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.
Fig 2: KLK co-expression in PDAC tissues. (a) On RNA sequencing, KLK1, KLK6, KLK7, KLK8, KLK10 and KLK11 were identified as highly upregulated in PDAC tissues. Yellow dots represent individual PDAC patients. (b) KLK6, KLK7, KLK8, KLK10 and KLK11 were significantly upregulated in PDAC compared to normal pancreas tissues (p = 0.05), with KLK8 showing a trend (p = 0.0939). Yellow dots represent individuals. (c) Pearson correlation coefficients indicating a significant correlation between KLK10 and KLK6 (R2 = 0.61), KLK7 (R2 = 0.65), KLK8 (R2 = 0.55) and KLK11 (R2 = 0.7) levels (p = 0.05). Purple dots represent individual patients. (d) On whole-genome sequencing, KLK6, KLK7, KLK8, KLK10 and KLK11 were identified as a highly upregulated gene cluster in PDAC compared to normal pancreas tissues. Rectangles represent individual patients; red—upregulation; blue—downregulation; black—not differentially expressed. (e) Kaplan–Meier analysis indicated that high KLK6 (p = 0.0025), KLK7 (p = 0.021), KLK10 (p = 0.00045) and KLK11 (p = 0.0085) expression levels were significantly associated with a shorter 5-year survival than low expression levels.
Fig 3: Picrasidine J regulates cell metastasis by downregulating protease KLK-10 in HNSCC cancer cells. (A) Cells treated with 100 μM of picrasidine J were collected and measured metastatic-related protein expression using Human Protease Array Kit (Cat. ARY021B, R&D Systems Inc.). (B,C) KLK-10 measured using Western blot assay after picrasidine J treatment (0, 25, 50, or 100 μM), with β-actin as internal control. (D,E) KLK-10 level measured after transfection with si-NC (negative control siRNA) or si-KLK-10 siRNA using a Western blot assay, with β-actin as internal control. (F,G) Ca9-22 and (H,I) FaDu cells were transfected with si-NC or si-KLK-10 siRNA after treatment with or without picrasidine J (100 μM). After 0, 3, 6, and 24 h, a wound healing assay was used to analyze Ca9-22 and FaDu cells. * p < 0.05; # p < 0.05, compared to picrasidine J only. Scale bar: 100 μm.
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