Fig 1: Blockade of the Gal-9/Tim-3 pathway ameliorates motor dysfunction and dopaminergic neuronal loss in MPTP-treated mice. (A–C) The pole test, balance beam test, and rotation test were performed in WT, Gal-9 KO, and WT mice pre-administered with Tim-3 antibody. Data are presented as means ± SEM. Two-way ANOVA followed by Tukey’s post-hoc test (n = 8 mice per group). (D,E) Representative immunoblots (D) and quantification (E) of TH in SN lysates from WT, Gal-9 KO, and WT mice pre-administered with Tim-3 antibody. Data are presented as means ± SEM. Two-way ANOVA followed by Tukey’s post-hoc test (n = 4 mice per group). (F) Representative immunostaining of TH in the SNpc of WT, Gal-9 KO, and WT mice pre-administered with Tim-3 antibody. Scale bars, 200 µm for upper panel, 100 µm for magnification. (G) Stereological counts of TH+ neurons in the SNpc. Data are presented as means ± SEM. Two-way ANOVA followed by Tukey’s post-hoc test (n = 4 mice per group). (H) Representative TH staining in the striatum. Scale bars, 500 µm for upper panel, 200 µm for magnification. (I) Quantification of TH immunoreactivity in the striatum. Data are presented as means ± SEM. Two-way ANOVA followed by Tukey’s post-hoc test (n = 4 mice per group). (J–M) The levels of H2O2, CAT, GPx, and MDA in the SN of WT, Gal-9 KO, and WT mice pre-administered with Tim-3 antibody after PBS or MPTP treatment. Data are presented as means ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 4 mice per group). *p < 0.05, **p < 0.005, ***p < 0.0005.
Fig 2: MPP+ promotes microglial activation and Gal-9 expression. (A) The mRNA levels of IL-6, IL-1ß, TNF-a, MIP-1a, and Arg-1 were measured by RT-PCR in BV2 cells treated with MPP+ (500 µM) for 24 h. Data are presented as the means ± SEM. Unpaired Student’s t-test (n = 4 independent experiments). (B) Representative immunoblots and quantification of Gal-9 and Tim-3 in lysates from BV2 cells treated with different concentrations of MPP+ (0, 50, 100, and 500 µM) for 48 h. Data are presented as means ± SEM. One-way ANOVA followed by Tukey’s post-hoc test (n = 4). (C) The mRNA levels of Gal-9 in BV2 cells were assessed by RT-PCR after treatment with MPP+ (500 µM) for 24 h. Data are presented as means ± SEM. Unpaired Student’s t-test (n = 4). (D) Quantification of Gal-9 levels in the culture medium of MPP+-treated BV2 cells by ELISA. Data are presented as the means ± SEM. Unpaired Student’s t-test (n = 3). *p < 0.05, **p < 0.005, ***p < 0.0005.
Fig 3: Gal-9 exacerbates oxidative stress induced by MPP+. (A,B) The levels of ROS and H2O2 in SH-SY5Y cells exposed to MPP+ (500 µM) and Gal-9 (16 nM) for 24 h. n = 4 independent experiments. (C,D) The activity of catalase (CAT) and glutathione peroxidase (GPx) in SH-SY5Y cells exposed to MPP+ or MPP+ plus Gal-9 (n = 4 independent experiments). (E) The levels of malondialdehyde (MDA) in SH-SY5Y cells exposed to MPP+ and Gal-9. Data are presented as means ± SEM. One-way ANOVA followed by Tukey’s post-hoc test (n = 4 independent experiments). *p < 0.05, **p < 0.005, ***p < 0.0005.
Fig 4: CD8+ TILs are major antitumor effector cells in CTCL.a Pie charts show the distribution of TCRaß clonotypes of conventional CD4+ T cells (Tconvs), CD4+ regulatory T cells (Tregs) and CD8+ T cells from the 10× Genomics dataset based on clonal frequency. T cells with TCR clonotype frequency =3 are defined as clonally expanded T cells. b UMAP plots show all reactive CD8+ TILs from the 10× Genomics dataset after re-clustering with the expression of canonical exhaustion markers, including PDCD1, HAVCR2, CTLA4, LAG3, TIGIT and TCF7. The color scale represents normalized expression. Gray to red: low to high expression. c (Left) Pie charts show the distribution of TCRaß clonotypes of reactive CD8+ TILs in the TCyEM and TCM groups based on clonal frequency. T cells with TCR clonotype frequency =3 are defined as clonally expanded T cells. (Right) Bar plot shows the proportion of Exlow and Exhigh CD8+ TILs in the TCyEM and TCM groups. ***p < 0.001. P values were calculated using Pearson’s chi-square test. P = 2.20 × 10-16. d Violin plots show the expression levels of MHC-I molecules, including HLA-A, HLA-B, HLA-C, HLA-E and HLA-F, as well as LGALS9 in malignant T cells in the TCyEM and TCM groups (log2 fold-change >0.5, ***p < 0.001). P = 0 (all genes). e Representative immunofluorescence staining of TOX (green) and LGALS9 (red) on paraffin-embedded tissue samples from representative TCyEM (upper panel) and TCM (bottom panel) patients. DAPI (blue) was used to visualize cell nuclei. Scale bar = 50 µm. Results are representative of three different samples. Source data for (c) are provided in the Source Data file.
Fig 5: Gal-9 enhances neurotoxicity induced by MPP+ in vitro. (A) Cell viability was measured by CCK-8 assay in SH-SY5Y cells treated with PBS, Gal-9 (16 nM, 24 h), MPP+ (500 µM, 24 h), and MPP+ together with Gal-9. Data are presented as means ± SEM. One-way ANOVA followed by Tukey’s post-hoc test (n = 4 independent experiments). (B,C) Representative immunofluorescence images (B) and quantification (C) of apoptotic SH-SY5Y cells by TUNEL assay. Scale bar, 20 µm. Data are presented as means ± SEM. One-way ANOVA followed by Tukey’s post-hoc test (n = 4 independent experiments). (D,E) Representative images (D) and quantification (E) of TUNEL staining of primary cortical neurons treated with MPP+ (100 µM) and Gal-9 (16 nM) for 12 h. Scale bar, 20 µm. Data are presented as means ± SEM. One-way ANOVA followed by Tukey’s post-hoc test (s = 4 independent experiments). (F) Schematic for transferring conditioned medium into primary neurons. (G) The apoptosis of primary neurons cultured for 24 h with conditioned medium was detected by TUNEL assay. Scale bar, 20 µm. (H) Quantification of apoptotic primary cortical neurons cultured with conditioned medium. Data are presented as means ± SEM. One-way ANOVA followed by Tukey’s post-hoc test (n = 4 independent experiments). *p < 0.05, **p < 0.005, ***p < 0.0005.
Supplier Page from Abcam for Anti-galectin 9/Gal-9 antibody [EPR22214]