Fig 2: ARV-825 and JQ1 upregulate BACE1 levels in H4-APP751 cells. Cells were treated with ARV-825 (nM; 4, 20, 100, 500, and 2500) or JQ1 (nM; 4, 20, 100, 500, and 2500) for 18 h. Cell lysates were collected and applied to WB to detect BACE1 and NCT levels, with β-actin utilized as a control protein. A and B, WB analysis for effects of ARV-825. C and D, WB analysis for effects of JQ1. Mean ± SEM; n = 3; one-way ANOVA followed by Dunnett’s test; ∗/∗∗/∗∗∗/∗∗∗∗ showed significance; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗, p < 0.0001. NCT, nicastrin.

Fig 3: Enhanced aortic γ-secretase levels with elastin deficiency.(A and C) Lysates were analyzed by Western blotting for γ-secretase subunits NCT, PSEN1, PSEN2, and PEN2 and for GAPDH. In A, haSMCs were pretreated with Scr or siELN RNA, and in C, aortas of 2 WT or Eln–/– mice at P0.5 were pooled. (B and D) Densitometry of protein bands in A and C relative to GAPDH and normalized to Scr in B or WT in D (n = 3). *P < 0.05; **P < 0.01 by Student’s t test vs. Scr (B) or WT (D). Data are averages ± SD. (E and F) Ascending aortic cross sections of WT and Eln–/– mice at P0.5 stained for α-smooth muscle actin (SMA, marker of SMCs), nuclei (DAPI), and either PSEN1 in E or PSEN2 in F. n = 3 mice. Lu, lumen. Scale bars: 25 μm.

Fig 4: Analysis of pathological Tau proteins by JQ1 in 3D-AD human neural cell culture model. The 3D-AD human neural culture cells were differentiated for 5 weeks and then treated with JQ1 (250 nM and 500 nM) or vehicle (DMSO) for 2 weeks and applied to AD-related protein analysis, including WB (A–C), MSD-Aβ (D–E) and MSD-Tau for total and p-Tau (Thr231)(F) and WB for p-Tau (Ser396) (G–H). A–C, WB analysis for APP (by G12A antibody), BACE1, NCT and β-actin (as a control protein) in cell lysates and sAPPα/β proteins in medium. n = 5 to 6. D, MSD-Aβ analysis for soluble Aβ levels in medium. n = 5. E, MSD-Aβ analysis for insoluble Aβ42 from the matrix-gel extraction. n = 4. F, MSD-Tau analysis for Tau-/p-Tau (Thr231) levels in cell lysates. n = 4. G–H, WB analysis for p-Tau (Ser396) levels in cell lysates. The antibodies for p-Tau (Ser396) and β-actin (as a control protein) were utilized. n = 3. Mean ± SEM; ∗/∗∗/∗∗∗∗ showed significance; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗, p < 0.0001. NCT, nicastrin.

Fig 5: Results of biological activity assays. (A) The morphology of the U251-APP cells treated with or without compounds (5 μM or 20 μM), gemfibrozil (Gem, 50 μM, a positive control), or Dinacilib (Dina, 2.5 μM, a positive control) for 24 h. (B) Level of extracellular Aβ42 in the culture medium of U251-APP cells treated with compounds, Gem, or DMSO (control), determined by ELISA. (C–E) Levels of pTau217, pTau396 and pTau181 in the U251-APP cells treated with compounds, Dina, or DMSO (control) determined by ELISA. (F–K) Western blot assays showing the protein levels of CDK5, pCDK5, and GSK3β in the U251-APP cells treated with or without compounds. A representative Western blot result (F,H,J) and quantification of protein levels (G,I,K) based on three independent experiments. (L–Q) Western blot assays showing the protein levels of BACE1, NCSTN, PSEN2, and PSEN1 in the U251-APP cells treated with or without compounds. A representative Western blot result (L,N,P) and quantification of protein levels (M,O,Q) based on three independent experiments. (R) A proposed potential role of 1 against AD by downregulating BACE1, NCSTN, CDK5, and GSK3β-mediated pathways, resulting in Aβ42 reduction and decreased pTau217. Data are presented as the means ± SD; ns, not significant; ***, p < 0.001; **, p < 0.01; and *, p < 0.05; one-way ANOVA with Bonferroni’s post hoc test.