis of the number of phagocytic F4/80⁺ macrophages per field ( n = 3 animals). D Representative immunofluorescence images showing the localization of macrophages (F4/80, green) and FGF4 (red) in spinal cord sections from sham and injured mice at 1, 3, 7, 10, and 14 dpi. Nuclei are stained with DAPI (blue). Scale bars: 500 μm and 100 μm (magnified insets). E Quantitative analysis of the mean fluorescence intensity of FGF4 ( n = 4 animals). F Pearson’s correlation analysis between the fluorescence signals of F4/80 and FGF4 across the examined time points ( n = 4 animals). G Representative Western blot images of FGF4 protein expression in spinal cord tissue lysates from sham and injured mice at the indicated time points. β-actin served as the loading control. H Densitometric quantification of relative FGF4 protein levels normalized to β-actin ( n = 4 animals). * p < 0.05, ** p < 0.01, n.s. = not significant$
Figure Legend Snippet: Time-dependent evolution of myelin debris phagocytosis by macrophages and endogenous FGF4 expression following spinal cord injury. A Representative immunofluorescence images of transverse spinal cord sections at the lesion epicenter from sham-operated mice and at 1, 3, 7, 10, 14, and 42 days post-injury (dpi). Sections were stained for macrophages (F4/80, green), myelin debris (MBP; red), and nuclei (DAPI, blue). Scale bars: 500 μm and 100 μm (magnified insets). B Quantitative analysis of MBP-positive area fraction (%) ( n = 3 animals). C Quantitative analysis of the number of phagocytic F4/80⁺ macrophages per field ( n = 3 animals). D Representative immunofluorescence images showing the localization of macrophages (F4/80, green) and FGF4 (red) in spinal cord sections from sham and injured mice at 1, 3, 7, 10, and 14 dpi. Nuclei are stained with DAPI (blue). Scale bars: 500 μm and 100 μm (magnified insets). E Quantitative analysis of the mean fluorescence intensity of FGF4 ( n = 4 animals). F Pearson’s correlation analysis between the fluorescence signals of F4/80 and FGF4 across the examined time points ( n = 4 animals). G Representative Western blot images of FGF4 protein expression in spinal cord tissue lysates from sham and injured mice at the indicated time points. β-actin served as the loading control. H Densitometric quantification of relative FGF4 protein levels normalized to β-actin ( n = 4 animals). * p < 0.05, ** p < 0.01, n.s. = not significant

Techniques Used: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot, Control

FGF4 augments myelin debris phagocytosis in BMDMs and reduces neuronal apoptosis. A Representative images of Oil Red O staining in BMDMs treated with myelin debris (MD) and either phosphate-buffered saline (PBS, MD + PBS group) or fibroblast growth factor 4 (FGF4, MD+FGF4 group). Scale bars: 100 μm and 30 μm (magnified inset). B Quantitative analysis of the Oil Red O-positive area normalized to the total cell area ( n = 4 wells). C Representative immunofluorescence images of BMDMs in the MD + PBS and MD+FGF4 groups at 6, 12, 18, and 24 h post-stimulation. Cells were stained for the macrophage marker F4/80 (red), myelin basic protein (MBP, green), and nuclei (DAPI, blue). Scale bar: 100 μm. D Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). E Representative Western blot images showing the protein levels of MBP in BMDM lysates from the Con, MD + PBS, and MD+FGF4 groups. β-actin was used as a loading control. F Densitometric quantification of relative MBP protein levels normalized to β-actin ( n = 3 wells). G Representative immunofluorescence images of primary cortical neurons treated with conditioned medium from different groups of BMDMs, stained with the neuronal marker Tuj1 (green) and the nuclear marker DAPI (blue). Scale bar: 50 μm. H Sholl analysis of neuronal branching complexity. The number of neurite intersections with concentric circles at increasing distances from the soma is plotted for each group ( n = 3 wells). I Representative Western blot images showing the protein levels of the anti-apoptotic protein Bcl-2 and the apoptotic marker Cleaved Caspase-3 in primary cortical neurons treated with conditioned medium from different groups of BMDMs. β-actin was used as a loading control. J Densitometric quantification of relative Bcl-2 protein levels normalized to β-actin ( n = 3 wells). K Densitometric quantification of relative Cleaved Caspase-3 protein levels normalized to β-actin ( n = 3 wells). L Representative flow cytometry plots of primary cortical neurons stained with Annexin V and propidium iodide (PI) after treatment with conditioned media collected from different groups of BMDMs to assess apoptosis. M Quantitative analysis of the total apoptotic cell rate (sum of early and late apoptotic cells, n = 3 wells). * p < 0.05, ** p < 0.01, n.s. = not significant

Figure Legend Snippet: FGF4 augments myelin debris phagocytosis in BMDMs and reduces neuronal apoptosis. A Representative images of Oil Red O staining in BMDMs treated with myelin debris (MD) and either phosphate-buffered saline (PBS, MD + PBS group) or fibroblast growth factor 4 (FGF4, MD+FGF4 group). Scale bars: 100 μm and 30 μm (magnified inset). B Quantitative analysis of the Oil Red O-positive area normalized to the total cell area ( n = 4 wells). C Representative immunofluorescence images of BMDMs in the MD + PBS and MD+FGF4 groups at 6, 12, 18, and 24 h post-stimulation. Cells were stained for the macrophage marker F4/80 (red), myelin basic protein (MBP, green), and nuclei (DAPI, blue). Scale bar: 100 μm. D Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). E Representative Western blot images showing the protein levels of MBP in BMDM lysates from the Con, MD + PBS, and MD+FGF4 groups. β-actin was used as a loading control. F Densitometric quantification of relative MBP protein levels normalized to β-actin ( n = 3 wells). G Representative immunofluorescence images of primary cortical neurons treated with conditioned medium from different groups of BMDMs, stained with the neuronal marker Tuj1 (green) and the nuclear marker DAPI (blue). Scale bar: 50 μm. H Sholl analysis of neuronal branching complexity. The number of neurite intersections with concentric circles at increasing distances from the soma is plotted for each group ( n = 3 wells). I Representative Western blot images showing the protein levels of the anti-apoptotic protein Bcl-2 and the apoptotic marker Cleaved Caspase-3 in primary cortical neurons treated with conditioned medium from different groups of BMDMs. β-actin was used as a loading control. J Densitometric quantification of relative Bcl-2 protein levels normalized to β-actin ( n = 3 wells). K Densitometric quantification of relative Cleaved Caspase-3 protein levels normalized to β-actin ( n = 3 wells). L Representative flow cytometry plots of primary cortical neurons stained with Annexin V and propidium iodide (PI) after treatment with conditioned media collected from different groups of BMDMs to assess apoptosis. M Quantitative analysis of the total apoptotic cell rate (sum of early and late apoptotic cells, n = 3 wells). * p < 0.05, ** p < 0.01, n.s. = not significant

