Fig 2: Enhanced aortic ?-secretase levels with elastin deficiency.(A and C) Lysates were analyzed by Western blotting for ?-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 a-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 3: RO4929097 (RO) inhibits TGFß and Notch signalling in Müller cells stimulated by TGFß1. Müller cells were cultured in normal (control, Ctl) and test media supplemented with recombinant human TGFß1 (10 ng/ml), with or without RO (10 µM) for 18 hours. (A and B) TGFß1 treatment upregulated expression of TGFß receptors including TGFß R1 and 2, endogenous TGFß1 (A) and p-Smad3 (B) while this effect was significantly inhibited by RO. (C and D) TGFß1 also significantly upregulated expression of ?-secretase proteinases including nicastrin, presenilin 1 and 2, presenilin enhancer 2 (PEN2) as well as Notch downstream effectors including Hes1 and Hes5, while this effect was inhibited by RO treatment. *P<0.05, **P<0.01 and ***P<0.001, vs control (Ctl). †P<0.05, ‡P<0.01 and #P<0.0001 vs the corresponding groups without RO treatment. N=4/group. Independent repeats=2.

Fig 4: RO4929097 (RO) inhibits Notch and TGFß signalling in Müller cells stimulated by Notch ligands. MIO-M1 human Müller cells were cultured in normal (control, Ctl) and test media containing Notch ligands including Dll4 and Jagged1 (both 50 ng/ml), either with or without RO (10 µM) for 18 hours. (A) Treatment of Müller cells with Dll4 and Jag1 upregulated the expression of ?-secretase proteinases including nicastrin, presenilin 1 and 2 as well as presenilin enhancer 2 (PEN2), all of which were significantly inhibited by the selective ?-secretase inhibitor RO. (B and C) Treatment of Müller cells with Dll4 and Jag1 also upregulated the expression of TGFß1, TGFß receptors 1 and 2 (TGFß R1 and TGFß R2, (B) and p-Smad3 (C), all of which were significantly inhibited by RO treatment. *P<0.05, **P<0.01 and ***P<0.001, vs control (Ctl). †P<0.05, ‡P<0.01 and §P<0.001 vs the corresponding groups without RO treatment. N=4/group. Independent repeats=2.

Fig 5: Inhibition of expression of components of the ?-secretase complex by cowanin (2) in HPB-ALL cells. (A) Schematic representation of ?-secretase complex. ?-Secretase is composed of four membrane proteins: presenilin1 (PS1), nicastrin (NCT), anterior pharynx defective 1 (APH-1) and presenilin enhancer 2 (PEN-2). (B,C) Western blot analysis of nicastrin, APH-1, PS1 and PEN-2 expression in HPB-ALL cells after treatment with cowanin (2). HPB-ALL cells (1 × 106 cells/dish) were incubated in a 6-cm dish for 72 h at 37 °C with 2. The effect on protein expression was analyzed by Western blotting. ß-Actin was used as internal control. (D) MG132 abrogated the reduction of nicastrin expression by cowanin (2). HPB-ALL cells (1 × 106 cells/dish) were incubated in a 6-cm dish for 12 h at 37 °C with 2. The effect on protein expression was analyzed by Western blotting. ß-Actin was used as internal control. (E,F) Relative protein levels of nicastrin mature and immature analyzed by densitometry. Data are presented as the mean with SD from three independent experiments. *P < 0.05 and **P < 0.01 are considered statistically significant. P values were analyzed by Student’s t test.