Journal: bioRxiv

Article Title: Single-cell profiling and zebrafish avatars reveal LGALS1 as immunomodulating target in glioblastoma

doi: 10.1101/2023.04.27.538517

Figure Lengend Snippet: A Volcano plot depicting differentially expressed genes in LBT003 and all other GSCCs (Left: downregulated genes in LBT003; right: upregulated genes in LBT003.) Y-axis denotes -log10(adjusted p -value) while x-axis shows log2(avgFC) values. Cut-offs were set at -log10(adjusted p -value) > 1.3 and abs(log2(avgFC)) > 1.5. B Violin plot showing LGALS1 expression levels in GSCCs. C Representative double immunofluorescence images showing co-expression of SOX2 (magenta) and GAL1 (cyan) in GBM tissue from LBT003 and LBT070. For enhanced visualization, a binary mask was generated from the SOX2 + cells and multiplied with the image of GAL1 staining in Fiji to exclude GAL1 staining in non-tumor cells. Scale bars: 100 μm. D Mean fluorescence intensity values for GAL1 staining in SOX2 + cells in GBM tissue samples, normalized using Z -scores within each sample. n = 6 tumor samples derived from 5 different patients. E Representative maximum-projections of a z stack of the head region of a Tg(mpeg1:mCherryF) ump2 ; Tg(kdrl:lynEYFP) zebrafish embryo with a GFP-labeled LBT070 LGALS1 KO tumor, at 1 dpi (left panel) and 5 dpi (right panel). GBM tumor cells are shown in green, macrophages in red, and blood vessels in blue. Scale bars: 50 μm. F Tumor volume at the start of 1 dpi and 5 dpi time-lapse movies. ( n = 5-12 zebrafish embryos per group, with an average of 9.5 embryos per group). G Number of round macrophages at the start of 1 dpi and 5 dpi time-lapse movies. ( n = 5-12 zebrafish embryos per group, with an average of 9.5 embryos per group). H Number of ramified macrophages at the start of 1 dpi and 5 dpi time-lapse movies. ( n = 5-12 zebrafish embryos per group, with an average of 9.5 embryos per group). I Boxplot of macrophage distance to the tumor of round macrophages within 30 μm of the tumor, at the start of 1 dpi and 5 dpi time-lapse movies. ( n = 39-290 macrophages per group, with an average of 128.5 macrophages per group; boxes stand for 50% of the data and minima/maxima are indicated by the line ends). J Boxplot of macrophage distance to the tumor of ramified macrophages within 30 μm of the tumor, at the start of 1 dpi and 5 dpi time-lapse movies. ( n = 13-40 macrophages per group, with an average of 25.5 macrophages per group; boxes stand for 50% of the data and minima/maxima are indicated by the line ends). Data information: In F-H, data are presented as mean ± SD, and the p -values were calculated by mixed-effects model, followed by Šidák’s multiple comparisons correction. ns ≥ 0.05, * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. In I+J, the p -values were calculated by two-way ANOVA, followed by pairwise testing with Benjamini-Hochberg correction.

Article Snippet: LBT070 cells and LBT070 LGALS1 KO cells were stained with anti-GAL1 antibody (Cell Signaling Technology, 13888, 1:375) and staining was evaluated using the Operetta High Content Imaging System (PerkinElmer) (See Fig S8C).

Techniques: Expressing, Immunofluorescence, Generated, Staining, Fluorescence, Derivative Assay, Labeling

Journal: iScience

Article Title: Pro-α-cell-derived β-cells contribute to β-cell neogenesis induced by antagonistic glucagon receptor antibody in type 2 diabetic mice

doi: 10.1016/j.isci.2022.104567

Figure Lengend Snippet: Immunofluorescent analysis of α-cell regression to the progenitor state in the pancreatic tissues of T2D mice treated with GCGR mAb or IgG control for 4 weeks (A–C) Representative photograph showing β-gal (α-cell lineage-tracing marker) + Ngn3 + cells (A), β-gal + Glut2 + cells (B), and β-gal + Pdx1 + cells (C) in α-cell lineage-tracing T2D mice that were induced by HFD + STZ. The arrows indicate co-labeled cells. The cells in the small box are enlarged at the right of the image. Scale bar = 50 μm. See also Figures S5 .