Techniques Used: Staining, Saline, Immunofluorescence, Marker, Western Blot, Control, Flow Cytometry

FGF4 improves in vivo myelin debris resolution and attenuates lipid accumulation in phagocytes after SCI. A Representative immunofluorescence images of spinal cord sections at 7 and 14 dpi, comparing the SCI group to the FGF4 group. Sections were stained for macrophages (F4/80, red), myelin basic protein (MBP, green), and nuclei (DAPI, blue). The boxed regions magnified in the corresponding right panels. Scale bars: 50 μm, 20 μm (magnified). B Quantitative analysis of the MBP + area within F4/80 + regions at 7 dpi ( n = 5 animals). C Quantitative analysis of the MBP + area within F4/80 + regions at 14 dpi ( n = 5 animals). D Transmission electron microscopy (TEM) images of macrophages in the spinal cord lesion area at 7 dpi. Representative images from the SCI group and the FGF4 group are shown. The boxed regions are magnified on the right, revealing degraded myelin debris (indicated by red arrows). Scale bars: 5 μm, 1 μm (magnified). E Quantitative analysis of the number of degraded myelin debris per macrophage from the TEM images ( n = 3 animals). F Representative images of Oil Red O staining in spinal cord sections at 7, 14, and 42 dpi, comparing the SCI group and the FGF4 group. Lipid deposits are stained red. Higher-magnification views of the boxed regions displayed on the right. Scale bars: 1000 μm, 200 μm (magnified). G-I Quantitative analysis of the Oil Red O-positive lesion area (mm²) at 7 (G) , 14 (H) , and 42 (I) dpi for the indicated groups ( n = 4 animals). * p < 0.05, ** p < 0.01

Figure Legend Snippet: FGF4 improves in vivo myelin debris resolution and attenuates lipid accumulation in phagocytes after SCI. A Representative immunofluorescence images of spinal cord sections at 7 and 14 dpi, comparing the SCI group to the FGF4 group. Sections were stained for macrophages (F4/80, red), myelin basic protein (MBP, green), and nuclei (DAPI, blue). The boxed regions magnified in the corresponding right panels. Scale bars: 50 μm, 20 μm (magnified). B Quantitative analysis of the MBP + area within F4/80 + regions at 7 dpi ( n = 5 animals). C Quantitative analysis of the MBP + area within F4/80 + regions at 14 dpi ( n = 5 animals). D Transmission electron microscopy (TEM) images of macrophages in the spinal cord lesion area at 7 dpi. Representative images from the SCI group and the FGF4 group are shown. The boxed regions are magnified on the right, revealing degraded myelin debris (indicated by red arrows). Scale bars: 5 μm, 1 μm (magnified). E Quantitative analysis of the number of degraded myelin debris per macrophage from the TEM images ( n = 3 animals). F Representative images of Oil Red O staining in spinal cord sections at 7, 14, and 42 dpi, comparing the SCI group and the FGF4 group. Lipid deposits are stained red. Higher-magnification views of the boxed regions displayed on the right. Scale bars: 1000 μm, 200 μm (magnified). G-I Quantitative analysis of the Oil Red O-positive lesion area (mm²) at 7 (G) , 14 (H) , and 42 (I) dpi for the indicated groups ( n = 4 animals). * p < 0.05, ** p < 0.01

Techniques Used: In Vivo, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy

FGF4 enhances cholesterol efflux, suppresses inflammasome activation and pyroptosis, and promotes the repair of lysosomal function after SCI. A Representative Western blot images of proteins related to cholesterol efflux (ABCA1), inflammasome activation (NLRP3, Cleaved Caspase-1, IL-1β), pyroptosis (GSDMD), and lysosomal function (Pro-CTSD, Cleaved-CTSD) in spinal cord tissue lysates from sham-operated mice and mice subjected to SCI with or without FGF4 treatment, assessed at 7 and 14 dpi. β-actin was used as a loading control. B–G Quantification of relative protein levels normalized to β-actin: ABCA1 (B) , NLRP3 (C) , GSDMD (D) , IL-1β (E) , Cleaved Caspase-1 (F) , The ratio of Cleaved-CTSD to Pro-CTSD (G) ( n = 4 animals). H Representative immunofluorescence staining for CTSD (green) and the macrophage marker F4/80 (red) in spinal cord sections. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. I Representative immunofluorescence staining for ABCA1 (green) and F4/80 (red) in spinal cord sections. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. J Quantification of the mean fluorescence intensity of CTSD ( n = 5 animals). K Quantification of the mean fluorescence intensity of ABCA1 ( n = 5 animals). L Representative immunohistochemical staining of CASP-1 and NLRP3 in spinal cord tissues from the indicated groups at 7 dpi. Scale bar: 100 μm. M Quantification of relative CASP-1 intensity (Integrated Density/Area, normalized to Sham, n = 5 animals). N Quantification of relative NLRP3 intensity (Integrated Density/Area, normalized to Sham, n = 5 animals). * p < 0.05, ** p < 0.01

