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: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in D-gal-treated cardiomyocytes. (A) Relative expression levels of p16 and p21 in H9c2 cells treated with 2.5, 5 and 10 g/l of D-gal were analyzed using western blot analysis. *P<0.05, **P<0.01, ***P<0.001 vs. control. Transfection with Ov-adiponectin promotes the expression of AdipoR1 and APPL1 in D-gal-treated cardiomyocytes. (B) Relative mRNA expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed via RT-qPCR. ***P<0.001 vs. control; ###P<0.001 vs. D-gal + Ov-NC. (C) Relative protein expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed using RT-qPCR. ***P<0.001 vs. control; ##P<0.01 vs. D-gal + Ov-NC. (D) Relative mRNA expression levels of AdipoR1 and APPL1 in H9c2 cells before and after transfection with Ov-adiponectin plasmids were detected using RT-qPCR. ***P<0.001 vs. control; ###P<0.001 vs. D-gal + Ov-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; D-gal, D-galactose; Ov-, overexpression.

Fig 4: 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.

Fig 5: Radiation enhances the expression of key proteins in the physiological endocytosis pathway, possibly mediating intracellular delivery of morpholino into cancer cells. (a) Immunoblot of T98G cells showing that radiation affected the levels of endocytosis-related proteins caveolin-1, clathrin and dynamin 2, whereas others were unchanged (early endosome antigen (EEA), adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1)); (b) Immunohistochemistry shown that radiation (6 Gy) affected caveolin-1 expression level and location in H460 non-small cell lung carcinoma; (c) Intracellular delivery of Green 3′-carboxyfluorescein morpholinos (1 μM; Gene Tool, LLC) in T98G cells 2 days after 1 Gy radiation. Red: wheat germ agglutinin (WGA) plasma membrane stain; (d) Scheme and principle of how anti-MGMT oligonucleotide (AMON) works to block O6-methylguanine DNA methyltransferase (MGMT) protein expression. rab, Ras-related protein; RT, radiotherapy.