rmgal 9 protein  (R&D Systems)

Journal: Aging Cell

Article Title: Galectin‐9 high Neutrophils Exacerbate Radiation‐Induced Frailty

doi: 10.1111/acel.70448

Figure Lengend Snippet: Single‐cell sequencing reveals a subpopulation of hyperactive GAL‐9 high MA2 neutrophils in circulation. (A) Scheme of single‐cell sequencing of circulating, splenic, and bone marrow neutrophils in mice. Spleen, bone marrow, and peripheral blood were taken from mice in the sham group, 20 days post‐radiation, and 80 days post‐radiation. 40% c‐Kit + progenitor cells and 60% Gr‐1 + neutrophils were sorted from the spleen. Gr‐1 + neutrophils were sorted from the peripheral blood. The bone marrow cells were the total number of cells that were flushed out of the bone marrow lumen. (B) UMAP projection delineating neutrophil, preNeu (G1‐G2), immatureNeu (IM1‐IM3), matureNeu (MA1‐MA2). (C) Changes in the ratio of neutrophils according to the different groups. (D) The composition of each population of circulating, splenic, and bone marrow neutrophils by circle plots. (E) Differential number/strength of interactions with each population of neutrophils in the local radiation group relative to the sham group by CellChat analysis (red means higher, blue means lower, and the width of the line represents the interaction strength). (F) Pathway enrichment between MA1 and MA2 neutrophils by bubble plots. (G–I) (G) Glycolysis score, (H) neutrophil activation score, and (I) maturation score for each population of neutrophils by violin plots. (J) The main neutrophil interaction pathways by CellChat analysis for the sham group and 80 days post‐radiation. (K) The main Outgoing signaling patterns of each population of neutrophils in the sham group and 80 days post‐radiation by CellChat analysis. (L) Lgals9 expression in neutrophils UMAP of mice (Figure ). (M) Galectin pathway network's enrichment intensity of the sham group and 80 days post‐radiation in each population of neutrophils by CellChat analysis. (N) Lgals9 expression in the sham group, 20 days post‐radiation, and 80 days post‐radiation by violin plots.

Article Snippet: Intraperitoneal injection (i.p.) of the following drugs: recombinant mouse GAL‐9 (rmGAL‐9) protein (200 μg, R&D Systems #3535‐GA‐050), anti‐GAL‐9 antibody (1 mg, BioXCell#BE0218), or IgG isotype control (1 mg, Abcam #ab172730).

Techniques: Single Cell, Sequencing, Activation Assay, Expressing

Journal: Aging Cell

Article Title: Galectin‐9 high Neutrophils Exacerbate Radiation‐Induced Frailty

doi: 10.1111/acel.70448

Figure Lengend Snippet: GAL‐9 high neutrophils are crucial mediators for inducing frailty after local radiation injury. (A) Circulating GAL‐9 high neutrophils at different time post‐radiation. n = 4. (B) The survival rate of circulating neutrophils in the sham group and circulating GAL‐9 high neutrophils 80 days post‐radiation was analyzed by the CCK‐8 kit. (C–E) Cultured circulating neutrophils in the sham group and GAL‐9 high neutrophils 80 days post‐radiation were treated with media with 10 mg/mL PGN for 2 h, after which (C) ROS, (D) phagocytic rate, and (E) IFN‐γ were quantified. n = 5–6. (F) Circulating IFN‐γ protein level by ELISA kits. n = 5. (G) Cultured circulating neutrophils in the sham group and GAL‐9 high neutrophils 80 days post‐radiation were treated with media with 50 mg/mL PGN for 4 h, after which MPO and NETs were visualized by cell smears. n = 4. (H–K) Representative plots and statistics of (H, J) GAL‐9 high neutrophil and (I, K) NETs expression in multiple organs by immunofluorescence. n = 4. (L, M) Bone marrow neutrophils in the sham group and 80 days post‐radiation were treated with media or 10 mg/mL PGN for 2 h, after which (L) ROS and (M) IFN‐γ were quantified. n = 6. (N) Representative plots of HE staining of bone marrow in the sham group and 80 days post‐radiation. (O) The frequence of bone marrow endothelial cells (CD45 − Ter119 − CD31 + CD105 − ), SECs (CD45 − Ter119 − CD31 + CD105 + ), and stromal cells (CD45 − Ter119 − CD31 − CD105 + ) at different time post‐radiation. n = 3. (P–R) Assessment Scheme of different groups of bone marrow macrophages phagocytosed neutrophils by confocal live cell station imaging in vitro. (P) The scheme, (Q) the survival area statistics of neutrophils, and (R) the representative plots of phagocytosis in different groups at different time were shown. Data are presented as mean ± SD; each dot represents an individual animal from at least 2–4 independent experiments that used male and female mice. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analyses were performed using unpaired Student's t ‐test (B–G and J–M), one‐way ANOVA (A), and two‐way ANOVA (O).

Article Snippet: Intraperitoneal injection (i.p.) of the following drugs: recombinant mouse GAL‐9 (rmGAL‐9) protein (200 μg, R&D Systems #3535‐GA‐050), anti‐GAL‐9 antibody (1 mg, BioXCell#BE0218), or IgG isotype control (1 mg, Abcam #ab172730).