Figure Legend Snippet: FGF4 enhances cholesterol efflux, suppresses inflammasome activation and pyroptosis, and promotes the repair of lysosomal function after SCI. A Representative Western blot images of proteins related to cholesterol efflux (ABCA1), inflammasome activation (NLRP3, Cleaved Caspase-1, IL-1β), pyroptosis (GSDMD), and lysosomal function (Pro-CTSD, Cleaved-CTSD) in spinal cord tissue lysates from sham-operated mice and mice subjected to SCI with or without FGF4 treatment, assessed at 7 and 14 dpi. β-actin was used as a loading control. B–G Quantification of relative protein levels normalized to β-actin: ABCA1 (B) , NLRP3 (C) , GSDMD (D) , IL-1β (E) , Cleaved Caspase-1 (F) , The ratio of Cleaved-CTSD to Pro-CTSD (G) ( n = 4 animals). H Representative immunofluorescence staining for CTSD (green) and the macrophage marker F4/80 (red) in spinal cord sections. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. I Representative immunofluorescence staining for ABCA1 (green) and F4/80 (red) in spinal cord sections. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. J Quantification of the mean fluorescence intensity of CTSD ( n = 5 animals). K Quantification of the mean fluorescence intensity of ABCA1 ( n = 5 animals). L Representative immunohistochemical staining of CASP-1 and NLRP3 in spinal cord tissues from the indicated groups at 7 dpi. Scale bar: 100 μm. M Quantification of relative CASP-1 intensity (Integrated Density/Area, normalized to Sham, n = 5 animals). N Quantification of relative NLRP3 intensity (Integrated Density/Area, normalized to Sham, n = 5 animals). * p < 0.05, ** p < 0.01

Techniques Used: Activation Assay, Western Blot, Control, Immunofluorescence, Staining, Marker, Fluorescence, Immunohistochemical staining

FGF4 treatment improves locomotor functional recovery following SCI. A Representative footprint images of hindlimb steps from the Sham, SCI, and FGF4 groups at 42 dpi. B Basso Mouse Scale (BMS) scores for hindlimb motor function assessed at 1, 3, 7, 10, 14, 21, 28, 35, and 42 dpi in the Sham, SCI, and FGF4 groups ( n = 6 animals). C Representative kinematic analysis of hindlimb locomotion. Color-coded simulate limb movement, and joint angle curves for the hip, knee, and ankle joints are plotted. D Quantitative analysis of the number of successful plantar steps ( n = 6 animals). E Quantitative analysis of the height of the trunk above the ground (mm, n = 6 animals). F Representative motor evoked potential (MEP) traces recorded from the Sham, SCI, and FGF4 groups at 42 dpi. G Quantification of MEP amplitude (mV, n = 6 animals). ** p < 0.01

Figure Legend Snippet: FGF4 treatment improves locomotor functional recovery following SCI. A Representative footprint images of hindlimb steps from the Sham, SCI, and FGF4 groups at 42 dpi. B Basso Mouse Scale (BMS) scores for hindlimb motor function assessed at 1, 3, 7, 10, 14, 21, 28, 35, and 42 dpi in the Sham, SCI, and FGF4 groups ( n = 6 animals). C Representative kinematic analysis of hindlimb locomotion. Color-coded simulate limb movement, and joint angle curves for the hip, knee, and ankle joints are plotted. D Quantitative analysis of the number of successful plantar steps ( n = 6 animals). E Quantitative analysis of the height of the trunk above the ground (mm, n = 6 animals). F Representative motor evoked potential (MEP) traces recorded from the Sham, SCI, and FGF4 groups at 42 dpi. G Quantification of MEP amplitude (mV, n = 6 animals). ** p < 0.01

Techniques Used: Functional Assay

FGFR1 inhibition abrogates FGF4-mediated PI3K/AKT activation and phagocytic enhancement in macrophages. A Representative Oil Red O staining of BMDMs under PD173074 (FGFR1 inhibitor) treatment. Scale bar: 30 μm. Representative immunofluorescence images stained for myelin debris (MBP, green), macrophages (F4/80, red), and nuclei (DAPI, blue). Boxed regions are magnified below. Scale bars: 100 μm, 30 μm (magnified). B Quantitative analysis of the Oil Red O-positive area expressed as a percentage (%) of the total cell area ( n = 6 wells). C Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). D Representative Western blot images of phosphorylated FGFR1 (p-FGFR1), phosphorylated PI3K (p-PI3K), total PI3K, phosphorylated AKT (p-AKT), total AKT, MBP, and β-actin in BMDM lysates under PD173074 treatment. E–H Densitometric quantification of relative protein levels: p-FGFR1 (E) ( n = 4 wells), p-PI3K/PI3K ratio (F) ( n = 3 wells), p-AKT/AKT ratio (G) ( n = 3 wells), MBP (H) ( n = 3 wells). All data normalized to β-actin. * p < 0.05, ** p < 0.01

Figure Legend Snippet: FGFR1 inhibition abrogates FGF4-mediated PI3K/AKT activation and phagocytic enhancement in macrophages. A Representative Oil Red O staining of BMDMs under PD173074 (FGFR1 inhibitor) treatment. Scale bar: 30 μm. Representative immunofluorescence images stained for myelin debris (MBP, green), macrophages (F4/80, red), and nuclei (DAPI, blue). Boxed regions are magnified below. Scale bars: 100 μm, 30 μm (magnified). B Quantitative analysis of the Oil Red O-positive area expressed as a percentage (%) of the total cell area ( n = 6 wells). C Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). D Representative Western blot images of phosphorylated FGFR1 (p-FGFR1), phosphorylated PI3K (p-PI3K), total PI3K, phosphorylated AKT (p-AKT), total AKT, MBP, and β-actin in BMDM lysates under PD173074 treatment. E–H Densitometric quantification of relative protein levels: p-FGFR1 (E) ( n = 4 wells), p-PI3K/PI3K ratio (F) ( n = 3 wells), p-AKT/AKT ratio (G) ( n = 3 wells), MBP (H) ( n = 3 wells). All data normalized to β-actin. * p < 0.05, ** p < 0.01

Techniques Used: Inhibition, Activation Assay, Staining, Immunofluorescence, Western Blot

