Fig 2: EGF signaling contributes to KLK8-induced activation of PI3K-Akt-mTOR signaling pathway and KLK8-induced pro-proliferation and anti-apoptotic effects in pancreatic cancer cells. Mia-paca-2 and Panc-1 cells were treated with EGFR antagonist AG1478 (100 nM). Twenty-four hours later, cells were harvested for measuring cell protein level, proliferation and apoptosis. (A) Expression level of EGF in Mia-paca-2 and Panc-1 cells were evaluated by ELISA assay. (B) Levels of key proteins in PI3K-AKT-mTOR signaling pathways were examined in Mia-paca-2 and Panc-1 cells using western blot (C) Cell proliferation was detected by CCK8 assay in Mia-paca-2 and Panc-1 cells. (D) Colony formation was detected in Mia-paca-2 and Panc-1 cells. (E) Demonstrated the quantified data of cell colonies. (F) Cell apoptosis was determined by Annexin V-FITC and PI double staining analysis performed by flow cytometry. Representative flow cytometry images were shown. (G) Demonstrated the quantified data of cell apoptosis. (H) Pro-apoptotic markers cleaved caspase-3, cleaved caspase-9 and Bax assessed by western blot. Data were presented as the mean ± SEM (n=3). **p <0.01 vs Lv-control; p < 0.05, ##p < 0.01 vs Lv-KLK8.

Fig 3: XL44 induces apoptosis in an hRpn13-dependent manner and reduces hRpn13Pru levels.a–c Lysates from RPMI 8226 WT a–c, trRpn13-MM2 a, b or trRpn13-MM2 with reintroduced hRpn13 (b, trRpn13-MM2/hRpn13) treated for 24 h with 20 μM (a and b) or indicated concentration c of XL44 or DMSO (control) were immunoprobed as indicated. *, cleaved caspase-9. Cartoon (a, bottom) displaying the hRpn13 domains (purple circles), including hRpn2-binding Pru and UCHL5-binding DEUBAD, with the intrinsically disordered region as a black line. A red ‘X’ denotes the deletion of DEUBAD from hRpn13Pru. d Immunoblots as indicated of lysates from RPMI 8226 cells treated for indicated times with 20 μM XL44 or DMSO (0 h, vehicle control). Abundance (mean ± SD) plotted against XL44 concentration (c, μM) or time (d, hours) derived as a percentage by intensity ratio of hRpn13Pru normalized to β-actin (IhRpn13Pru/Iβ-actin)sample divided by DMSO-treated values and multiplied by 100 for two replicates. Data fit by ‘[Inhibitor] vs. normalized response - Variable slope’ (c) or ‘One phase decay’ (d) in GraphPad Prism9. DepC50 (c) and d1/2 (d) values are included, as are values for XL5-VHL-221. e MTT assay for RPMI 8226 WT (blue) or trRpn13-MM2 (light blue) cells treated for 48 h with indicated XL44 concentration. Data represent mean ± SD of n = 6 biological replicates. Viability calculated as (λ570)sample/(λ570)control*100 (%). f As in e, but with MCF7 (green), OVCAR-4 (purple) or OVCAR-5 (orange) cells. Data represent mean ± SD of n = 3 biological replicates. g MTS assay of human fibroblast 1634 (grey) or HS5 (BMSCs) (black) cells treated for 48 h with indicated XL44 concentrations. Data represent mean ± SD of n = 4 biological replicates. IC50 values in e, f analyzed by ‘[Inhibitor] vs. normalized response - Variable slope’ in GraphPad Prism9. h, LDH activity measured by CQUANT LDH Cytotoxicity Assay Kit for RPMI 8226 WT (blue) or trRpn13-MM2 (light blue) cells treated for 24 h with indicated XL44 concentration. Data represent mean ± SD of n = 4 biological replicates. Cytotoxicity calculated as ((LDH activity)sample-(LDH activity)spontaneous))/((LDH activity)Maximum-(LDH activity)spontaneous))*100 (%). Source data are provided as a Source Data file.

Fig 4: pp-DY inhibits cancer cell proliferation by inducing apoptosis and S-phase arrest. (A) Flow cytometric analysis of apoptotic cells was performed by using annexin V staining. Green square indicates populations of apoptotic cells. **P < 0.01, student's t-test. Means ± S.D., n = 3. (B) Effect of PCA on the expression level of proteins involved in apoptosis pathway. Cleaved form of caspase 9 and caspase 3. Full gels are shown in Supplementary Fig. 2. (C) Flow cytometric analysis of cell cycle was performed by using PI staining. **P < 0.01, student's t-test. Means ± S.D., n = 3. (D) Effect of PCA on the expression level of proteins involved in cell cycle pathway. p16INK4A (p16), p21Cip1 (p21) and phopho-ERK (p-ERK). Full gels are shown in Supplementary Fig. 2.

Fig 5: KLK8 overexpression inhibited apoptosis of PDAC cells. KLK8 overexpression was induced with recombinant Lentivirus infection (Lv-KLK8) in human pancreatic cancer cell lines Mia-paca-2 (A, B) and Panc-1 (C, D), and an empty adenovirus served as control (Lv-Control). Cell apoptosis was determined by Annexin V-FITC and PI double staining analysis performed by flow cytometry. Representative flow cytometry images were shown (A, C). (B, D) demonstrated the quantified data of cell apoptosis. (E) pro-apoptotic markers cleaved caspase-3, cleaved caspase-9 and Bax assessed by western blot. Data are expressed as the mean ± SEM (n=3). **p < 0.01 vs Lv-control.