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. 2024 Aug 15;16(9):2146–2169. doi: 10.1038/s44321-024-00117-y

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) The polymerization of F-actin in neutrophils was observed under a fluorescent microscope by staining with phalloidin (cytoskeleton, green) and Hoechst 33342 (nuclei, blue). The cells were observed at 2.5 h after stimulation with tPA (10 μg/ml), tPA (10 μg/ml) + HRG (0.5 μM), and LPS (10 μg/ml). Scale bar = 20 µm. (B) Quantitative analysis of F-actin immunofluorescence intensity under different conditions (n = 6–8). (C) Schematic diagram illustrating the experimental design. Transwell membrane inserts were coated with collagen IV and fibronectin, and hCMEC/D3 cells were seeded onto them followed by 4 h of hypoxia-glucose deprivation. Neutrophils pretreated with different factors (tPA: 10 μg/ml, HRG: 0.1 μM, HRG: 0.5 μM, and HRG: 1 μM) for 2 h were added to the upper chamber. After 2 h (D) (n = 8), 4 h (E) (n = 7–8), and 8 h (F) (n = 7–8) of culture, the number of neutrophils in the lower chamber was counted. Migration percentage = the number of neutrophils in the lower chamber/the total number of neutrophils added to the upper chamber. (G) The line graph summarizes the migrated percentage of neutrophils at 2 h, 4 h, and 8 h (n = 6–8). (H) The average migration rate of neutrophils during the 0–2 h, 2–4 h, and 4–8 h of the transwell assay. Data information: Data are presented as mean ± SEM; two-tailed Kruskal–Wallis H test is used in (B, D–F). Source data are available online for this figure.