Fig 2: Association between p62, autophagy status, and glypican 3 (GPC3) expression in HCC cell lines and normal hepatocytes. (A) A panel of HCC cell lines and primary human hepatocytes (PHH) immunostained for p62. Scale bars = 20 µm. (B) Relative optical density (OD) of p62 immunostaining per representative view field. The results are expressed as the mean ± standard deviation (SD). ***P < 0.001. For each cell line 10 view fields were quantified. (C) Representative Western blot of whole cell extracts for p62 and loading control. (D) The images of DQ-Red BSA uptake and processing in autophagy-deficient (Huh-7, Huh-7PX, and Huh-7.5) and an autophagy-competent (HLE) HCC cell lines ± Torin1 treatment (200 nM). Scale bars = 20 µm. (E) Representative Western blot analysis of p62 degradation after Torin1 treatment in autophagy-deficient (Huh-7, Huh-7PX, and Huh-7.5) and an autophagy-competent (HLE) HCC cell lines. (F) GPC3 and glypican 1 (GPC1) immunostaining in autophagy-deficient (Huh-7, Huh-7PX, and Huh-7.5) and an autophagy-competent (HLE) HCC cell lines. Scale bars = 20 µm. (G) Relative OD of GPC3 and GPC1 immunostaining per representative view field. Data indicate mean ± SD. ***P < 0.001. (H) Representative Western blot analysis showing the expression of GPC3 and GPC1 in autophagy-deficient (Huh-7, Huh-7PX, and Huh-7.5) and an autophagy-competent (HLE) HCC cell lines. The data are resulted in three independent experiments.

Fig 3: HS and CS chains are responsible for endocytosis of 3D8 scFv. At 24 h post-transfection with plasmids encoding SDC2-GFP, HA-GPC3, CD44-GFP, or HA-BCAN, HeLa cells were treated with 500 mIU/ml heparinase III or 500 mIU/ml chondroitinase ABC for 2 h at 37 °C. Then, cells were treated for 6 h at 37 °C with 3D8 scFv, followed by immunoprecipitation with anti-GFP, anti-GPC3, anti-CD44, or anti-HA antibodies. Proteins in the extract (Input; 10%) and pulled-down fractions (IP) were analyzed by immunoblotting.

Fig 4: Glypican 3 (GPC3)-enriched exosomes are release exclusively from autophagy-deficient hepatocellular carcinoma (HCC) cells. (A) Cell compartment breakdown of 129 proteins identified by proteomic analysis of exosomes isolated from autophagy-deficient Huh-7.5. (B) Representative immunogold-transmission electron microscopy images of exosomes labeled against glypican 1 (GPC1) and GPC3 after isolation from autophagy-deficient (Huh-7.5) and autophagy-competent (HLE) HCC lines. (C) Fluorescence histograms of bead-captured exosomes from Huh-7.5 and HLE stained for GPC1. (D) Fluorescence histograms of bead-captured exosomes from Huh-7.5 and HLE stained for GPC3. (E) Background fluorescence of bead-captured exosomes with primary antibody omitted. (F). Representative Western blot analysis of GPC1 and GPC3 in purified exosomes isolated from Huh-7.5 and HLE cultures. All experiments were in triplicate.

Fig 5: Immunohistochemical staining of p62 and glypican 3 (GPC3) in cirrhotic livers with HCC. (A and D) Histologic sections of HCC with adjacent liver cirrhosis (H&E, ×200). (B and C) The expression of p62 in HCC and adjacent cirrhotic livers (various etiologies) with semiquantitative scoring for expression of the entire cohort. (E and F) Representative expression of GPC3 in HCC and adjacent cirrhotic livers (various etiologies) with semiquantitative scoring for expression of the entire cohort. Original image magnification was 200×. Data indicate mean ± standard error of mean and analyzed by Student’s t-test. ***P < 0.001.