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-glucagon (1:800; Cell Signaling Technology, Boston, MA, USA; RRID: AB_659831), mouse monoclonal anti-glucagon (1:400; Sigma-Aldrich; RRID: AB_259852), mouse monoclonal anti-insulin (1:800; Sigma-Aldrich; RRID: AB_260137), rabbit monoclonal anti-cytokeratin 19 (1:200; Abcam, Cambridge, UK; RRID: AB_2281020), rabbit polyclonal anti-RFP (1:200; Abcam; RRID: AB_945213), rabbit polyclonal anti-β-gal (1:100; Abcam; RRID: AB_2920785), mouse monoclonal anti-Ngn3 (1:50; Santa Cruz, CA, USA; RRID: AB_ 10988579), mouse monoclonal anti-Glut2 (1:100; Santa Cruz; RRID: AB_2890905), rabbit polyclonal anti-Pdx1 (1:200; Abcam; RRID: AB_777179), rabbit monoclonal anti-Nkx6.1 (1:400; Abcam; RRID: AB_2754979), rabbit polyclonal anti-PC1/3 (1:400; Millipore, Darmstadt, Germany; RRID: AB_1977441), rabbit polyclonal anti-C-peptide (1:400; Cell Signaling Technology; RRID: AB_10691857), rabbit anti-GCGR (Proteintech, Rosemont, IL, USA; RRID: AB_2880634).

Techniques: Control, Marker, Labeling

Journal: iScience

Article Title: Pro-α-cell-derived β-cells contribute to β-cell neogenesis induced by antagonistic glucagon receptor antibody in type 2 diabetic mice

doi: 10.1016/j.isci.2022.104567

Figure Lengend Snippet: Immunofluorescent analysis of α-to-β cell conversion in the pancreatic tissues of T2D mouse models treated with GCGR mAb or IgG control for 4 weeks (A and B) Quantification of glucagon + insulin + cells in db/db mice (A) and HFD + STZ-induced T2D mice (B) as shown in ( Figures 2 A and 2C). (C and D) Representative image of an islet immunostained with β-gal (α-cell lineage-tracing marker) and insulin (C), and quantification of β-gal + insulin + cells (D) in α-cell lineage-tracing T2D mice that were induced by HFD + STZ. The arrows indicate co-labeled cells. The cells in the small box are enlarged at the right of the image. Scale bar = 50 μm. n = 5 sections/mouse multiplied by 6 mice/group in db/db mice, n = 3 sections/mouse multiplied by 9 mice/group in HFD + STZ-induced T2D mice, and n = 3 sections/mouse multiplied by 5 mice/group in the α-cell lineage-tracing T2D mice. Data represent the mean ± SEM. Statistical analysis was conducted by Student’s t -test. ∗∗∗p < 0.001 vs. control. See also Figures S5 , , and .

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-glucagon (1:800; Cell Signaling Technology, Boston, MA, USA; RRID: AB_659831), mouse monoclonal anti-glucagon (1:400; Sigma-Aldrich; RRID: AB_259852), mouse monoclonal anti-insulin (1:800; Sigma-Aldrich; RRID: AB_260137), rabbit monoclonal anti-cytokeratin 19 (1:200; Abcam, Cambridge, UK; RRID: AB_2281020), rabbit polyclonal anti-RFP (1:200; Abcam; RRID: AB_945213), rabbit polyclonal anti-β-gal (1:100; Abcam; RRID: AB_2920785), mouse monoclonal anti-Ngn3 (1:50; Santa Cruz, CA, USA; RRID: AB_ 10988579), mouse monoclonal anti-Glut2 (1:100; Santa Cruz; RRID: AB_2890905), rabbit polyclonal anti-Pdx1 (1:200; Abcam; RRID: AB_777179), rabbit monoclonal anti-Nkx6.1 (1:400; Abcam; RRID: AB_2754979), rabbit polyclonal anti-PC1/3 (1:400; Millipore, Darmstadt, Germany; RRID: AB_1977441), rabbit polyclonal anti-C-peptide (1:400; Cell Signaling Technology; RRID: AB_10691857), rabbit anti-GCGR (Proteintech, Rosemont, IL, USA; RRID: AB_2880634).

Techniques: Control, Marker, Labeling