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Animal study verifying the effect of AE in mitigating EBI of SAH. (A) The complete timeline of an experimental plan, schematic diagram, and actual skull base image of the SAH/Sham model in this animal experiment. Some elements are created in BioRender (2026); https://BioRender.com/vwwir98 . (B) SAH grading score of each group in this stage. n = 24. (C) Modified Garcia score of each group, n = 24. (D) The water content of the left and right brains in different groups at this stage. n = 6. (E and F) Apoptosis of bleeding focus cortical neurons was detected by TUNEL assay (red), NeuN (green), and DAPI (blue) immunofluorescence. n = 6. Scale bars, 50 μm. (G) Bars show changes in BBs in SAH mice. n = 24. (H to N) Western blotting images and quantitative data of relative expression level of Il-1b, Il-6, Nos2, <t>Il-4,</t> <t>Il-10,</t> and Tgfb1 in the ipsilateral cortex after SAH. n = 6. (O) The coronal sections were stained with hematoxylin and eosin (H&E) for histological evaluation AE-regulated in SAH. n = 6. Scale bars, 50 μm. (P) Histopathological changes detected by Nissl’s staining in hippocampus and the hemorrhagic cortex. n = 6. Scale bars, 50 μm. In (B), (C), and (G), data were represented as median (interquartile range), and P values were calculated using Kruskal–Wallis H with Dunn test. In (D), (F), and (I) to (N), data were represented as mean ± SD, and P values were calculated using one-way ANOVA with Tukey multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. The original Western blot images are shown in Data . Detailed experimental data and statistical results, including Z and P values for Kruskal–Wallis test and F and P values for ANOVAs, are provided in Data to .
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Animal study verifying the effect of AE in mitigating EBI of SAH. (A) The complete timeline of an experimental plan, schematic diagram, and actual skull base image of the SAH/Sham model in this animal experiment. Some elements are created in BioRender (2026); https://BioRender.com/vwwir98 . (B) SAH grading score of each group in this stage. n = 24. (C) Modified Garcia score of each group, n = 24. (D) The water content of the left and right brains in different groups at this stage. n = 6. (E and F) Apoptosis of bleeding focus cortical neurons was detected by TUNEL assay (red), NeuN (green), and DAPI (blue) immunofluorescence. n = 6. Scale bars, 50 μm. (G) Bars show changes in BBs in SAH mice. n = 24. (H to N) Western blotting images and quantitative data of relative expression level of Il-1b, Il-6, Nos2, Il-4, Il-10, and Tgfb1 in the ipsilateral cortex after SAH. n = 6. (O) The coronal sections were stained with hematoxylin and eosin (H&E) for histological evaluation AE-regulated in SAH. n = 6. Scale bars, 50 μm. (P) Histopathological changes detected by Nissl’s staining in hippocampus and the hemorrhagic cortex. n = 6. Scale bars, 50 μm. In (B), (C), and (G), data were represented as median (interquartile range), and P values were calculated using Kruskal–Wallis H with Dunn test. In (D), (F), and (I) to (N), data were represented as mean ± SD, and P values were calculated using one-way ANOVA with Tukey multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. The original Western blot images are shown in Data . Detailed experimental data and statistical results, including Z and P values for Kruskal–Wallis test and F and P values for ANOVAs, are provided in Data to .

Journal: Research

Article Title: Aerobic Exercise-Induced TGF-β Receptor Reprogramming Disrupts Neutrophil–Microglia Crosstalk to Attenuate Early Brain Injury after Subarachnoid Hemorrhage

doi: 10.34133/research.1301

Figure Lengend Snippet: Animal study verifying the effect of AE in mitigating EBI of SAH. (A) The complete timeline of an experimental plan, schematic diagram, and actual skull base image of the SAH/Sham model in this animal experiment. Some elements are created in BioRender (2026); https://BioRender.com/vwwir98 . (B) SAH grading score of each group in this stage. n = 24. (C) Modified Garcia score of each group, n = 24. (D) The water content of the left and right brains in different groups at this stage. n = 6. (E and F) Apoptosis of bleeding focus cortical neurons was detected by TUNEL assay (red), NeuN (green), and DAPI (blue) immunofluorescence. n = 6. Scale bars, 50 μm. (G) Bars show changes in BBs in SAH mice. n = 24. (H to N) Western blotting images and quantitative data of relative expression level of Il-1b, Il-6, Nos2, Il-4, Il-10, and Tgfb1 in the ipsilateral cortex after SAH. n = 6. (O) The coronal sections were stained with hematoxylin and eosin (H&E) for histological evaluation AE-regulated in SAH. n = 6. Scale bars, 50 μm. (P) Histopathological changes detected by Nissl’s staining in hippocampus and the hemorrhagic cortex. n = 6. Scale bars, 50 μm. In (B), (C), and (G), data were represented as median (interquartile range), and P values were calculated using Kruskal–Wallis H with Dunn test. In (D), (F), and (I) to (N), data were represented as mean ± SD, and P values were calculated using one-way ANOVA with Tukey multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. The original Western blot images are shown in Data . Detailed experimental data and statistical results, including Z and P values for Kruskal–Wallis test and F and P values for ANOVAs, are provided in Data to .

