axl inhibitor r428  (ApexBio)

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Gas6 improves the inflammatory micro-environment to promote tissue repair and locomotor recovery. (A) Immunofluorescence analysis of CD68 + (red, Alexa Fluor 594) cells and GFAP (green, FITC) immunopositivity in the injured spinal cord after SCI ( n = 3). CD68 + cells and the immunopositivity of GFAP were increased after SCI and were significantly decreased with additional Gas6. After addition of Gas6 and R428, CD68 + cells and the immunopositivity of GFAP were increased compared with the Gas6 group. Scale bars: 100 μm (upper), 20 μm (lower). (B) Quantification of CD68 + cells and GFAP immunopositivity in A ( n = 3). (C) Hematoxylin-eosin and Nissl staining of the spinal cord in different groups. The cavity area was decreased in the Gas6 group. The number of Nissl bodies was increased in the Gas6 group. (D) Quantification of hematoxylin-eosin and Nissl staining in different groups ( n = 5). (E) Behavioral character images showing hindlimb movement in different groups. Rats in the Gas6 group grabbed and stood up more easily than those in the other groups. (F) Footprint analysis of different groups. Rats in the Gas6 group stepped more easily than those in the other group. Blue indicates the forelimb and red indicates the hindlimb. (G) BBB scores in different groups ( n = 5). The data are presented as the means ± SD. * P < 0.05, ** P < 0.01, vs . SCI group (one-way analysis of variance followed by Bonferroni post hoc test). A.U.: Arbitrary unit; Axl: AXL receptor tyrosine kinase; BBB: Basso, Beattie, and Bresnahan locomotion scale; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; Gas6: growth arrest specific protein 6; GFAP: glial fibrillary acidic protein; R428: AXL inhibitor; SCI: spinal cord injury.

Article Snippet: Then, before surgery, rats were injected intrathecally with 2 μg of Gas6 (NovoProtein) daily for 7 days, with or without AXL inhibitor, R428 (Glpbio) (3.5 mg/kg, dissolved in PBS, intrathecal injection), as described in a previous study (Zhou et al., 2021).

Techniques: Immunofluorescence, Staining

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Gas6 inhibits cytotoxic phenotype polarization of astrocytes through suppression of YAP activation. (A) KEGG pathways of differentially expressed genes in naive, reactive and scar-forming astrocytes. (B) Immunofluorescence analysis of YAP1 (pink, Alexa Fluor 647) and GFAP (green, FITC) at the injured site of the spinal cord after SCI. The immunopositivities of YAP1 and GFAP were significantly decreased in the Gas6 group. Scale bars: 100 μm (upper), 20 μm (lower). (C) Quantification of YAP1 and GFAP immunopositivities in B. (D) Western blot analysis of YAP1 phosphorylation in astrocytes with Gas6 and R428 treatment. (E, F) Western blot and immunofluorescence analyses of YAP1 (green, FITC) nuclear localization in astrocytes after Gas6 and R428 treatment. The nuclear localization of YAP1 in astrocytes was significantly decreased in Gas6 group. Scale bar: 20 μm. (G) Quantification of YAP1 immunopositivity in F. The data are presented as the means ± SD from one representative experiment of three independent experiments performed in triplicate. ** P < 0.01 (one-way analysis of variance followed by Bonferroni post hoc test). A.U.: Arbitrary unit; Axl: AXL receptor tyrosine kinase; CE: cytoplasm expression; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; Gas6: growth arrest specific protein 6; GFAP: glial fibrillary acidic protein; KEGG: Kyoto Encyclopedia of Genes and Genome; LPS: lipopolysaccharide; NE: nuclear expression; NT: non-treatment; p-YAP1: phospho-YAP1; R428: AXL inhibitor; SCI: spinal cord injury; YAP1: YES-associated protein 1.

Article Snippet: Then, before surgery, rats were injected intrathecally with 2 μg of Gas6 (NovoProtein) daily for 7 days, with or without AXL inhibitor, R428 (Glpbio) (3.5 mg/kg, dissolved in PBS, intrathecal injection), as described in a previous study (Zhou et al., 2021).

Techniques: Activation Assay, Immunofluorescence, Western Blot, Expressing

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Gas6 inhibits inflammation-related signaling pathways to suppress pro-inflammatory phenotype polarization. (A) Enrich GO_BP terms of different pathways genes in microglia after SCI. (B) Immunofluorescence analysis of phospho-p65 (p-p65, red, Alexa Fluor 647) immunopositivity in CD68 + (green, FITC) cells in the injured spinal cord after SCI. The immunopositivity of p-p65 in CD68 + cells was significantly decreased in Gas6 group Scale bars: 100 μm (upper), 20 μm (lower). (C) Immunofluorescence analysis of phospho-Stat3 (p-Stat3, FITC) immunopositivity in CD68 + cells (FITC) in the injure dspinal cord after SCI. The immunopositivity of p-Stat3 in CD68 + cells was significantly decreased in the Gas6 group. Scale bars: 100 μm (upper), 20 μm (lower). (D) Quantification of p-p65 immunopositivity and CD68 + cells in B. (E) Quantification of p-Stat3 immunopositivity and CD68 + cells in C. (F) Western blot analysis of p65 and Stat3 phosphorylation in microglia after Gas6 and R428 treatment. (G, H) Western blot and immunofluorescence analyses of p65 (green, FITC) and Stat3 (green, FITC) nuclear localization in microglia after Gas6 and R428 treatment. The immunopositivity of nuclear localization p65 and Stat3 were decreased in the Gas6 group. Scale bar: 20 μm. (I, J) Quantification of p65 and Stat3 immunopositivities in H. The data are presented as the means ± SD from one representative experiment of three independent experiments performed in triplicate. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Bonferroni post hoc test). A.U.: Arbitrary unit; Axl: AXL receptor tyrosine kinase; BP: biological process; CE: cytoplasm expression; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; Gas6: growth arrest specific protein 6; GO: Gene Ontology; LPS: lipopolysaccharide; NE: nuclear expression; p-p65: phospho-p65; p-Stat3: phospho-Stat3; R428: AXL inhibitor; SCI: spinal cord injury; Stat3: signal transducer and activator of transcription 3.

Article Snippet: Then, before surgery, rats were injected intrathecally with 2 μg of Gas6 (NovoProtein) daily for 7 days, with or without AXL inhibitor, R428 (Glpbio) (3.5 mg/kg, dissolved in PBS, intrathecal injection), as described in a previous study (Zhou et al., 2021).

Techniques: Immunofluorescence, Western Blot, Expressing