Fig 2: Effect of overexpression of wild-type APPL1 on cell survival and proliferation in DAT-treated HepG2 cells.(A) Overexpression of wild-type APPL1 on cell viability. (B) Overexpression of wild-type APPL1 on cell proliferation. (C) Representative images of cell apoptosis by overexpression of wild-type APPL1 (×400). (D) Quantitative analysis of cell apoptosis in (C). (E) Impact of overexpression of wild-type APPL1 on cleaved caspase-3 levels. APPL1, adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1; DAT, diallyl trisulfide. **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. DAT-treated control group.

Fig 3: Transient formation of actin domain structures upon insulin treatment in H4IIEC3 cells(A) After transfection of the LifeAct-EGFP plasmid and serum starvation, live-cell time-lapse imaging of H4IIEC3 upon insulin treatment was performed. Images were acquired every 3 min after 1 μM insulin stimulation. Arrows indicate the actin structures. In the lower column, the period when the structures were observed in each cell are shown as arrows. Bar = 5 μm. (B) Serum-starved H4IIEC3 cells were stimulated with 1 μM insulin for 15 min, fixed, permeabilized, and stained with antibodies against Akt, phosphorylated Akt at T308 (Akt pT308), phosphorylated Akt at S473 (Akt pS473), phosphorylated GSK3β at S9 (GSK3β pS9), mTOR, phosphorylated mTOR at S2448 (mTOR pS2448), phosphorylated Erk at T202 and Y204 (Erk pT202Y204), EEA1, APPL1, phosphorylated insulin receptor at Y999 (IR pY999), or phosphorylated insulin receptor at Y1185 (IR pY1185) (green) and rhodamine-conjugated phalloidin (Actin, orange). Bar = 5 μm.

Fig 4: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in aged mouse plasma and myocardial tissue. (A) Relative expression levels of p16 and p21 in myocardial tissues of young mice (aged 2 months) and aged mice (aged 15 months) were detected by western blotting. (B) Relative mRNA expression levels of adiponectin, AdipoR1 and APPL1 in the plasma of young mice and aged mice were detected by reverse transcription-quantitative PCR. (C) Relative expression levels of adiponectin, AdipoR1 and APPL1 in myocardial tissues of young mice and aged mice were analyzed using western blot analysis. **P<0.01, ***P<0.001 vs. young mice. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1.

Fig 5: Effect of overexpression of wild-type APPL1 on phosphorylation of STAT3, Akt, and Erk1/2 in DAT-treated HepG2 cells.(A) Representative Western blot images. (B) Quantitative analysis of the ratio of phosphorylated proteins to the related-proteins. APPL1, adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1; DAT, diallyl trisulfide; OE, overexpression; Endo, endogenous; WT, wild type. **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. DAT-treated control group.