Article Snippet: The membrane was blocked at room temperature for 1 h with 5% skim milk or bovine serum albumin, and then incubated overnight at 4 °C with the following primary antibodies: Rabbit anti-Lrg1 polyclonal antibody (Proteintech, 13224-1-AP, diluted 1:100); Rabbit anti-Il-1b polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB0002); Rabbit anti-interleukin 6 (Il-6) polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB0062); Rabbit anti-interleukin 4 (Il-4) polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB3904); Rabbit anti-interleukin 10 (Il-10) polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB0104); Rabbit anti-Tgfb1 polyclonal antibody (ABclonal, A16640); Rabbit anti-nitric oxide synthase 2 (Nos2) polyclonal antibody (Proteintech, 22226-1-AP); Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Proteintech, 10494-1-AP); Rabbit anti-Alk1 polyclonal antibody (Huabio, HA722529); Rabbit anti-Alk5 polyclonal antibody (Immunoway, YM8858); Rabbit anti-Tgfbr2 polyclonal antibody (Immunoway, YM8220); Rabbit anti-Eng polyclonal antibody (Proteintech, 10862-1-AP); Rabbit anti-Mothers Against Decapentaplegic Homolog 1/5 (Smad1/5) polyclonal antibody (immunoway, YT4325); Rabbit anti-Smad2/3 polyclonal antibody (Wanleibio, wl01520); Rabbit anti-phospho-Smad1/5 (p-Smad1/5) polyclonal antibody (Huabio, HA722566); Rabbit anti-p-Smad2/3 polyclonal antibody (Wanleibio, WL02305); Rabbit anti-DYKDDDDK-tag monoclonal antibody (Proteintech, 20543-1-AP); Rabbit anti-His-tag polyclonal antibody (Proteintech, 10001-0-AP); Rabbit anti-HA-Tag(26D11) polyclonal antibody (Proteintech, 51064-2-AP); Rabbit anti-myelocytomatosis oncogene (Myc)-tag monoclonal antibody (Abcom, ab9106); Rabbit anti-glutathione S-transferase (GST)-tag monoclonal antibody (ABclonal, AE006); and Rabbit anti-V5-tag polyclonal antibody (Proteintech, 14440-1-AP).

Techniques: Modification, TUNEL Assay, Immunofluorescence, Western Blot, Expressing, Staining

Primary peripheral neutrophil and microglia interactions based on wild-type and knockout mice. (A) Western blotting images demonstrated the effect of SB431541 treatment on the activation status of the TGF-β signaling pathway in SAH regulated by AE. (B to G) Quantitative analysis of relative expression level changes in Alk1, Alk5, Smad1/5, Smad2/3, p-Smad1/5, and p-Smad2/3 in the AE-regulated ipsilateral cortex in SAH, with the treat of SB431541. n = 6. (H) Schematic diagram of coculture experiments between primary peripheral neutrophils and primary microglia. (I) Immunofluorescent staining of primary microglia from wt/ko mice cocultured with primary neutrophils from wt/ko mice for Lpl/Sec24a (red), Iba1 (green), and DAPI (blue). Scale bars, 10 μm. (J) Immunofluorescent staining of primary peripheral neutrophils from wild-type and knockout mice for Lrg1 (red), Ly6G (green), and DAPI (blue). Scale bars, 10 μm. (K) Western blotting images of primary microglia from wt/ko mice cocultured with primary neutrophils from wt/ko mice on pro-inflammatory and anti-inflammatory factors and TGF-β signaling pathway. (L to W) Quantitative analysis of relative expression level of IL-1β, IL-6, Nos2, IL-4, IL-10, Tgfb1, Alk1, Alk5, Smad1/5, Smad2/3, p-Smad1/5, and p-Smad2/3 in the primary microglia cocultured with peripheral neutrophils. n = 3. (X) Quantitative analysis of the proportion of Lpl+/Iba1+ cells in the immunofluorescent staining of primary microglia in the primary cell coculture experiment. n = 3. (Y) Quantitative analysis of the proportion of Sec24a+/Iba1+ cells in the immunofluorescent staining of primary microglia in the primary cell coculture experiment. n = 3. In (B) to (G) and (L) to (Y), data were represented as mean ± SD, and P values were calculated using one-way ANOVA with Tukey multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. The original Western blot images are shown in Data . Detailed experimental data and statistical results, including F and P values for ANOVAs, are provided in Data to .

