Fig 1: CTSS is produced in macrophage and transported by exosomes. Western blot analysis of CTSS in macrophage cell lysates and medium after PA (500 µM) stimulation for 24 h (A). Western blot analysis of exosome-specific proteins in exosomes and cell lysates detected 24 h after palmitic acid (PA, 500 µM) stimulation of macrophage (B). Electron microscopy of exosomes (C). (Scale bars = 500 nm) Exosome-derived CTSS from PA (500 µM) stimulated macrophage (D). QRT-PCR (E) and western blot (F) analysis of CTSS gene knockdown efficiency in macrophage. Data are shown as the mean ± SEM, n = 3, ANOVA. *P < 0.05. Detection of CTSS in exosomes from PA (500 µM) stimulated macrophage; macrophage were transfected with shCTSS, scr transfection as the control (G)
Fig 2: Transcription factor IRF5 is responsible for CTSS expression in proinflammatory macrophage. Western blot analysis (A) for CTSS expression in macrophage with increased concentrations of palmitic acid (PA, 0, 100, 200, 300, 400, 500 µM). Western blot (B) and qRT-PCR (C) analysis for the expression of IRF family gene expression under 500 µM PA. JASPAR (http://jaspar.genereg.net/) prediction of the IRF5 transcription binding site in the CTSS promoter (D). Dual-luciferase reporter system was performed with WT and MUT CTSS promoter with IRF5 overexpression (E). qRT-PCR and western blot analysis (F) for the efficiency of IRF5 gene knockdown in macrophage. An electrophoretic mobility shift assay (EMSA) was used to assess the IRF5 and CTSS interaction (G). qRT-PCR (H) and western blot analysis (I) for CTSS expression when transfected with scr or shIRF5-1. Data are shown as the mean ± SEM, n = 3, ANOVA. *P < 0.05, **P < 0.05
Fig 3: CTSS is up-regulated in peripheral blood of hyperlipidemic pancreatitis (HP) patients and in an HP mouse model. Amylase level (A), lipase level (B), TG level (C), FFA level (D), CTSS level (E), IL-1ß (F), TNF-a (G) and IL-10 (H) levels in the peripheral blood of HP patients. Data are shown as the mean ± SEM, HP (NHP = 57), control (nn = 7), ANOVA. **P < 0.01. Linear correlation analysis of CTSS and FFA (I), CTSS and IL-1ß (J)
Fig 4: Macrophage exosome-derived CTSS induced pyroptosis in vitro. Western blot analysis of caspase-1, GSDMD, pro-IL-1ß, IL-1ß, ASC, and caspase-8 in acinar cells, which are treated with EXO-CTSS or EXO-vector and transfected with scr or shGSDMD (A). Immunofluorescence staining for GSDMD, caspase-1, caspase-8, and ASC in acinar cells are treated with EXO-CTSS or EXO-vector, and transfected with scr or shGSDMD (B). The location and level of GSDMD, caspase-1, caspase-8, and ASC are shown by red fluorescence, and nucleiare stained with DAPI. QRT-PCR and western blot analysis (C) for the efficiency of GSDMD gene knockdown in acinar cells, scr as the control. Acinar cells viability over time in control, EXO-vector + scr, EXO-CTSS + scr, and EXO-CTSS + shGSDMD (D). TUNEL assay for apoptosis was performed in acinar cells after EXO-CTSS treatment; cells were transfected with scr or shGSDMD. (Scale bars = 100 µm). (E). Data are shown as the mean ± SEM, n = 3, ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 5: Macrophage exosome-derived CTSS induced acinar cell injury via trypsinogen and caspase-1. QRT-PCR and western blot analysis (A) of CTSS overexpression in macrophage, empty vector are used as the control. Pancreatic acinar cell viability over time in control, EXO-vector + scr, EXO-CTSS + scr, EXO-CTSS + shCaspase1, and EXO-CTSS + shtrypsinogen (B). QRT-PCR (C) and western blot analysis (D) for caspase-1 and trypsinogen expression of acinar cells after EXO-CTSS stimulation. TUNEL assay for acinar cells apoptotic after EXO-CTSS treatment, with empty vector control, and normal blank control. (Scale bars = 100 µm) (E). QRT-PCR and western blot analysis (F) of the efficiency of caspase-1 and trypsinogen gene knockdown in acinar cells, with scr control. TUNEL assay to assess apoptosis after EXO-CTSS treatment in acinar cells, which are transfected with scr, shCaspase1, or shTrypsinogen, no transfection is the blank control. (Scale bars = 100 µm) (G). Data are shown as the mean ± SEM, n = 3, ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001
Supplier Page from Abcam for Human Cathepsin S ELISA Kit (CTSS)