Fig 1: Screen BACE2 as a candidate of csGRP78 downstream molecule. a Heat map shows the differential proteins acquired by mass spectrometry. Anti-GRP78 versus Isotype, blue indicates downregulation while red indicates upregulation. b mRNA profile distinguishes PN GBMs from MES GBMs in TCGA database. c Compared downregulated proteins in anti-GRP78 versus isotype with upregulated genes in MES-subtype GBMs, overlapping total 6 genes. d Statistical analysis for differential expression of these 6 candidates in mRNA profiling of MES and PN GSCs (GSE67089). e BACE2 mRNA expression in normal tissues, low-grade glioma (LGG) and GBM from TGCA database. f BACE2 mRNA expression in TCGA database grouped by subtype. g Survival curve and log-rank test for GBM patients stratified by the BACE2 expression from TCGA database. h GSEA indicates expression of BACE2 positively correlated with MES subtype, while negatively with PN subtype. i GSEA shows BACE2 has the strong correlation with NF-κB pathway. j Correlation between expression of BACE2 with selected MES and PN signatures in TCGA database. k Correlation analysis for expression of BACE2 and GRP78 in TCGA database. l western blot analysis for BACE2 in GSC11, 8–11,20 and 267. *p < 0.05, **p < 0.01, ***p < 0.001
Fig 2: Plasma sVEGFR3 is a superior marker for BACE2 activity.(A) Plasma sVEGFR3 and (B) sSEZ6L levels in mice, fed with diet supplemented with 0 (black), 0.002%, and 0.1% of verubecestat (blue) (n = 4). (C) Photographs of the fur pigmentation of the corresponding mice. (D and E) Relative ELISA quantifications of sVEGFR3 in plasma and CSF Aβ40 as well as Aβ42 levels of (D) verubecestat-treated NHPs (n = 4) before and after treatment and (E) clinical trial participants treated with atabecestat (n = 9) or placebo (n = 4). Human Aβ1-40 (Aβ40) and Aβ1-42 (Aβ42) CSF data for the selected individuals were extracted from a previous publication (41). NHP data were normalized to the predose mean; human data are expressed as postdose/predose ratio for each individual. All dot plots were normalized on the control or predose mean, respectively, and (A and B) depict the SD alongside the calculated P values, calculated by 1-way ANOVA, followed by Bonferroni’s multiple-comparison test (A and B), paired t test (D), or unpaired t test (E). *P < 0.05; **P < 0.01; ****P < 0.0001.
Fig 3: Identification of VEGFR3 as a BACE2 substrate candidate.(A) Volcano plot of proteomic analysis of murine plasma from WT and B2KO mice (n = 6). VEGFR3 (FLT4) is highlighted in red. (B) Normalized VEGFR3 LFQ intensities extracted from A. (C) MSD-assay quantifications of sVEGFR3 in the same plasma samples. (D) Immunoblot detection of sVEGFR3 ectodomain in mouse plasma from A, using nonreducing and reducing conditions. (E) Volcano plot of proteomic analysis of murine plasma from an independent B2KO line (n = 9) compared with WT (n =9) and (F) the extracted normalized LFQ values. Volcano plots of the proteomic analyses of Bace1/Bace2 double-knockout (BDKO) mice (n =9) compared with the WT line (n = 9) (G) (corresponding extracted LFQ intensities of sVEGFR3 in F) and B1KO (n = 9) compared with an individual control WT line (n = 9) (H). (I) Normalized LFQ values extracted from H. (J) Localization of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The signal peptide is shown in rose, the ectodomain is indicated in blue, the intracellular domain in green, and the transmembrane domain in yellow. Two sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots (A, E, G, and H). Proteins with P < 0.05 are shown as red circles. Extracted LFQ quantifications (B, F, and I) of VEGFR3 with significance after FDR correction are labeled with plus signs. All dot plots were normalized on the WT mean and depict mean and SD. MSD-assay data (C) additionally depicts the P value calculated by unpaired t test. ****P < 0.0001.
Fig 4: Expression of BACE2 in the hippocampus of the brains of wild-type and APP/PS1mice after 12 weeks of exposure to fluoride. WT, wild-type; LF, low fluoride; HF, high fluoride. The levels were determined by Western blotting and are the values presented are means ± SD (n = 10). *P < 0.05 in comparison to WT control; #P<0.05 in comparison to APP/PS1 mice, as determined by analysis of variance (ANOVA), followed by least significant differences post hoc test
Fig 5: Knockdown of BACE2 in MES GSCs restrains stemness and radioresistance. a Western blotting analysis for MES-related signatures CD44, YKL40 and pathways p65, p-p65 and C/EBPβ after silencing BACE2 in GSC20 and 267. b Left, images of spheres formation assay after GSC20 and 267 transfected with lentiviral shBACE2 or shNT and right, results were quantified with diameter of tumor spheres. Scale bar, 500 μm. c In vitro limiting dilution assay for GSC20 and 267 expressing lentiviral shBACE2 or shNT. d Cell cycle analysis for G2/M phase cells of GSC20 and 267 in control, 3 Gy radiation only or combined with shBACE2 groups. e Flowcytometry detected apoptosis of GSC20 and 267 in control, 3 Gy radiation only or combined with shBACE2 groups. f Bioluminescence imaging for xenografts grouped as control, shBACE2 and/or radiation at the indicated time points. The radiation was delivered at day 7 after GSCs implantation. g Survival curve for each group, and the statistical significance was tested by log-rank test. h Represented images of H&E staining in each group. The magnification was marked in blue number. Error bar indicates at least three independent experiments and data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001
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