Techniques: CCK-8 Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining, Imaging, In Vitro

Journal: Aging Cell

Article Title: Galectin‐9 high Neutrophils Exacerbate Radiation‐Induced Frailty

doi: 10.1111/acel.70448

Figure Lengend Snippet: GAL‐9 high neutrophils adoptive‐transfer assay and TCGA database analysis. (A–G) Assessment of the effect of circulating neutrophils in sham group and 80 days post‐radiation adoptive‐transfer to recipients, and the neutrophils were marked by Dil. (A) The scheme was shown. Representative flow plots and frequency of Dil + neutrophils in (B) circulation and (C) bone marrow, and the frequency of bone marrow (D) CMP cells, (E) CLP cells, (F) macrophages and their (G) polarization state in different groups of recipients. n = 4–7. (H–J) K‐M curve analysis of GAL‐9 high neutrophils‐related genes in (H) GBM, (I) THYM and (J) LGG in TCGA database. p ‐value was shown. Data are presented as mean ± SD, each dot represents an individual animal from at least 2–3 independent experiments that used male and female mice. ** p < 0.01, *** p < 0.001. Statistical analyses were performed using unpaired Student's t ‐test (B–F), two‐way ANOVA (G) and log‐rank test (H–J). p ‐value was shown.

Article Snippet: Intraperitoneal injection (i.p.) of the following drugs: recombinant mouse GAL‐9 (rmGAL‐9) protein (200 μg, R&D Systems #3535‐GA‐050), anti‐GAL‐9 antibody (1 mg, BioXCell#BE0218), or IgG isotype control (1 mg, Abcam #ab172730).

Techniques: Adoptive Transfer Assay

Journal: Aging Cell

Article Title: Galectin‐9 high Neutrophils Exacerbate Radiation‐Induced Frailty

doi: 10.1111/acel.70448

Figure Lengend Snippet: GAL‐9 protein is an important regulatory molecule in neutrophil hyperactivity. (A) Intracellular GAL‐9 protein expression of circulating neutrophils in the sham group and 80 days post‐radiation. n = 6. (B) Circulating GAL‐9 protein level by ELISA kits. n = 8. (C, D) (C) Scheme and (D) the IFN‐γ expression of circulating neutrophils in the sham group under different conditions. n = 3. (E–M) (E) Scheme of the IFN‐γ and NETs expression of circulating neutrophils in the sham group by treatment with (F, H, K) circulating serum at 80 days post‐radiation and (F, I, L) culture supernatant of GAL‐9 high neutrophils. (F, J, M) IFN‐γ and NETs expression of GAL‐9 high neutrophils 80 days post‐radiation under different stimuli. n = 4–5. (N–Q) Assessment of the effect of GAL‐9 protein on the polarization of bone marrow macrophages under different conditions. (N) The scheme and the effect of (O) rmGAL‐9 protein, (P) culture supernatant of GAL‐9 high neutrophils, and (Q) circulating serum at 80 days post‐radiation were shown. n = 3. (R–S) Representative plots and statistics of bone marrow CD47 + neutrophils in the sham group and 80 days post‐radiation. n = 4. (T‐BB) Assessment of the reversal effect of GAL‐9 intervention in mice. (T) Scheme of the administration of anti‐GAL‐9 and rmGAL‐9 proteins in the local radiation group and the sham group, respectively. (U) Circulating GAL‐9 high neutrophils, bone marrow (V) CMP cells, (W) CLP cells, (X) non‐immune cells, (Y) macrophages, and (Z, AA) their polarization state and (BB) frailty index score were shown after different treatments in the local radiation group and the sham group. n = 3–5. Data are presented as mean ± SD; each dot represents an individual animal from at least 2–4 independent experiments that used male and female mice. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analyses were performed using unpaired Student's t ‐test (A, B, S, U), one‐way ANOVA (D, G–M, V–AA), and two‐way ANOVA (O–Q).

Article Snippet: Intraperitoneal injection (i.p.) of the following drugs: recombinant mouse GAL‐9 (rmGAL‐9) protein (200 μg, R&D Systems #3535‐GA‐050), anti‐GAL‐9 antibody (1 mg, BioXCell#BE0218), or IgG isotype control (1 mg, Abcam #ab172730).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Aging Cell

