Fig 1: (A, left panel) Serum-starved CD-HIF were treated with 100 µg/mL CDSE for 2 hours. Serum-starved CCD-18Co colonic fibroblasts were treated with 10 ng/mL TGF-ß1 for 2 hours. Conditioned media were loaded to Proteome Profiler Human Protease Arrays (ARY021B; R&D Systems). A Bio-Rad ChemiDoc Imaging system captured the images. The rectangles highlighted the cathepsin S expression. (A, right panel) Quantification of cathepsin S signals (B7-8) and control signals (A1-2 and E1-2) using Bio-Rad Image Lab Software. Results were pooled from 3 independent experiments. Student t test was used to compare no serum exosome and CDSE groups. (B, left panel) Colonic cathepsin S mRNA expression in 40 non-IBD, 52 UC, 28 non-stricturing CD, and 15 stricturing CD patients was determined by real-time reverse transcription polymerase chain reaction. Ordinary one-way ANOVA test did not find any significant differences. (B, middle panel) Colonic cathepsin S mRNA (CTSS) expression in 43 CD patients is not correlated with colonic elafin mRNA expression. (B, right panel) Fresh human colonic tissues from 4 colon cancer patients were incubated in serum-free RPMI1640 media with or without 100 µg/mL CDSE. Two hours later, elafin (1 µg/mL) was added and incubated for 24 hours. Ordinary one-way ANOVA test did not find any significant differences. (C) Cathepsin S activity assay was performed by incubating 2 µL of CS substrate (200 µmol/L final concentration), 94 µL CS reaction buffer, 2 µL cathepsin S inhibitor provided by the assay kit, 1 µL cathepsin S (0.4 µg/mL final concentration), and 1 µL elafin (0.5–10 µg/mL final concentration) at 37oC for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). Results were pooled from 3 independent experiments. Ordinary one-way ANOVA with Tukey test. (D) Fresh human colonic tissues were pretreated with 100 µg/mL CDSE for 2 hours, followed by elafin 1 µg/mL for 2 hours. Conditioned media were collected. Each piece of tissue was homogenized in 500 µL CS cell lysis buffer. Next, 50 µg of tissue lysate supernatants in 50 µL CS lysis buffer or 50 µL of conditioned media were mixed with 2 µL CS substrates (200 µmol/L final concentration) and 48 µL CS reaction buffer and incubated for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). n = 6 patients. Ordinary one-way ANOVA with Tukey test. (E) CD-HIF in 96-well plates were pretreated with 100 µg/mL CDSE for 2 hours, followed by elafin 1 µg/mL for 2 hours. Conditioned media were collected. Cells were then lysed in 200 µL/well CS cell lysis buffer. Next, 50 µg of cell lysates in 50 µL CS lysis buffer or 50 µL of conditioned media were mixed with 2 µL of CS substrate (200 µmol/L final concentration) and 48 µL CS reaction buffer and incubated at 37oC for 1 hour. Cathepsin S activity was represented by relative fluorescence units (RFU). Results were pooled from 6 independent experiments. Ordinary one-way ANOVA with Tukey test. (F) Serum-starved CD-HIF were pretreated with either 0.1% TFA r 0.4 µg/mL cathepsin S (1183-CY-010; R&D Systems) for 30 minutes, followed by 100 µg/mL CDSE. Two hours later, elafin (1 µg/mL) was added and incubated for 24 hours. ProCOL1A1 protein was determined by ELISA. Results were pooled from 4 experiments. Ordinary one-way ANOVA with Tukey test.