Human breast cancer MCF7, MDA-MB-175VII, BT474 and MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC). MCF7 and BT474 cells were cultured in RPMI-1640 medium (Nacalai Tesque, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Nichirei Biosciences, Inc.). MDA-MB-175VII and MDA-MB-231 cells were maintained in Leibovitz's L-15 medium (Thermo Fisher Scientific, Inc.) containing 10% heat-inactivated FBS. All of the media contained 100 IU/ml of penicillin and 100 µg/ml of streptomycin (both from Thermo Fisher Scientific, Inc.). Cells were maintained at 37°C in an incubator with a controlled humidified atmosphere consisting of 95% air and 5% Co₂.

Cells were seeded at a density of 5×10⁴ cells/ml and allowed to attach on the bottom of culture plate. Twenty-four hours after seeding, cells were transfected with siRNA using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. siRNA for E2F5 (siRNA ID s4417; cat. no. 4392420; Thermo Fisher Scientific, Inc.), siRNA for TP53 (cat. no. sc-29435; Santa Cruz Biotechnology, Inc.), and control siRNA (cat. no. 4390843; Thermo Fisher Scientific, Inc.) were used in the present study.

MCF7 and MDA-MB-231 cells were seeded in 96-well culture plates at a density of 5×10⁴ cells/ml and transfected with E2F5 siRNA or control siRNA 24 h after seeding. At the indicated time-points, viability of the transfected cells was measured by the standard WST-8 assay using Cell Count Reagent CF (Nacalai Tesque, Inc.). One tenth volume of WST8 solution was added to culture medium and incubated for 1 h at 37°C in Co₂ incubator. Then absorbance of culture medium at OD 450 nm was measured.

For fluorescent-activated cell sorting (FACS) analysis, MCF7 and MDA-MB-231 cells were seeded in 6-well culture plates at a density of 1×10⁵ cells/ml and transfected with E2F5 siRNA or control siRNA 24 h after seeding. Cells were cultured for 4 days, and then both floating and attached cells were collected for FACS analysis. Cells were washed in PBS and fixed in 70% ethanol overnight at 4°C. After washing in PBS, cells were suspended in PBS containing 0.1% FBS, 25 µg/ml propidium iodide (Sigma-Aldrich; Merck KGaA) and 200 µg/ml of RNase A (Sigma-Aldrich; Merck KGaA), and incubated for 15 min at room temperature. Subsequently, the cells were subjected to FACS analysis (FACSCalibur; BD Biosciences).

Total RNA was extracted from cells using RNeasy mini kits (Qiagen, Inc.) according to the manufacturer's instructions. For cDNA synthesis, aliquots of 500 ng of total RNA were reverse-transcribed using iScript cDNA synthesis system (Bio-Rad Laboratories, Inc.). Real-time RT-qPCR for E2F5 and β-actin was performed using Premix Ex Taq Perfect Real Time (Takara Βio, Inc.) with TaqMan Pre-Developed Assay Reagents (Thermo Fisher Scientific, Inc.), Hs00231092_m1 for E2F5 and Hs99999903_m1 for β-actin. The reaction was carried out at 95°C for 5 sec and 60°C for 20 sec, for total of 40 cycles. Real-time RT-qPCR for p21^WAF1, BAX, NOXA and PUMA was performed using SYBR Premix Ex Taq™ (Takara Bio, Inc.) according to the manufacturer's recommendations. The primers used were as follows: p21^WAF1, 5′-GCAGACCAGCATGACAGATTT-3′ (sense) and 5′-GGATTAGGGCTTCCTCTTGGA-3′ (antisense); BAX, 5′-TTGCTTCAGGGTTTCATCCA-3′ (sense) and 5′-AGACACTCGCTCAGCTTCTTG-3′ (antisense); NOXA, 5′-GCAGAGCTGGAAGTCGAGTG-3′ (sense) and 5′-GAGCAGAAGAGTTTGGATATCAG-3′ (antisense); PUMA, 5′-GACGACCTCAACGCACAGTA-3′ (sense) and 5′-AGGAGTCCCATGATGAGATTGT-3′ (antisense). The reaction for p21^WAF1 and BAX was carried out at 95°C for 5 sec, 55°C for 10 sec and 72°C for 10 sec, for total of 40 cycles. The reaction for NOXA and PUMA was carried out at 95°C for 5 sec and 60°C for 20 sec, for total of 40 cycles. Assessments were performed three times. A mixture of cDNA generated from total RNA of MCF7 and MDA-MB-231 cells was used as a reference. A cDNA mixture dilution series was prepared and used for real-time RT-qPCR as templates to obtain a standard curve for each gene. The housekeeping gene β-actin was used as an internal reference.

Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Nacalai Tesque, Inc.) and phosphatase inhibitor cocktail (Nacalai Tesque, Inc.), followed by a brief sonication (BIORUPTOR UDC250; Cosmo Bio). Protein concentration of the lysates was measured using Bio-Rad DC kits (Bio-Rad Laboratories, Inc.). The lysates containing 15 µg protein/lane were separated by 4–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto Immobilon-P membranes (EMD Millipore). Membranes were blocked with Blocking-one (Nacalai Tesque, Inc.) overnight at 4°C, and incubated with anti-E2F5 (dilution 1:500; cat. no. GTX129491; GeneTex, Inc.), anti-TP53 (DO1; dilution 1:1,000; cat. no. sc-126; Santa Cruz Biotechnology, Inc.), anti-phospho-TP53 at Ser-15 (dilution 1:1,000; product no. 9284; Cell Signaling Technology, Inc.), anti-p21^WAF1 (dilution 1:500; cat. no. sc-756; Santa Cruz Biotechnology, Inc.), anti-BAX (dilution 1:1,000; cat. no. 2772; Cell Signaling Technology, Inc.), anti-PARP (dilution 1:1,000; product no. 9542; Cell Signaling Technology, Inc.) or with anti-β-actin antibody (dilution 1:5,000; product no. A5441; Sigma-Aldrich; Merck KGaA) at 4°C. After 24 h of incubation, the membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBS-T), followed by incubation with horseradish peroxidase-conjugated secondary antibodies for mouse (dilution 1:2,000; cat. no. NA931V) or for rabbit (dilution 1:2,000; cat. no. NA934V; both GE Healthcare Life Sciences) for 1 h at room temperature. The membranes were washed extensively with TBS-T, and treated with Chemi-Lumi-One Super (Nacalai Tesque, Inc.) to visualize immunoreactivity using LAS4000 (Fujifilm). Intensity of the bands for TP53 and phosphorylated TP53 were measured by ImageJ software ver. 1.48 (National Institutes of Health).

Statistical analyses to examine the significance of differences between paired data were performed by Student's t-test, and one-way ANOVA followed by post hoc Tukey test was used to examine significance of differences among multiple data. All of the statistical analyses were performed using JMP software ver. 11.2 (SAS Institute, Inc.). Data are presented as the means ± SD from at least three independent experiments. In all analyses, P<0.05 was considered to indicate a statistically significant difference.

To clarify the potential role of E2F5 in the development and/or the progression of breast cancer, the expression level of E2F5 in the triple-negative-type breast cancer MDA-MB-231 cells, HER2-positive-type breast cancer BT474 cells, plus the luminal-type breast cancer MCF7 and MDA-MB-175VII cells, was examined. As revealed in Fig. 1, real-time RT-qPCR analysis demonstrated that there were no significant differences in the E2F5 expression levels among the cell lines examined. MDA-MB-231 (triple-negative-type) and MCF7 (luminal-type) cells were used for further experiments.

siRNA-mediated knockdown of E2F5 in MCF7 and MDA-MB-231 cells was then performed. Under the experimental conditions, E2F5 mRNA and protein expression levels were efficiently downregulated in MCF7 (Fig. 2A and C) and MDA-MB-231 cells (Fig. 2B and D). Next, the possible effects of E2F5 downregulation on cell proliferation were examined. As revealed in Fig. 2E and F, downregulation of E2F5 expression caused a significant decrease in the proliferation rates of MCF7 and MDA-MB-231 cells, indicating that E2F5 may regulate proliferation of breast cancer cells regardless of their subtype.

