Fig 1: MGST3-mediated regulation of BACE1 is dependent on RGS4.A–C, analysis of immunofluorescent images of MGST3 (red) and RGS4 (green) in SH-SY5Y cells transfected with the scrambled control (siNC) or MGST3 siRNA (siMGST3) for 48 h (100× objective lens, 10× eyepieces, the scale bar represents 1 μm, n = 9). MSGT3 and RGS4 are relatively colocalized in some area (A and B), whereas RGS4 signal is significantly reduced by MSGT3 knockdown (C). D and E, representative Western blots (D) and quantification (E) of RGS4 in SH-SY5Y cells transfected with the scrambled control (siNC) or MGST3 siRNA (siMGST3) for 48 h (n = 3 independent experiments). F and G, representative Western blots (F) and quantification (G) of BACE1 protein levels in SH-SY5Y cells transfected with the scrambled control (siNC) or MGST3 siRNA (siMGST3) for 24 h, followed by treatment of dimethyl sulfoxide or RGS4 inhibitor CCG50014 (30 nM) for 24 h (n = 3 independent experiments). H and I, representative Western blots (H) and quantification (I) of BACE1 protein levels in SH-SY5Y cell scoped with the scrambled control (siNC) or MGST3 siRNA (siMGST3) cotransfected with RGS4 siRNA (siRGS4) for 48 h (n = 3 independent experiments). Data are expressed as means ± SD. n.s. no significant difference. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. BACE1, beta-site amyloid precursor protein cleaving enzyme 1; MGST, microsomal glutathione transferase 3; RGS4, regulator of G-protein signaling 4.
Fig 2: Antiviral agents reduce the HSV1-induced increases in β-amyloid-related enzymes.Vero cells were infected with HSV1 SC16 at an MOI of 1 for 16 hours. Cells were treated with 0 µM, 50 µM, 100 µM or 200 µM acyclovir (ACV), penciclovir (PCV) or foscarnet (FOS) which was present throughout infection. After fixation the slides were tested for (A) β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and (B) nicastrin. Images for no antiviral, 50 µM ACV, 50 µM PCV and 200 µM FOS are shown. Scale bar: 50 µm.
Fig 3: Effect of Humulus japonicus (HJ) on APP processing and degrading enzyme. Western blot analysis and quantitative analysis for (A–E) APP and APP metabolites and (F–I) a disintegrin and metalloproteinase 10 (ADAM10), β-site APP-cleaving enzyme 1 (BACE1), neprilysin (NEP) and insulin degrading enzyme (IDE) in the cortex homogenates of non-Tg control (Non-Tg), vehicle-treated APP/PS1 transgenic (Tg-APP/PS1) control (Tg-Con), and HJ-treated Tg-APP/PS1 (Tg-HJ) mice. *p<0.05, significant differences from Tg-control, as shown by the Student's t-test. Data are presented as the means ± SEM.
Fig 4: AP2S1 knockdown reduces APP protein and promotes LE‐lyso fusion in APP/PS1 mice. (A) Schematic diagram shows the time course of animal manipulations. (B) Representative immunofluorescent images show GFP‐tagged AAVs are successively delivered to the hippocampus. Scale bar: 100 μm. (C) Representative Western blots (top) and quantification (bottom) of APP, sAPPβ, BACE1, AP2S1 and CTFs protein levels in the hippocampus. WT‐C/WT‐S, wild‐type mice injected with AAV control or AP2S1 shRNA; AD‐C/AD‐S, APP/PS1 mice injected with AAV control or AP2S1 shRNA (n = 5 for each group). (D) Representative immunofluorescent images show APP protein intensity in the hippocampus of WT‐C, WT‐S, AD‐C and AD‐S (n = 4 for each group). Green: APP; Blue: DAPI (nuclear marker). Scale bar: 75 μm. (E) Representative immunofluorescent image and quantification of Aβ intensity in AD‐C and AD‐S (n = 3 for each group). Red: Aβ; Blue: DAPI (nuclear marker). Scale bar: 75 μm. (F) The soluble and insoluble Aβ42 and Aβ40 levels in the hippocampus of AD‐C and AD‐S, respectively (n = 5 for each group). (G and H) Representative immunofluorescent images and quantification, which show the colocalization of APP with Rab9/Lamp1 in the hippocampus of AD‐C and AD‐S (n = 4 for each group). Scale bar: 20 μm. (I) Representative transmission electron micrographs of the hippocampus of AD‐C and AD‐S (left). Quantification per cell profile that exhibits an increase in average number of endo‐lysosomes fusion in AD‐S are shown on the right (n = 3 for each group). LE, late endosome; LY, lysosome; N, nucleus. Scale bar: 1 μm. n.s., non‐significant, *p < 0.05, **p < 0.01
Fig 5: Motor reflex behavior. A) Climbing assay measurement of motor reflex behavior of elav; APP; BACE heterozygous flies, as compared to elav; +; + heterozygous flies and w; APP:BACE heterozygous flies. Parental strains indicated. B) Modulation of elav; APP:BACE heterozygous flies' motor reflex behavior by γ-secretase inhibitor, L-685,458. Genotypes and treatment indicated. * indicates p<0.05. n = 50–100 flies. Error bars represent standard error.
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