Transcriptomic profiling reveals that FGF4 reshapes macrophage receptor and lipid-handling gene expression and identifies Clec10a as a key downstream effector. A Summary of differentially expressed genes (DEGs) identified by RNA-seq analysis among the Control, MD, and MD+FGF4 groups. BMDMs were pretreated with FGF4 for 2 h followed by exposure to MD for 24 h. B Heatmap displaying the expression patterns of genes related to phagocytosis across the indicated groups. C Volcano plot illustrating the significance and magnitude of gene expression changes for phagocytosis-related genes between the MD+FGF4 and MD groups. D-E Gene Ontology (GO) biological process enrichment analysis (D) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (E) of the phagocytosis-related DEGs. F–K Validation of RNA-seq data by quantitative RT-PCR (qRT-PCR). Relative mRNA expression levels of Fcgr4 (F) , Cd163 (G) , Irf4 (H) , Ccl6 (I) , Scd1 (J) , and Clec10a (K) in BMDMs from the Control, FGF4, MD, and MD+FGF4 groups. Data were normalized to β-actin and presented as relative to the Control group ( n = 3 wells). L Representative Western blot images showing Clec10a protein expression in BMDM lysates. β-actin was used as a loading control. M Densitometric quantification of relative Clec10a protein levels normalized to β-actin ( n = 3 wells). * p < 0.05, ** p < 0.01

Figure Legend Snippet: Transcriptomic profiling reveals that FGF4 reshapes macrophage receptor and lipid-handling gene expression and identifies Clec10a as a key downstream effector. A Summary of differentially expressed genes (DEGs) identified by RNA-seq analysis among the Control, MD, and MD+FGF4 groups. BMDMs were pretreated with FGF4 for 2 h followed by exposure to MD for 24 h. B Heatmap displaying the expression patterns of genes related to phagocytosis across the indicated groups. C Volcano plot illustrating the significance and magnitude of gene expression changes for phagocytosis-related genes between the MD+FGF4 and MD groups. D-E Gene Ontology (GO) biological process enrichment analysis (D) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (E) of the phagocytosis-related DEGs. F–K Validation of RNA-seq data by quantitative RT-PCR (qRT-PCR). Relative mRNA expression levels of Fcgr4 (F) , Cd163 (G) , Irf4 (H) , Ccl6 (I) , Scd1 (J) , and Clec10a (K) in BMDMs from the Control, FGF4, MD, and MD+FGF4 groups. Data were normalized to β-actin and presented as relative to the Control group ( n = 3 wells). L Representative Western blot images showing Clec10a protein expression in BMDM lysates. β-actin was used as a loading control. M Densitometric quantification of relative Clec10a protein levels normalized to β-actin ( n = 3 wells). * p < 0.05, ** p < 0.01

Techniques Used: Gene Expression, RNA Sequencing, Control, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot

Clec10a is essential for FGF4-mediated enhancement of myelin debris phagocytosis. A Representative Oil Red O staining of BMDMs transfected with LV-shClec10a. Scale bar: 30 μm. Representative immunofluorescence images of BMDMs stained for Clec10a (purple), MBP (green), F4/80 (red), and DAPI (blue). Boxed regions are magnified below. Scale bars: 100 μm, 30 μm (magnified). B Quantitative analysis of the Oil Red O-positive area expressed as a percentage (%) of the total cell area ( n = 5 wells). C Quantitative analysis of the mean fluorescence intensity of Clec10a in BMDMs ( n = 5 wells). D Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). E Representative Western blot images of MBP and Clec10a protein expression in BMDM lysates following LV-shClec10a transfection. β-actin was used as a loading control. F Densitometric quantification of relative MBP protein levels normalized to β-actin ( n = 3 wells). G Densitometric quantification of relative Clec10a protein levels normalized to β-actin ( n = 3 wells). H Representative flow cytometry plots of primary cortical neurons stained with Annexin V and PI after treatment with conditioned media collected from the differently treated BMDM groups. I Quantitative analysis of the total apoptotic cell rate ( n = 3 wells). J Representative immunofluorescence images of primary cortical neurons treated with conditioned medium from different groups of BMDMs, stained with the neuronal marker Tuj1 (green) and the nuclear marker DAPI (blue). Boxed regions are magnified below. Scale bars: 50 μm, 20 μm (magnified). K Sholl analysis of neuronal branching complexity. The number of neurite intersections with concentric circles at increasing distances from the soma is plotted for each group ( n = 3 wells). * p < 0.05, ** p < 0.01

Figure Legend Snippet: Clec10a is essential for FGF4-mediated enhancement of myelin debris phagocytosis. A Representative Oil Red O staining of BMDMs transfected with LV-shClec10a. Scale bar: 30 μm. Representative immunofluorescence images of BMDMs stained for Clec10a (purple), MBP (green), F4/80 (red), and DAPI (blue). Boxed regions are magnified below. Scale bars: 100 μm, 30 μm (magnified). B Quantitative analysis of the Oil Red O-positive area expressed as a percentage (%) of the total cell area ( n = 5 wells). C Quantitative analysis of the mean fluorescence intensity of Clec10a in BMDMs ( n = 5 wells). D Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). E Representative Western blot images of MBP and Clec10a protein expression in BMDM lysates following LV-shClec10a transfection. β-actin was used as a loading control. F Densitometric quantification of relative MBP protein levels normalized to β-actin ( n = 3 wells). G Densitometric quantification of relative Clec10a protein levels normalized to β-actin ( n = 3 wells). H Representative flow cytometry plots of primary cortical neurons stained with Annexin V and PI after treatment with conditioned media collected from the differently treated BMDM groups. I Quantitative analysis of the total apoptotic cell rate ( n = 3 wells). J Representative immunofluorescence images of primary cortical neurons treated with conditioned medium from different groups of BMDMs, stained with the neuronal marker Tuj1 (green) and the nuclear marker DAPI (blue). Boxed regions are magnified below. Scale bars: 50 μm, 20 μm (magnified). K Sholl analysis of neuronal branching complexity. The number of neurite intersections with concentric circles at increasing distances from the soma is plotted for each group ( n = 3 wells). * p < 0.05, ** p < 0.01

Techniques Used: Staining, Transfection, Immunofluorescence, Fluorescence, Western Blot, Expressing, Control, Flow Cytometry, Marker

