Fig 1: Expression of cathepsin B.Representative IHC stained images of CTSB, including 2 LGCA samples (A, C) and their patient-match NC tissues (B, D), and 2 HGCA samples (E, G) and their patient-matched NC tissues (F, H). Nuclei were counterstained with hematoxylin (A-H, blue); original magnification: 400x. CTSB mRNA expression levels were measured by RT-qPCR (I) in 3 LGCA and 3 HGCA primary cell lines that were sorted into EpCAMHigh (+) and EpCAMLow (-) subpopulations, and the average mRNA abundance from triplicate values relative to a pool of NC tissues are displayed with error bars representing standard deviation. CTSB protein expression by cell lines was detected by WB (J; 24 kDa and 27 kDa); positive control = HepG2 cells. Adapted from [38] under a CC BY license, with permission from Munro MJ et al. 2021.
Fig 2: SULF2 is associated with expression of lysosome-related proteins. Western blot analyses show protein expressions of LAMP1, cathepsin D, and cathepsin B (left column) according to SULF2 status in Huh7 and Hep3B cells. Common markers for apoptosis (right column) are also assessed. Abbreviations: Cas-8, caspase-8; Cas-9, caspase-9; CTSB, cathepsin B; CTSD, cathepsin D; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMP1, lysosome-associated membrane protein 1; SULF2, sulfatase 2.
Fig 3: SULF2 is associated with cathepsin D (A) and cathepsin B (B) activity. Abbreviations: CTSB, cathepsin B; CTSD, cathepsin D; SULF2, sulfatase 2.
Fig 4: The lysosomal enzyme cathepsin D shows reduced proteolytic activity in F508del macrophages. (A) Cathepsin D (CTSD) activity in wild-type (WT) and F508del macrophages either non-infected (NT) or treated with pepstatin (20 µg/ml). Data show mean fluorescence intensity (MFI) of Bodipy-pepstatin normalized to the number of cells. Data represent mean ± SEM (n = 4 biological replicates). Statistical analysis was done using two-way ANOVA; **, p = 0.01; ns, non-significant. (B) CTSD activity in F508del macrophages NT (plotted in A) or treated with Teza + Iva. Data show MFI of Bodipy-pepstatin normalized to the number of cells. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was done using one-way ANOVA; *, p = 0.0.5. (C) Cathepsin B (CTSB) activity in WT and F508del either NT or treated with CTSB inhibitor. Data show mean fluorescence intensity per 10 µg of total protein. Data represent mean ± SEM (n = 5 biological replicates). Statistical analysis was performed using two-way ANOVA; **p = 0.01; ***p = 0.001; ns, non-significant. (D) CTSB activity in F508del either NT (plotted in C) or treated with Teza+Iva for 24 h. Data show MFI per 10 µg of total protein. Data represent mean ± SEM (n = 4 biological replicates). Statistical analysis was performed using paired t-test; *, p = 0.05.
Fig 5: The influence of estrogen-regulated calcium influx in Ishikawa and Hec-1A cell line lysosome. (A) An E2-BSA significantly promoted the LAMP1 expression in Ishikawa cells with rapid effect. (B) Inhibition of extracellular calcium influx reduced lysosomal response. (C) Effect of E2-BSA on lysosomes. The lysosomes were labeled by 5 nM Lyso-Tracker Red (LTR) and examined confocal. Magnification 63 ×. Scale bar 25 or 5µm. (D) Ishikawa cells stained with 5 µg/ml acridine orange (AO) for 15 min were then treated with E2-BSA and imaged under a confocal microscope (scale bar: 25 µm). (E) E2-BSA treatment causes differential regulation in Ishikawa or HEC-1A cells. The activities of CTSB were measured by the fluorometric method using commercially available kits. All data are presented as the means ± SE. n = 3–4. (F,G) E2-BSA stimulated external calcium influx can activate the nuclear translocation of TFEB. (H) Effect of TFEB nuclear shift after E2-BSA treatment. All data are presented as the means ± SE. n = 50 for cell numbers and n = 3 for replicated. ns, non significant; **p < 0.01 compared with the control group.
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