Fig 2: LAPTM4B inhibition reverses the effect of SULF2-induced changes in autophagy. (A) Western blot analysis shows decreased LAMP1 and increased cathepsin D in SULF2-positive Huh7 cells and SULF2-transfected Hep3B cells that are depleted of LAPTM4B. (B) Confocal microscopic images show GFP-LC3 puncta according to LAPTM4B status in SULF2-positive Huh7 cells and SULF2-transfected Hep3B cells. (C) Merged immunofluorescence images show autophagosomes (anti-LC3B, red) and lysosomes (anti-LAMP1, green) according to LAPTM4B status in SULF2-positive Huh7 cells and SULF2-transfected Hep3B cells. The LC3 puncta-positive cells are counted by examining a total of 50 cells in 10 random fields (5 cells/field) and presented as bar graphs (mean ± SEM). The correlation coefficients of colocalization between autophagosomes and lysosomes of 150 cells (50 cells × 3 independent experiment) are presented as dot plots (mean ± SEM). Abbreviations: GFP, green fluorescent protein; LAMP1, lysosome-associated membrane protein 1; LAPTM4B, lysosome-associated protein transmembrane 4 beta; SEM, standard error of the mean; SULF2, sulfatase 2.

Fig 3: Working model for the SULF2-LAPTM4B-dependent autophagic pathway in HCC cells. Abbreviations: HCC, hepatocellular carcinoma; LAPTM4B, lysosome-associated protein transmembrane 4 beta; SULF2, sulfatase 2.

Fig 4: SULF2 inhibition is associated with LMP. (A) Merged immunofluorescence images show lysosomal enzyme (anti-cathepsin D, red) and lysosomes (anti-LAMP1, green) according to SULF2 status in the HCC cells. Huh7 cells transfected with scrambled shRNA show more colocalization (upper panel). Huh7 cells transfected with SULF2 shRNA show diffuse cytosolic staining of cathepsin D (lower panel). Control Hep3B cells show less colocalization (upper panel). Hep3B cells transfected with SULF2 plasmid show more colocalization (lower panel). (B) Merged immunofluorescence images show galectin-3 (anti-galectin-3, red) and lysosomes (anti-LAMP1, green) according to SULF2 status in the HCC cells. Huh7 cells transfected with scrambled shRNA show diffuse cytosolic staining for galectin-3 (upper panel). Huh7 cells transfected with SULF2 shRNA show punctate staining for galectin-3 colocalized with LAMP1 (lower panel). Control Hep3B cells and Hep3B cells transfected with SULF2 plasmid show diffuse cytosolic staining for galectin-3. The Huh7 cells showing diffuse cytosolic staining of cathepsin D or punctate staining of galectin-3 were counted by examining a total of 50 cells in 10 random fields (5 cells/field) and presented as a bar graph (mean ± SEM). Abbreviation: CTSD, cathepsin D.

Fig 5: LAPTM4B depletion induces LMP. (A) Merged immunofluorescence images show lysosomal enzyme (anti-cathepsin D, red) and lysosomes (anti-LAMP1, green) according to LAPTM4B status in SULF2-positive Huh7 cells and SULF2-transfected Hep3B cells. (B) Merged immunofluorescence images show galectin-3 (anti-galectin-3, red) and lysosomes (anti-LAMP1, green) according to LAPTM4B status in SULF2-positive Huh7 cells and SULF2-transfected Hep3B cells. The cells showing diffuse cytosolic staining of cathepsin D or punctate staining of galectin-3 were counted by examining a total of 50 cells in 10 random fields (5 cells/field) and presented as a bar graph (mean ± SEM). Abbreviations: CTSD, cathepsin D; LAMP1, lysosome-associated membrane protein 1; LAPTM4B, lysosome-associated protein transmembrane 4 beta; LMP, lysosomal membrane permeabilization; SEM, standard error of the mean; SULF2, sulfatase 2.