Clec10a acts intracellularly to facilitate myelin debris processing rather than through surface-level recognition. A Representative immunofluorescence images of BMDMs pretreated with or without FGF4 for 2 h, followed by incubation with Dil-labeled myelin debris (red) at 4 °C for 30 min to block internalization and assess surface binding. Cells were stained for Clec10a (green) and DAPI (blue). Scale bar, 200 μm. B Quantification of Clec10a/Dil-myelin colocalization expressed as Pearson’s correlation coefficient ( n = 4 wells). C Representative immunofluorescence images of BMDMs pretreated with FGF4 for 2 h and then incubated with Dil-myelin debris at 37 °C for 5, 15, 30, 60 min, and 6 h. Cells were stained for Clec10a (green), RAB5 (purple), and DAPI (blue). Scale bar: 100 μm. D–F Representative images showing colocalization of Dil-myelin (red) with Rab5 (green) (D) , Rab7 (green) (E) , or Lamp1 (green) (F) in BMDMs pretreated with or without FGF4 and incubated with myelin debris at 37 °C for 15 min. Nuclei are stained with DAPI (blue). Scale bars: 200 μm. G–I Quantitative analysis of Pearson’s correlation coefficients for Rab5/Dil-myelin (G) , Rab7/Dil-myelin (H) , and Lamp1/Dil-myelin ( n = 3 wells) (I) . * p < 0.05, ** p < 0.01

Figure Legend Snippet: Clec10a acts intracellularly to facilitate myelin debris processing rather than through surface-level recognition. A Representative immunofluorescence images of BMDMs pretreated with or without FGF4 for 2 h, followed by incubation with Dil-labeled myelin debris (red) at 4 °C for 30 min to block internalization and assess surface binding. Cells were stained for Clec10a (green) and DAPI (blue). Scale bar, 200 μm. B Quantification of Clec10a/Dil-myelin colocalization expressed as Pearson’s correlation coefficient ( n = 4 wells). C Representative immunofluorescence images of BMDMs pretreated with FGF4 for 2 h and then incubated with Dil-myelin debris at 37 °C for 5, 15, 30, 60 min, and 6 h. Cells were stained for Clec10a (green), RAB5 (purple), and DAPI (blue). Scale bar: 100 μm. D–F Representative images showing colocalization of Dil-myelin (red) with Rab5 (green) (D) , Rab7 (green) (E) , or Lamp1 (green) (F) in BMDMs pretreated with or without FGF4 and incubated with myelin debris at 37 °C for 15 min. Nuclei are stained with DAPI (blue). Scale bars: 200 μm. G–I Quantitative analysis of Pearson’s correlation coefficients for Rab5/Dil-myelin (G) , Rab7/Dil-myelin (H) , and Lamp1/Dil-myelin ( n = 3 wells) (I) . * p < 0.05, ** p < 0.01

Techniques Used: Immunofluorescence, Incubation, Labeling, Blocking Assay, Binding Assay, Staining

FGF4 activates FGFR1–PI3K/AKT signaling in macrophages to upregulate intracellular Clec10a, which promotes phagosome maturation and endolysosomal trafficking of internalized myelin debris from Rab5⁺ early endosomes to Rab7⁺ late endosomes and Lamp1⁺ lysosomes. This process restores lysosomal membrane stability and CTSD-dependent degradative capacity, enhances myelin debris clearance and ultimately creates a permissive microenvironment for neuronal survival and spinal cord repair. The schematic is created using BioRender

Figure Legend Snippet: FGF4 activates FGFR1–PI3K/AKT signaling in macrophages to upregulate intracellular Clec10a, which promotes phagosome maturation and endolysosomal trafficking of internalized myelin debris from Rab5⁺ early endosomes to Rab7⁺ late endosomes and Lamp1⁺ lysosomes. This process restores lysosomal membrane stability and CTSD-dependent degradative capacity, enhances myelin debris clearance and ultimately creates a permissive microenvironment for neuronal survival and spinal cord repair. The schematic is created using BioRender

Techniques Used: Membrane

Image Search Results

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: Time-dependent evolution of myelin debris phagocytosis by macrophages and endogenous FGF4 expression following spinal cord injury. A Representative immunofluorescence images of transverse spinal cord sections at the lesion epicenter from sham-operated mice and at 1, 3, 7, 10, 14, and 42 days post-injury (dpi). Sections were stained for macrophages (F4/80, green), myelin debris (MBP; red), and nuclei (DAPI, blue). Scale bars: 500 μm and 100 μm (magnified insets). B Quantitative analysis of MBP-positive area fraction (%) ( n = 3 animals). C Quantitative analysis of the number of phagocytic F4/80⁺ macrophages per field ( n = 3 animals). D Representative immunofluorescence images showing the localization of macrophages (F4/80, green) and FGF4 (red) in spinal cord sections from sham and injured mice at 1, 3, 7, 10, and 14 dpi. Nuclei are stained with DAPI (blue). Scale bars: 500 μm and 100 μm (magnified insets). E Quantitative analysis of the mean fluorescence intensity of FGF4 ( n = 4 animals). F Pearson’s correlation analysis between the fluorescence signals of F4/80 and FGF4 across the examined time points ( n = 4 animals). G Representative Western blot images of FGF4 protein expression in spinal cord tissue lysates from sham and injured mice at the indicated time points. β-actin served as the loading control. H Densitometric quantification of relative FGF4 protein levels normalized to β-actin ( n = 4 animals). * p < 0.05, ** p < 0.01, n.s. = not significant

Article Snippet: Recombinant proteins and small molecules, including FGF4 (HY-P7014), PD173074 (HY-10321), and N-acetyl-D-galactosamine (HY-128852), were procured from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: FGF4 augments myelin debris phagocytosis in BMDMs and reduces neuronal apoptosis. A Representative images of Oil Red O staining in BMDMs treated with myelin debris (MD) and either phosphate-buffered saline (PBS, MD + PBS group) or fibroblast growth factor 4 (FGF4, MD+FGF4 group). Scale bars: 100 μm and 30 μm (magnified inset). B Quantitative analysis of the Oil Red O-positive area normalized to the total cell area ( n = 4 wells). C Representative immunofluorescence images of BMDMs in the MD + PBS and MD+FGF4 groups at 6, 12, 18, and 24 h post-stimulation. Cells were stained for the macrophage marker F4/80 (red), myelin basic protein (MBP, green), and nuclei (DAPI, blue). Scale bar: 100 μm. D Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). E Representative Western blot images showing the protein levels of MBP in BMDM lysates from the Con, MD + PBS, and MD+FGF4 groups. β-actin was used as a loading control. F Densitometric quantification of relative MBP protein levels normalized to β-actin ( n = 3 wells). G Representative immunofluorescence images of primary cortical neurons treated with conditioned medium from different groups of BMDMs, stained with the neuronal marker Tuj1 (green) and the nuclear marker DAPI (blue). Scale bar: 50 μm. H Sholl analysis of neuronal branching complexity. The number of neurite intersections with concentric circles at increasing distances from the soma is plotted for each group ( n = 3 wells). I Representative Western blot images showing the protein levels of the anti-apoptotic protein Bcl-2 and the apoptotic marker Cleaved Caspase-3 in primary cortical neurons treated with conditioned medium from different groups of BMDMs. β-actin was used as a loading control. J Densitometric quantification of relative Bcl-2 protein levels normalized to β-actin ( n = 3 wells). K Densitometric quantification of relative Cleaved Caspase-3 protein levels normalized to β-actin ( n = 3 wells). L Representative flow cytometry plots of primary cortical neurons stained with Annexin V and propidium iodide (PI) after treatment with conditioned media collected from different groups of BMDMs to assess apoptosis. M Quantitative analysis of the total apoptotic cell rate (sum of early and late apoptotic cells, n = 3 wells). * p < 0.05, ** p < 0.01, n.s. = not significant

