Fig 2: PTTG1 is highly expressed in pancreatic cancer. (a) PTTG1 expression levels were analyzed using the GEPIA website. red, pancreatic adenocarcinoma tissues (n = 179); gray, normal tissues (n = 171). (b) Western blot was performed to analyze PTTG1 expression in pancreatic cancer cells including Sw1990, Capan1, Panc1, Mia-PaCa2 and PDAC cell lines HPNE. (c) The high expression of PTTG1 was associated with poorer overall survival and disease-free survival in patients with pancreatic cancer (n = 89). The data from three repeated experiments were used for the statistical analysis. Error bar, mean ± SD; *p < 0.05, **p < 0.01.

Fig 3: XL44 reduces protein levels of PCLAF, its downstream target PTTG1 and RRM2.a Volcano plot from quantitative TMT proteomics analysis performed in triplicate for RPMI 8226 cell lysates treated for 8 h with 20 μM XL44 or DMSO (control). p-values calculated by two-tailed two-sample equal variance t-test. Dashed lines indicate -log100.001 (horizontal), log20.5 (vertical) and log22 (vertical). Proteins reduced at ≥0.5-fold with p-values < 0.001 indicated by green dots. b–c Repeated experiments of Fig. 5a or Fig. 5c immunoblotting for cleaved caspase-9 b, hRpn13 b, PCLAF, PTTG1, or RRM2 with mouse b, c or rabbit b primary and secondary antibodies. d Lysates from RPMI 8226 cells treated with indicated XL44 concentration or DMSO (control) for 24 h immunoprobed for PCLAF, RRM2 (rabbit) and β-actin. e Repeated experiments of Fig. 5d with inclusion of immunoblots for PCLAF, PTTG1 and RRM2 (mouse and rabbit). f Immunoblots of RPMI 8226 cell lysates treated for indicated times with 20 μM XL44 or DMSO (0 h, control) detecting PCLAF, RRM2 or β-actin. Percentage (%) in d–f calculated as the ratio of intensities for PCLAF d–f, RRM2 d–f, or hRpn13Pru e normalized to β-actin (IPCLAF/RRM2/hRpn13Pru/Iβ-actin)sample, divided by DMSO control and multiplied by 100. Percentage (%) of PCLAF (orange), RRM2 (light blue) d–f or hRpn13Pru (black) e plotted against XL44 concentration (d, μM) or time (e, f, hours) and fit by ‘[Inhibitor] vs. normalized response - Variable slope’ d and ‘One phase decay’ e–f in GraphPad Prism9. DepC50, d and d1/2 f values are included. “#” in b, c and e indicates a non-specific band detected by anti-RRM2 mouse antibodies. g Graphical display of the correlation (p-value < 0.0001) between high (yellow) and low (blue) PCLAF (top panel) or RRM2 (bottom panel) expression levels with overall survival for patients with myeloma. h Immunoblots with antibodies against RRM2 (1 or 30 min) or PCLAF of streptavidin-bound XL44B or biotin (negative control) following incubation with RPMI 8226 cell lysates and subsequent wash (top panels) and corresponding input are included (lower panels). Experiments in 6b, 6c, 6e and 6 h were performed twice; 6d and 6 f were performed once. Source data are provided as a Source Data file.

Fig 4: PTTG1 promotes proliferation of pancreatic cancer cells. (a) Western blot was used to measure the protein level of PTTG1 and PCNA in panc1 cells transfected with si-PTTG1 #1 and #2. (b) Cell proliferation curves of Panc1 with PTTG1 knockdown using si-PTTG1 #1 and #2. (c) Clone formation of Panc1 cells with PTTG1 knockdown using si-PTTG1 #1 and #2. (d) Western blot was used to measure the protein level of PTTG1 and PCNA in Mia-Paca2 cells with PTTG1 overexpression. (e) Cell proliferation curves of Mia-Paca2 with PTTG1 overexpression. (f) Clone formation of Mia-Paca2 cells with PTTG1 overexpression. All the experiments were repeated at least three times. Error bar, mean ± SD; *p < 0.05, **p < 0.01.

Fig 5: PTTG1 promotes migration and invasion of pancreatic cancer cells. (a) Wound healing assay was performed to detect migration in Panc1 cells with PTTG1 knockdown. (b) The transwell assay was performed to analyze the invasion of Panc1 cells with PTTG1 knockdown. (c) Western blot was performed to evaluate the expression of E-cadherin and N-cadherin in Panc1 cells. (d) Wound heal assay was performed to detect migration in Mia-Paca2 cells with PTTG1 overexpression. (e) The transwell assay was performed to analyze the invasion of Mia-Paca2 cells with PTTG1 overexpression. (f) Western blot was performed to evaluate the expression of E-cadherin and N-cadherin in in Mia-Paca2 cells. Three repeated experiments were used for analysis. Error bar, mean ± SD; *p < 0.05, **p < 0.01.