Fig 2: A. annua extract rescued neuronal cell apoptosis in 3xTg AD mice via regulating Hippo/YAP signaling. (A) Apoptosis determined by TUNEL staining in the cortex (scale bars 50 μm). (B) Quantification of western blot form (A). (C) The expression of Bax, Bcl2, caspase 3 and cleaved caspase-3 and GAPDH was detected by Western blot. (D) Quantification of western blot from (C). (E) Western blot was used to assess the expression of p-YAP, YAP, EDTA2, GAPDH. (F) Quantification of western blot from (E). (G) The downstream proteins expression of YAP such as Survivin and PML was detected by western blot. (H) Quantification of western blot from (G). (I) The upstream proteins expression of YAP including p-MST1(Ser410), p-LAST1/2(Ser909/872), p-MOB1(Thr35) and GAPDH was assessed by western blot. (J) Quantification of western blot from (I) (* p < 0.05, ** p < 0.005 and *** p < 0.0005; # p < 0.05 and ## p < 0.005 were considered statistically significant; ns was not considered significantly different. * Representative comparison between 3xTg-Ctrl and WT-Ctrl; # representative comparison between 3xTg-Ctrl and 3xTg+extract).

Fig 3: AXL promotes Yes‐associated protein (YAP) nuclear translocation and transcriptional regulation via dephosphorylation. A, P‐YAP was detected after AXL plasmid transfection for 48 h. B, P‐YAP was detected after treatment with 2% FBS for 12 h and rhGas6 for 1 h. C, After AXL inhibitor BGB324 treatment for 24 h, p‐YAP expression was assessed. D, Cells were treated with 2% FBS for 12 h and 1000 ng/mL rhGas6 for 1 h. Confocal immunofluorescence assay was used to visualize YAP localization. Scale bars: 20 μm. E, The localization of YAP was assessed after treatment with 2 μmol/L BGB324 for 24 h. Scale bar: 20 μm. F, Nuclear and cytoplasmic YAP proteins were separated and analyzed after treatment with 2% FBS for 12 h and 1000 ng/mL rhGas6 for 1 h. G, YAP expression in nucleus and cytoplasm were analyzed after 2 μmol/L BGB324 treatment for 24 h. H, Nuclear and cytoplasmic YAP were determined after si‐AXL transfection with 2% FBS for 48 h and 1000 ng/mL rhGas6 treatment for 1 h. I, CCN2 and AREG relative mRNA expression was measured after AXL plasmid transfection for 24 h. J, CCN2‐promoter activities were detected after cotransfection of si‐YAP with 2% FBS for 24 h and 1000 ng/mL rhGas6 treatment for 1 h. K, CCN2‐promoter activities were analyzed after cotransfection of YAPWT or YAPS127A plasmid with 2% FBS for 24 h and 1000 ng/mL rhGas6 treatment for 1 h. L, P‐LATS1 and p‐MOB1 were determined after treatment with 2% FBS for 12 h and 1000 ng/mL rhGas6 for 1 h. *P < .05, **P < .01, ***P < .001, according to Student t test

Fig 4: Human MOB1(wt) or MOB1(K104/K105E), but not MOB1(D63V) or MOB1(D63V/K104E/K105E), expression suppresses elevated Yki activity in mats null mutant wing imaginal disk clones. Analysis of Ex expression in wing imaginal disks of the indicated genotypes (wt wild-type, DV D63V, KE K104E/K105E, DVKE D63V/K104E/K105E). All panels show third instar wing imaginal disks containing mats mutant clones marked by the absence of GFP expression (green in b, g, l, q, v). GFP-negative mats null clones are indicated by dashed lines. Maximum intensity projections of XY confocal stacks are shown in red to visualize Expanded (Ex) labeling (red in a, f, k, p, u). X–Z confocal sections through the same wing imaginal disks were acquired c, h, m, r, w. Nuclei are stained with DAPI in blue. Ex (d, i, n, s, x) and GFP (e, j, o, t, y) protein levels are indicated. All scale bars are 20 µm. As expected, mats null clones displayed increased Yki activity as judged by elevated Ex levels a–e. Expression of MOB1(wt) f–j or MOB1(K104E/K105E) p–t suppresses Yki activity to normal levels in mats mutant clones (compare i, s with d), while expression of MOB1(D63V) k–o or MOB1(D63V/K104E/K105E) u–y does not (compare n, x with d and i)

Fig 5: A. annua extract’s protective effect occurs via regulating YAP signaling in PC12 cells. PC12 cells were treated with (30 μg/mL, 100 μg/mL, 300 μg/mL and 1000 μg/m) extract for 24 h. (A) Western blot analysis of YAP, P-YAP2 and GAPDH expression. (B) Quantification of A, extract increased the expression of YAP and decreased the phosphorylation of YAP in a dose-dependent manner. (C) Immunofluorescence of YAP (scale bars 5 μm). (D) The expression of TEAD2, Survivin, PML and GAPDH were detected by western blot. (E) Quantification of western blot from (D), extract promoted the expression of TEAD2 and Survivin in PC12 cells, and decreased the expression of PML. (F) Western blot analysis of p-MST1(Ser410), p-LATS1/2(Ser909/872) and p-MOB1(Thr35) expression. (G) Quantification of western blot from (F), extract inhibited the phosphorylation of MST1 and LATS1/2, while no effect the expression of p-MOB1(Thr35). (* p < 0.05, ** p < 0.005 and *** p < 0.0005 were considered statistically significant. * Representative comparison between Ctrl group and extract groups). (H) PC12 cells were incubated with 8 μM Aβ1–42 with or without extract at different concentrations for 24 h. Western blot analysis of the effect of extract on YAP, p-YAP and GAPDH. (I) Quantification of western blot from (H). (J) Immunofluorescence of YAP expression (scale bars 20 μm and scale bars 5 μm). (K) Western blot analysis of p-MST1(Ser410), p-LATS1/2(Ser909/872), p-MOB1(Thr35) and GAPDH expression. (L) Quantification of western blot from (K). (M) Western blot analysis of TEAD2, Survivin, PML and GAPDH. (N) Quantification of western blot from (M). (* p < 0.05, ** p < 0.005 and *** p < 0.0005; # p < 0.05, ## p < 0.005 and ### p < 0.0005 were considered statistically significant; ns was not considered significantly different. * Representative comparison between Ctrl and Aβ1–42, # representative comparison between Aβ1–42 and extract).