Techniques: Staining, Saline, Immunofluorescence, Marker, Western Blot, Control, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: FGF4 improves in vivo myelin debris resolution and attenuates lipid accumulation in phagocytes after SCI. A Representative immunofluorescence images of spinal cord sections at 7 and 14 dpi, comparing the SCI group to the FGF4 group. Sections were stained for macrophages (F4/80, red), myelin basic protein (MBP, green), and nuclei (DAPI, blue). The boxed regions magnified in the corresponding right panels. Scale bars: 50 μm, 20 μm (magnified). B Quantitative analysis of the MBP + area within F4/80 + regions at 7 dpi ( n = 5 animals). C Quantitative analysis of the MBP + area within F4/80 + regions at 14 dpi ( n = 5 animals). D Transmission electron microscopy (TEM) images of macrophages in the spinal cord lesion area at 7 dpi. Representative images from the SCI group and the FGF4 group are shown. The boxed regions are magnified on the right, revealing degraded myelin debris (indicated by red arrows). Scale bars: 5 μm, 1 μm (magnified). E Quantitative analysis of the number of degraded myelin debris per macrophage from the TEM images ( n = 3 animals). F Representative images of Oil Red O staining in spinal cord sections at 7, 14, and 42 dpi, comparing the SCI group and the FGF4 group. Lipid deposits are stained red. Higher-magnification views of the boxed regions displayed on the right. Scale bars: 1000 μm, 200 μm (magnified). G-I Quantitative analysis of the Oil Red O-positive lesion area (mm²) at 7 (G) , 14 (H) , and 42 (I) dpi for the indicated groups ( n = 4 animals). * p < 0.05, ** p < 0.01

Techniques: In Vivo, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: FGF4 enhances cholesterol efflux, suppresses inflammasome activation and pyroptosis, and promotes the repair of lysosomal function after SCI. A Representative Western blot images of proteins related to cholesterol efflux (ABCA1), inflammasome activation (NLRP3, Cleaved Caspase-1, IL-1β), pyroptosis (GSDMD), and lysosomal function (Pro-CTSD, Cleaved-CTSD) in spinal cord tissue lysates from sham-operated mice and mice subjected to SCI with or without FGF4 treatment, assessed at 7 and 14 dpi. β-actin was used as a loading control. B–G Quantification of relative protein levels normalized to β-actin: ABCA1 (B) , NLRP3 (C) , GSDMD (D) , IL-1β (E) , Cleaved Caspase-1 (F) , The ratio of Cleaved-CTSD to Pro-CTSD (G) ( n = 4 animals). H Representative immunofluorescence staining for CTSD (green) and the macrophage marker F4/80 (red) in spinal cord sections. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. I Representative immunofluorescence staining for ABCA1 (green) and F4/80 (red) in spinal cord sections. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. J Quantification of the mean fluorescence intensity of CTSD ( n = 5 animals). K Quantification of the mean fluorescence intensity of ABCA1 ( n = 5 animals). L Representative immunohistochemical staining of CASP-1 and NLRP3 in spinal cord tissues from the indicated groups at 7 dpi. Scale bar: 100 μm. M Quantification of relative CASP-1 intensity (Integrated Density/Area, normalized to Sham, n = 5 animals). N Quantification of relative NLRP3 intensity (Integrated Density/Area, normalized to Sham, n = 5 animals). * p < 0.05, ** p < 0.01

Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Staining, Marker, Fluorescence, Immunohistochemical staining

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: FGF4 treatment improves locomotor functional recovery following SCI. A Representative footprint images of hindlimb steps from the Sham, SCI, and FGF4 groups at 42 dpi. B Basso Mouse Scale (BMS) scores for hindlimb motor function assessed at 1, 3, 7, 10, 14, 21, 28, 35, and 42 dpi in the Sham, SCI, and FGF4 groups ( n = 6 animals). C Representative kinematic analysis of hindlimb locomotion. Color-coded simulate limb movement, and joint angle curves for the hip, knee, and ankle joints are plotted. D Quantitative analysis of the number of successful plantar steps ( n = 6 animals). E Quantitative analysis of the height of the trunk above the ground (mm, n = 6 animals). F Representative motor evoked potential (MEP) traces recorded from the Sham, SCI, and FGF4 groups at 42 dpi. G Quantification of MEP amplitude (mV, n = 6 animals). ** p < 0.01

Techniques: Functional Assay

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: FGFR1 inhibition abrogates FGF4-mediated PI3K/AKT activation and phagocytic enhancement in macrophages. A Representative Oil Red O staining of BMDMs under PD173074 (FGFR1 inhibitor) treatment. Scale bar: 30 μm. Representative immunofluorescence images stained for myelin debris (MBP, green), macrophages (F4/80, red), and nuclei (DAPI, blue). Boxed regions are magnified below. Scale bars: 100 μm, 30 μm (magnified). B Quantitative analysis of the Oil Red O-positive area expressed as a percentage (%) of the total cell area ( n = 6 wells). C Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). D Representative Western blot images of phosphorylated FGFR1 (p-FGFR1), phosphorylated PI3K (p-PI3K), total PI3K, phosphorylated AKT (p-AKT), total AKT, MBP, and β-actin in BMDM lysates under PD173074 treatment. E–H Densitometric quantification of relative protein levels: p-FGFR1 (E) ( n = 4 wells), p-PI3K/PI3K ratio (F) ( n = 3 wells), p-AKT/AKT ratio (G) ( n = 3 wells), MBP (H) ( n = 3 wells). All data normalized to β-actin. * p < 0.05, ** p < 0.01