Journal: Research

Article Title: Aerobic Exercise-Induced TGF-β Receptor Reprogramming Disrupts Neutrophil–Microglia Crosstalk to Attenuate Early Brain Injury after Subarachnoid Hemorrhage

doi: 10.34133/research.1301

Figure Lengend Snippet: Primary peripheral neutrophil and microglia interactions based on wild-type and knockout mice. (A) Western blotting images demonstrated the effect of SB431541 treatment on the activation status of the TGF-β signaling pathway in SAH regulated by AE. (B to G) Quantitative analysis of relative expression level changes in Alk1, Alk5, Smad1/5, Smad2/3, p-Smad1/5, and p-Smad2/3 in the AE-regulated ipsilateral cortex in SAH, with the treat of SB431541. n = 6. (H) Schematic diagram of coculture experiments between primary peripheral neutrophils and primary microglia. (I) Immunofluorescent staining of primary microglia from wt/ko mice cocultured with primary neutrophils from wt/ko mice for Lpl/Sec24a (red), Iba1 (green), and DAPI (blue). Scale bars, 10 μm. (J) Immunofluorescent staining of primary peripheral neutrophils from wild-type and knockout mice for Lrg1 (red), Ly6G (green), and DAPI (blue). Scale bars, 10 μm. (K) Western blotting images of primary microglia from wt/ko mice cocultured with primary neutrophils from wt/ko mice on pro-inflammatory and anti-inflammatory factors and TGF-β signaling pathway. (L to W) Quantitative analysis of relative expression level of IL-1β, IL-6, Nos2, IL-4, IL-10, Tgfb1, Alk1, Alk5, Smad1/5, Smad2/3, p-Smad1/5, and p-Smad2/3 in the primary microglia cocultured with peripheral neutrophils. n = 3. (X) Quantitative analysis of the proportion of Lpl+/Iba1+ cells in the immunofluorescent staining of primary microglia in the primary cell coculture experiment. n = 3. (Y) Quantitative analysis of the proportion of Sec24a+/Iba1+ cells in the immunofluorescent staining of primary microglia in the primary cell coculture experiment. n = 3. In (B) to (G) and (L) to (Y), data were represented as mean ± SD, and P values were calculated using one-way ANOVA with Tukey multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. The original Western blot images are shown in Data . Detailed experimental data and statistical results, including F and P values for ANOVAs, are provided in Data to .

Article Snippet: The membrane was blocked at room temperature for 1 h with 5% skim milk or bovine serum albumin, and then incubated overnight at 4 °C with the following primary antibodies: Rabbit anti-Lrg1 polyclonal antibody (Proteintech, 13224-1-AP, diluted 1:100); Rabbit anti-Il-1b polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB0002); Rabbit anti-interleukin 6 (Il-6) polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB0062); Rabbit anti-interleukin 4 (Il-4) polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB3904); Rabbit anti-interleukin 10 (Il-10) polyclonal antibody (Baijia Biotechnology Co., Ltd., IPB0104); Rabbit anti-Tgfb1 polyclonal antibody (ABclonal, A16640); Rabbit anti-nitric oxide synthase 2 (Nos2) polyclonal antibody (Proteintech, 22226-1-AP); Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Proteintech, 10494-1-AP); Rabbit anti-Alk1 polyclonal antibody (Huabio, HA722529); Rabbit anti-Alk5 polyclonal antibody (Immunoway, YM8858); Rabbit anti-Tgfbr2 polyclonal antibody (Immunoway, YM8220); Rabbit anti-Eng polyclonal antibody (Proteintech, 10862-1-AP); Rabbit anti-Mothers Against Decapentaplegic Homolog 1/5 (Smad1/5) polyclonal antibody (immunoway, YT4325); Rabbit anti-Smad2/3 polyclonal antibody (Wanleibio, wl01520); Rabbit anti-phospho-Smad1/5 (p-Smad1/5) polyclonal antibody (Huabio, HA722566); Rabbit anti-p-Smad2/3 polyclonal antibody (Wanleibio, WL02305); Rabbit anti-DYKDDDDK-tag monoclonal antibody (Proteintech, 20543-1-AP); Rabbit anti-His-tag polyclonal antibody (Proteintech, 10001-0-AP); Rabbit anti-HA-Tag(26D11) polyclonal antibody (Proteintech, 51064-2-AP); Rabbit anti-myelocytomatosis oncogene (Myc)-tag monoclonal antibody (Abcom, ab9106); Rabbit anti-glutathione S-transferase (GST)-tag monoclonal antibody (ABclonal, AE006); and Rabbit anti-V5-tag polyclonal antibody (Proteintech, 14440-1-AP).

Techniques: Knock-Out, Western Blot, Activation Assay, Expressing, Staining