Fig 1: Gene expression patterns of co-expressed gene set of RAD54L, cancer-specific survival, and progression-specific survival of BLC patients in Korean cohort. (A) Expression patterns of RAD54L gene and its correlated genes. A total of 1284 genes with expression patterns highly correlated with RAD54L were selected for cluster analysis. (B) Cancer-specific survival (CSS) and progression-specific survival (PSS) analyses according to RAD54L expression in BLC patients. BLC patients were divided into two subgroups: low RAD54L expression (Green) and high RAD54L expression (Blue).
Fig 2: E2F1 is involved in transcriptional activation of RAD54L gene. (A) Schematic diagram of RAD54L promoter vector map shown by E2F1 binding sequence. White boxes denote putative E2F1 #1(-767/-760), #2(-155/-148) binding sites. The first ATG is indicated by a black arrow. (B) Dual-luciferase reporter assays of RAD54L gene promoter vector were co-transfected with E2F1 overexpression vector (pE2F1) into 5637 and EJ cells. The pGL3-B (pGL3-Basic) was used as the control. Firefly luciferase activity was adjusted with the activity of Renilla luciferase as the internal control. Assays were performed with triplicate samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) ChIP analysis showed recruitment of E2F1 at E2F1-binding sites (#1, #2 and NTS; non-target sequence) on the promoter region of RAD54L. ns, no significant; *, P < 0.05.
Fig 3: E2F1 regulates RAD54L gene expression. (A) E2F1 protein expression levels and (B) protein band intensities in BLC cell lines including KU7, RT4, T24, 5637 and EJ cell lines were detected by western blotting analysis. The mRNA and protein levels of E2F1, RAD54L in (C) E2F1-transfected 5637 cells, and in (D) EJ cells (shE2F1, control cells; shNTS) with a stable E2F1 knockdown were analyzed by qRT-PCR and western blot. Data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001.
Fig 4: E2F1 and RAD54L expression levels are correlated with cancer progression in BLC patients. (A) Immunohistochemical analysis of early and late T stage BLC tissues was performed to detect E2F1 and RAD54L expression in representative patient samples. Scale bar: 100µm. (B) Progression-free survival in NMIBC patients (GSE13507, n = 102, P = 0.02 by log-rank test, left panel). Progression-free survival in NMIBC patients (GSE13507, n = 102, P = 0.04 by log-rank test, right panel). RLEL, RAD54L low and E2F1 low; RLEH, RAD54L low and E2F1 high; RHEL, RAD54L high and E2F1 low; RHEH, RAD54L high and E2F1 high.
Fig 5: RAD54L expression is induced in MMC-treated cells with E2F1 overexpression or knockdown. (A) Cells were incubated without or with 10 µM MMC for 4 h and allowed to recover for up to 24 h. Expression of E2F1 and RAD54L mRNAs and proteins in E2F1 transfected-5637 cells and in (B) EJ cells with a stable knockdown of E2F1 (shE2F1, control cells; shNTS) were analyzed by qRT-PCR and western blot, respectively. Data are presented as mean ± SD.*, P < 0.05; **, P < 0.01; ***, P < 0.001.
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