Figure 2.

Periplocin induces lysosomal damage in CRC cells. (A) TMT-based quantitative proteomics analysis was used to profile the global protein expression in SW480 cells treated with or without 0.50 μM periplocin for 24 h. The differentially expressed lysosome-associated proteins were enriched by gene set enrichment analysis (GSEA). (B) Immunoblotting analysis of LAMP1, LAMP2 and CTSD under 0.50 μM periplocin treatment for 24 h. m-CTSD, mature CTSD. (C and D) Representative images (C) and quantitative analysis (D) of immunohistochemical staining for LAMP1 in SW480 ×enografts from vehicle- or periplocin-treated mice. Scale bar: 50 μm. (E) Acridine orange (AO, 5 μM) was used to label the lysosomes of cells treated with or without 0.50 μM periplocin for 24 h. Green fluorescence indicating the leakage of lysosomal contents was detected by flow cytometry. (F) Immunofluorescent analysis of CTSB in cells treated with or without 0.50 μM periplocin for 24 h. Scale bar: 10 μm. (G) Quantitation of the relative fluorescent intensity of Magic Red staining in cells treated with or without 0.50 μM periplocin for 24 h. (H-J) Representative images (H) and quantitative analysis (I and J) of LysoSensor Yellow/Blue DND-160 staining in cells treated with or without 0.50 μM periplocin for 24 h. Scale bar: 10 μm. (K and L) Representative images (K) and quantitative analysis (L) of LysoTracker Red DND-99 staining in cells treated with or without 0.50 μM periplocin for 24 h. Scale bar: 10 μm. (M and N), Representative images (M) and quantitative analysis (N) for immunofluorescent staining of LGALS3 in cells treated with or without 0.50 μM periplocin for 24 h. Scale bar: 10 μm. (O) Immunofluorescent analysis of the colocalization of LGALS3 with LAMP1 in cells treated with or without 0.50 μM periplocin for 24 h. Scale bar: 10 μm. Results are representative of three independent experiments. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.