Since E2F5 knockdown resulted in a significant decrease in the proliferation rates of MCF7 and MDA-MB-231 cells, the possible effects of E2F5 knockdown on cell cycle distribution was examined. FACS analysis demonstrated a clear increase in the proportion of cells with sub-G1 DNA content, indicating that the proportion of dead cells was increased, in E2F5-knockdown MCF7 cells in comparison to control cells (Fig. 3A and C). In contrast, depletion of E2F5 had no detectable effect on the number of MDA-MB-231 cells with sub-G1 DNA content (Fig. 3B and D). Further analysis of these results indicated that the proportion of mitotic cells was marginally increased in E2F5-knockdown MDA-MB-231 cells relative to the control cells (Fig. 3D). These observations indicated that E2F5 may regulate breast cancer cell proliferation through distinct mechanisms in different types of cells.

Finally, the molecular mechanisms underlying the cell death mediated by E2F5 knockdown in MCF7 cells were examined. As MCF7 cells carry the wild-type TP53 (23) and MDA-MB-231 cells carry mutant TP53 (23), we postulated that knockdown of E2F5 in MCF7 induced cell death via the TP53-dependent pathway. The observation that the effects of E2F5 knockdown in BT474 cells carrying mutant TP53 (23) were similar to those in MDA-MB-231 cells (Fig. S1A) supported this theory. Real-time RT-qPCR experiments revealed that several TP53-target genes implicated in the induction of cell cycle arrest and/or cell death including p21^WAF1, BAX, NOXA and PUMA were significantly upregulated in E2F5-depleted MCF7 cells compared to control cells (Fig. 4A). In contrast, E2F5 gene silencing resulted in induction of p21^WAF1 and NOXA but not of BAX and PUMA in TP53-mutant MDA-MB-231 cells (Fig. 4B). In another TP53-mutant cell line BTB474, silencing of E2F5 induced all of the TP53 target genes aforementioned except PUMA, but to a lesser degree (Fig. S2A). To further confirm these results, immunoblotting analysis was performed. Consistent with the results of real-time RT-qPCR, E2F5 depletion promoted the expression of p21^WAF1 and BAX in MCF7 cells (Fig. 5A). The level of cleaved PARP was clearly increased, and the expression levels of TP53 and phosphorylated TP53 at Ser-15 were increased in E2F5-knockdown cells (Fig. 5A and C). The intensity ratio of phosphorylated TP53 to TP53 was also higher in E2F5-knockdown cells compared to control cells (Fig. 5C). In a sharp contrast to MCF7 cells, knockdown of E2F5 expression in MDA-MB-231 cells had a negligible effect on the expression levels of p21^WAF1 and BAX, and the expression levels of TP53 and phosphorylated TP53 at Ser-15 were not altered by E2F5 knockdown (Fig. 5B and D). Similar results as in MDA-MB-231 cells were also obtained in BT474 cells (Fig. S2B and C).

Since the results outlined above indicated that E2F5 regulates cellular proliferation and cell death in a TP53-dependent manner in MCF7 cells, the effects of E2F5 silencing in TP53-silenced MCF7 cells were next examined (Fig. 6A). As revealed in Fig. 6B, the viability of MCF7 cells transfected with both E2F5 siRNA and TP53 siRNA was almost the same as that of control cells. In addition, the proportion of double-knockdown cells with sub-G1 DNA was also the same as the control cells (Fig. 6C and D). Real-time RT-qPCR analysis demonstrated that induction of p21^WAF1, BAX and PUMA by E2F5 depletion was significantly suppressed by co-depletion of TP53 in comparison to siRNA-E2F5 (Fig. 7A). The level of NOXA expression was also significantly reduced in the double-knockdown cells compared to the cells in which only E2F5 was silenced, however the extent of the reduction was not as marked as in the other 3 genes (Fig. 7A). Consistent with these results, induction of p21^WAF1 and BAX and increase in the level of cleaved PARP in E2F5-silenced cells were strongly suppressed by co-transfection with TP53 siRNA (Fig. 7B).