Fig 2: Overexpression of QPCT promotes tumor angiogenesis. (A) Representative immunohistochemical results of QPCT, CD31 and CD34 in xenografts. Scale bar, 100 µm. (B) Representative results of HUVEC tube formation cultured with the supernatant of RCC cells overexpressing QPCT. (C) Representative results of HUVEC tube formation cultured with purified QPCT cytokines (rhQPCT). The group incubated with exogenous VEGF was used as the positive control group. Total tube length was calculated using ImageJ software. Results are presented as the mean ± SD. *P<0.05, **P<0.01. QPCT, glutaminyl peptide cyclotransferase; CM, conditioned medium.

Fig 3: In the sunitinib-non-responsive RCC tissues and plasma, QPCT expression is increased, and patients with RCC with a high QPCT expression have a poor response to sunitinib. (A) mRNA expression of QPCT in 20 pairs of sunitinib-non-responsive and -responsive RCC tissues. (B) Results of western blot analysis of QPCT protein in 20 pairs of sunitinib nonresponsive and responsive RCC tissues. (C) Representative immunohistochemical results of QPCT expression in sunitinib-non-responsive and -responsive RCC tissues (scale bar, 100 µm; left panel), and percentage of samples nonresponsive and responsive to sunitinib at different QPCT levels (right panel). (D) ELISA of plasma QPCT levels in patients with RCC at Jinling Hospital. (E) Kaplan-Meier analysis of PFS for all patients (P=0.0207) (left panel). Kaplan-Meier analysis of PFS in patients with a high QPCT expression (P=0.5125) (middle panel). Kaplan-Meier analysis of PFS in patients with low QPCT expression (P=0.0264) (right panel). The results are presented as the mean ± SD. *P<0.05. RCC, renal cell carcinoma; QPCT, glutaminyl peptide cyclotransferase; PFS, progression-free survival.

Fig 4: CTCF binds to the QPCT promoter region, negatively regulating its expression. (A) ChIP analysis demonstrated that CTCF binds to the promoter region of QPCT. (B) QPCT mRNA expression in ACHN and OS-RC-2 cells 72 h following CTCF knockdown, and in a control group (n=3). (C) QPCT protein expression in ACHN and OS-RC-2 cells 72 h following CTCF knockdown, and in a control group (n=3). (D) QPCT mRNA expression in CTCF overexpressed 786-O and KETR-3 cells and control cells (n=3). (E) QPCT protein expression in CTCF-overexpressing 786-O and KETR-3 cells and control cells (n=3). Results are presented as the mean ± SD. *P<0.05, **P<0.01. CTCF, CCCTC-binding factor; QPCT, glutaminyl peptide cyclotransferase.

Fig 5: QPCT enhances the stability of PIK3CA by reducing the degradation of PIK3CA ubiquitination. (A) Results of western blot analysis of QPCT and PIK3CA in QPCT-overexpressing 786-O and A498 cells and control cells. (B) Results of western blot analysis of QPCT and PIK3CA in OS-RC-2 and ACHN cells transfected with sh-QPCT or sh-NC. (C) Representative immunohistochemical results of QPCT and PIK3CA in xenografts. Scale bar, 100 µm. (D) Western blot analysis of PIK3CA in QPCT overexpressed 786-O cells and control cells treated with CHX and sunitinib (5 µm) for a different period of time. (E) Western blot analysis of PIK3CA ubiquitination in QPCT overexpressed 786-O and A498 cells and control cells after sunitinib (5 µM) treatment for 48 h. GAPDH was used as the load control. Results are presented as the mean ± SD. *P<0.05. QPCT, glutaminyl peptide cyclotransferase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; CHX, cycloheximide.