Techniques: Inhibition, Activation Assay, Staining, Immunofluorescence, Western Blot

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: Transcriptomic profiling reveals that FGF4 reshapes macrophage receptor and lipid-handling gene expression and identifies Clec10a as a key downstream effector. A Summary of differentially expressed genes (DEGs) identified by RNA-seq analysis among the Control, MD, and MD+FGF4 groups. BMDMs were pretreated with FGF4 for 2 h followed by exposure to MD for 24 h. B Heatmap displaying the expression patterns of genes related to phagocytosis across the indicated groups. C Volcano plot illustrating the significance and magnitude of gene expression changes for phagocytosis-related genes between the MD+FGF4 and MD groups. D-E Gene Ontology (GO) biological process enrichment analysis (D) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (E) of the phagocytosis-related DEGs. F–K Validation of RNA-seq data by quantitative RT-PCR (qRT-PCR). Relative mRNA expression levels of Fcgr4 (F) , Cd163 (G) , Irf4 (H) , Ccl6 (I) , Scd1 (J) , and Clec10a (K) in BMDMs from the Control, FGF4, MD, and MD+FGF4 groups. Data were normalized to β-actin and presented as relative to the Control group ( n = 3 wells). L Representative Western blot images showing Clec10a protein expression in BMDM lysates. β-actin was used as a loading control. M Densitometric quantification of relative Clec10a protein levels normalized to β-actin ( n = 3 wells). * p < 0.05, ** p < 0.01

Techniques: Gene Expression, RNA Sequencing, Control, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: Clec10a is essential for FGF4-mediated enhancement of myelin debris phagocytosis. A Representative Oil Red O staining of BMDMs transfected with LV-shClec10a. Scale bar: 30 μm. Representative immunofluorescence images of BMDMs stained for Clec10a (purple), MBP (green), F4/80 (red), and DAPI (blue). Boxed regions are magnified below. Scale bars: 100 μm, 30 μm (magnified). B Quantitative analysis of the Oil Red O-positive area expressed as a percentage (%) of the total cell area ( n = 5 wells). C Quantitative analysis of the mean fluorescence intensity of Clec10a in BMDMs ( n = 5 wells). D Quantitative analysis of the percentage of MBP + area within F4/80 + cells ( n = 5 wells). E Representative Western blot images of MBP and Clec10a protein expression in BMDM lysates following LV-shClec10a transfection. β-actin was used as a loading control. F Densitometric quantification of relative MBP protein levels normalized to β-actin ( n = 3 wells). G Densitometric quantification of relative Clec10a protein levels normalized to β-actin ( n = 3 wells). H Representative flow cytometry plots of primary cortical neurons stained with Annexin V and PI after treatment with conditioned media collected from the differently treated BMDM groups. I Quantitative analysis of the total apoptotic cell rate ( n = 3 wells). J Representative immunofluorescence images of primary cortical neurons treated with conditioned medium from different groups of BMDMs, stained with the neuronal marker Tuj1 (green) and the nuclear marker DAPI (blue). Boxed regions are magnified below. Scale bars: 50 μm, 20 μm (magnified). K Sholl analysis of neuronal branching complexity. The number of neurite intersections with concentric circles at increasing distances from the soma is plotted for each group ( n = 3 wells). * p < 0.05, ** p < 0.01

Techniques: Staining, Transfection, Immunofluorescence, Fluorescence, Western Blot, Expressing, Control, Flow Cytometry, Marker

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: Clec10a acts intracellularly to facilitate myelin debris processing rather than through surface-level recognition. A Representative immunofluorescence images of BMDMs pretreated with or without FGF4 for 2 h, followed by incubation with Dil-labeled myelin debris (red) at 4 °C for 30 min to block internalization and assess surface binding. Cells were stained for Clec10a (green) and DAPI (blue). Scale bar, 200 μm. B Quantification of Clec10a/Dil-myelin colocalization expressed as Pearson’s correlation coefficient ( n = 4 wells). C Representative immunofluorescence images of BMDMs pretreated with FGF4 for 2 h and then incubated with Dil-myelin debris at 37 °C for 5, 15, 30, 60 min, and 6 h. Cells were stained for Clec10a (green), RAB5 (purple), and DAPI (blue). Scale bar: 100 μm. D–F Representative images showing colocalization of Dil-myelin (red) with Rab5 (green) (D) , Rab7 (green) (E) , or Lamp1 (green) (F) in BMDMs pretreated with or without FGF4 and incubated with myelin debris at 37 °C for 15 min. Nuclei are stained with DAPI (blue). Scale bars: 200 μm. G–I Quantitative analysis of Pearson’s correlation coefficients for Rab5/Dil-myelin (G) , Rab7/Dil-myelin (H) , and Lamp1/Dil-myelin ( n = 3 wells) (I) . * p < 0.05, ** p < 0.01

Techniques: Immunofluorescence, Incubation, Labeling, Blocking Assay, Binding Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: FGF4 activates FGFR1 - PI3K/AKT signaling to enhance Clec10a-mediated intracellular myelin debris processing and promote spinal cord repair

doi: 10.1186/s12974-026-03743-0

Figure Lengend Snippet: FGF4 activates FGFR1–PI3K/AKT signaling in macrophages to upregulate intracellular Clec10a, which promotes phagosome maturation and endolysosomal trafficking of internalized myelin debris from Rab5⁺ early endosomes to Rab7⁺ late endosomes and Lamp1⁺ lysosomes. This process restores lysosomal membrane stability and CTSD-dependent degradative capacity, enhances myelin debris clearance and ultimately creates a permissive microenvironment for neuronal survival and spinal cord repair. The schematic is created using BioRender

