Fig 2: 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 3: RAD54L and E2F1 increase HR recovery activity when DSB is induced in BC cells. (A) Cells transfected with E2F1, RAD54L, or both plasmids were exposed to 10 μM mitomycin C for 4 h before the comet assay was performed. DNA damage was quantified based on tail length. (H, head of comet; T, tail of comet, Scale bar: 10 μm). (B) Tail length, (C) HR assay by U2OS DR-GFP. (D) HR frequency expressed as a percentage of GFP-positive cells in viable whole cell population. Data are presented as mean ± SD. *, P < 0.05; ***, P < 0.001.

Fig 4: 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.

Fig 5: 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.