Article Title: Galectin‐9 high Neutrophils Exacerbate Radiation‐Induced Frailty

doi: 10.1111/acel.70448

Figure Lengend Snippet: Skin eccDNA triggers JAK1/2‐STAT1 phosphorylation in splenic GMP cells to produce GAL‐9 high neutrophils. (A) Pathway enrichment of splenic and bone marrow neutrophils at 20 days post‐radiation by DecoupleR analysis. (B) Representative plots of spleen in the sham group and 20 days post‐radiation. (C, D) Phosphorylation levels of JAK1, JAK2, and STAT1 in (C) splenic and (D) bone marrow GMP cells in the sham group and 20 days post‐radiation. n = 4. (E) Pathway enrichment of DNA damage repair pathways in various populations of splenic neutrophils at 20 days post‐radiation by bubble plots. (F–H) Representative plots of eccDNA of (F) spleen, (G) skin, and (H) bone marrow in the sham group, 5, 10, and 20 days post‐radiation by agarose gel. (M), linear DNA marker. (I–M) Assessment of the dosing regimen for extracting different groups of circulating eccDNA for tail‐vein injection into recipients. (I) The scheme and phosphorylation levels of JAK1, JAK2, and STAT1 in (J) splenic and (K) bone marrow GMP cells, (L) circulating GAL‐9 high neutrophils, and (M) splenic GMP cells of recipients injected with Sham‐eccDNA or IR‐eccDNA. n = 4–6. (N–P) Assessment of the co‐culture of splenic GMP cells in the sham group with Sham‐eccDNA or IR‐eccDNA in vitro. (N) The scheme and phosphorylation levels of (O) JAK1, JAK2, and (P) STAT1 in splenic GMP cells under different treatment conditions. n = 5. (Q–S) Assessment of the effect of splenectomy and the sham group. (Q) Circulating GAL‐9 high neutrophils, (R) bone marrow GMP and CLP cells, and (S) Frailty index score after radiation in splenectomy and sham group. n = 4–5. Data are presented as mean ± SD; each dot represents an individual animal from at least 2–4 independent experiments that used male and female mice. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analyses were performed using unpaired Student's t ‐test (except R) and two‐way ANOVA (R).

Article Snippet: Intraperitoneal injection (i.p.) of the following drugs: recombinant mouse GAL‐9 (rmGAL‐9) protein (200 μg, R&D Systems #3535‐GA‐050), anti‐GAL‐9 antibody (1 mg, BioXCell#BE0218), or IgG isotype control (1 mg, Abcam #ab172730).

Techniques: Phospho-proteomics, Agarose Gel Electrophoresis, Marker, Injection, Co-Culture Assay, In Vitro

Journal: Aging Cell

Article Title: Galectin‐9 high Neutrophils Exacerbate Radiation‐Induced Frailty

doi: 10.1111/acel.70448

Figure Lengend Snippet: EccDNA shedding after skin radiation injury activates the JAK1/2‐STAT1 pathway in splenic GMP cells to induce the production of GAL‐9 high neutrophils. This unique population of hyperactive GAL‐9 high neutrophils is identified with characteristics of elevated NETs and IFN‐γ, etc. These neutrophils infiltrate into multiple organs to induce injuries, disrupt the bone marrow microenvironment, drive sustained bone marrow myeloid‐biased differentiation and polarization of bone marrow macrophages towards the M 1 state, and resist clearance by bone marrow macrophages. Highlight the ‘skin‐spleen‐bone marrow‐multiple organs’ axis drives the generation of GAL‐9 high neutrophils to exacerbate frailty.

Article Snippet: Intraperitoneal injection (i.p.) of the following drugs: recombinant mouse GAL‐9 (rmGAL‐9) protein (200 μg, R&D Systems #3535‐GA‐050), anti‐GAL‐9 antibody (1 mg, BioXCell#BE0218), or IgG isotype control (1 mg, Abcam #ab172730).

Techniques:

Journal: The Journal of biological chemistry

Article Title: ATXN3 functions as a tumor suppresser through potentiating Galectin-9-mediated apoptosis in human colon adenocarcinoma.

doi: 10.1016/j.jbc.2024.107415

Figure Lengend Snippet: Figure 5. Targeted ATXN3 deletion inhibits colon cancer growth in mice. A–C, WT or ATXN3-KO HCT116 cells were injected subcutaneously into RAG1 mutant mice (n = 10). Tumor growth curve (A), photograph (B), and weight (C) are shown. D and E, the protein expression of Galectin-9, Ki67, Cleaved Caspase-3 in (B) tumor was detected by IHC and measured relative expression by image J software(n = 5). Scale bar: 200 mm. F–H, HCT116 cells were injected subcutaneously into RAG1 mutant mice and treated with Galectin-9 recombinant protein (n = 10). Tumor growth curve (F), photograph (G), and weight (H) are shown. I and J, the protein expression of Galectin-9, Ki67, Cleaved Caspase-3 in (G) tumor was detected by IHC and measured relative expression by image J software(n = 4). Scale bar: 200 mm. K–M, tumor growth curve (K) of RAG1 mutant mice injected subcutaneously with WT or ATXN3-KO HCT116 cells and stabilize overexpression of Galectin-9 by lentivirus (n = 20). Tumor photograph (L) and tumor weight (M) are shown. A, C, F, H, E, and J, 2- tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001. L and M: ordinary 1-way ANOVA. *p < 0.05, **p < 0.01,***p < 0.001.

Article Snippet: In order to achieve in vivo overexpression of the recombinant protein Galectin-9, mice were administered intraperitoneal injections of Galectin-9 recombinant protein (R&D Systems) every 2 days, with an average dosage of 1 mg per injection.

Techniques: Injection, Mutagenesis, Expressing, Software, Recombinant, Over Expression