Techniques: Membrane

The differentiation block in I-BET-resistant (I-BETR) diapause-like ES cells. ( A ) Alkaline phosphatase (AP) levels in control and I-BETR ES cells. Scale bar, 100 µm. ( B ) Expression levels of the pluripotency ( left panel) or differentiation-inducing ( right panel) genes in control, 2i-treated, or I-BETR ES cells. RNA expression levels were quantified by qPCR. Values represent normalized mean ± SD. n = 3. ( C ) Colony morphology and/or alkaline phosphatase (AP) levels in control and I-BETR ES cells following vehicle or FGF4 triggering. Scale bar, 100 µm. The bar graph represents quantification of relative percentage of pluripotent colonies in different groups. Values represent ± SD. n = 9. (n.s.) No significance, (****) P < 0.0001, one-way ANOVA with Dunnett's multiple comparisons test. ( D ) Expression levels of selected pluripotency ( left ) or differentiation-inducing ( right ) genes in control and I-BETR ES cells treated or not treated with FGF4. Error bars indicate SD. n = 3. (n.s.) No significance, (****) P < 0.0001, one-way ANOVA with Dunnett's multiple comparisons test. ( E ) Withdrawal of I-BET (I-BETW) restores the control ES cell-like pluripotency gene expression pattern in I-BETR ES cells. Values represent gene expression levels normalized to the mean of control samples based on TPM values observed by bulk mRNA RNA-seq analysis . ( F ) Generation of chimeras by I-BETR ES cells. The I-BETR ES cells were incubated in I-BET-free medium for 12–14 h and injected into the C57BL/6J blastocysts. White coat color indicates the chimerism.

Journal: Genes & Development

Article Title: Transcriptional derepression of negative regulators of MAP kinase supports maintenance of diapause ES cells in the pluripotent state

doi: 10.1101/gad.353143.125

Figure Lengend Snippet: The differentiation block in I-BET-resistant (I-BETR) diapause-like ES cells. ( A ) Alkaline phosphatase (AP) levels in control and I-BETR ES cells. Scale bar, 100 µm. ( B ) Expression levels of the pluripotency ( left panel) or differentiation-inducing ( right panel) genes in control, 2i-treated, or I-BETR ES cells. RNA expression levels were quantified by qPCR. Values represent normalized mean ± SD. n = 3. ( C ) Colony morphology and/or alkaline phosphatase (AP) levels in control and I-BETR ES cells following vehicle or FGF4 triggering. Scale bar, 100 µm. The bar graph represents quantification of relative percentage of pluripotent colonies in different groups. Values represent ± SD. n = 9. (n.s.) No significance, (****) P < 0.0001, one-way ANOVA with Dunnett's multiple comparisons test. ( D ) Expression levels of selected pluripotency ( left ) or differentiation-inducing ( right ) genes in control and I-BETR ES cells treated or not treated with FGF4. Error bars indicate SD. n = 3. (n.s.) No significance, (****) P < 0.0001, one-way ANOVA with Dunnett's multiple comparisons test. ( E ) Withdrawal of I-BET (I-BETW) restores the control ES cell-like pluripotency gene expression pattern in I-BETR ES cells. Values represent gene expression levels normalized to the mean of control samples based on TPM values observed by bulk mRNA RNA-seq analysis . ( F ) Generation of chimeras by I-BETR ES cells. The I-BETR ES cells were incubated in I-BET-free medium for 12–14 h and injected into the C57BL/6J blastocysts. White coat color indicates the chimerism.

Article Snippet: For FGF4-driven ES cell differentiation, 10 ng/μL recombinant FGF4 (R&D Systems 235-F4) was added on day 0 together with 1 μg/μL heparin (Sigma-Aldrich H3149).

Techniques: Blocking Assay, Control, Expressing, RNA Expression, Gene Expression, RNA Sequencing, Incubation, Injection

Overview of the protocol steps. The complete directed differentiation process lasts for 37 days, including 28 days of culture in Matrigel. Two days after seeding the hPSCs, the first step involves adding Activin A for 3 days to generate definitive endoderm formation, followed by the addition of FGF4 and CHIRON for 4 more days to induce spheroid formation. The resulting spheroids are then embedded in Matrigel and cultured for 28 days, with a passage at day 14 to renew the Matrigel.

Journal: Biology of the Cell

Article Title: Generation of Intestinal and Colonic Organoids Derived From Human Pluripotent Stem Cells

doi: 10.1111/boc.70044

Figure Lengend Snippet: Overview of the protocol steps. The complete directed differentiation process lasts for 37 days, including 28 days of culture in Matrigel. Two days after seeding the hPSCs, the first step involves adding Activin A for 3 days to generate definitive endoderm formation, followed by the addition of FGF4 and CHIRON for 4 more days to induce spheroid formation. The resulting spheroids are then embedded in Matrigel and cultured for 28 days, with a passage at day 14 to renew the Matrigel.

Article Snippet: FGF4 (Human recombinant fibroblast growth factor 4 premium grade, Miltenyi Biotec, cat. no. 130‐109‐391) ▲CRITICAL .

Techniques: Cell Culture

Characterization of definitive endoderm induction and spheroids generation. (a) Immunofluorescence images for FOXA2 and SOX17 of definitive endoderm after 3 days of Activin A induction. (b) Immunofluorescence images of hindgut spheroids following 4 days of FGF4 and Chiron induction, characterized by CDX2 (intestinal marker) and E‐cadherin (epithelial marker) expression. Scale bars = 100 µm.

Journal: Biology of the Cell

Article Title: Generation of Intestinal and Colonic Organoids Derived From Human Pluripotent Stem Cells

doi: 10.1111/boc.70044

Figure Lengend Snippet: Characterization of definitive endoderm induction and spheroids generation. (a) Immunofluorescence images for FOXA2 and SOX17 of definitive endoderm after 3 days of Activin A induction. (b) Immunofluorescence images of hindgut spheroids following 4 days of FGF4 and Chiron induction, characterized by CDX2 (intestinal marker) and E‐cadherin (epithelial marker) expression. Scale bars = 100 µm.

Article Snippet: FGF4 (Human recombinant fibroblast growth factor 4 premium grade, Miltenyi Biotec, cat. no. 130‐109‐391) ▲CRITICAL .

Techniques: Immunofluorescence, Marker, Expressing

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