Human BC UMUC3 and EJ cell lines were purchased from the American Type Culture Collection (ATCC). EJ cells were authenticated by high-resolution small tandem repeat profiling and were confirmed mycoplasma-free before experiments began. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml), at 37°C in a balanced air humidified incubator with an atmosphere of 5% CO₂. Res cell lines were established via long term incubation (2 months) at 37°C with increasing concentration of cisplatin (Sigma-Aldrich; Merck KGaA) in a range of 0–4 µg/ml, and then were steadily grown in the presence of 2 µg/ml cisplatin. For PTX treatment, the Res cells or the parental UMUC3 and EJ cells were treated with 80 or 40 nM PTX (Sigma-Aldrich; Merck KGaA), respectively, for 3 days. For serum-starvation, cells were treated with serum-free medium for 48 h and then synchronized with 0.1 µM dexamethasone for 2 h to examine the circadian rhythm. Cells were incubated with serum-free medium for 24, 48 and 72 h, and the circadian proteins were examined via western blotting.

Cells were synchronized in circadian time via aspiration of media and replacement with fresh DMEM containing 0.1 µM dexamethasone (Sigma-Aldrich; Merck KGaA). The cells were synchronized for 2 h at 37°C, followed by replacing with fresh medium (circadian time 0; ZT0). The cells did not receive any further medium changes from this point until the time of harvest. Individual plates were harvested for total RNA at ZT0, ZT4, ZT8, ZT12, ZT16, ZT20, ZT24, ZT28, ZT32, ZT36, ZT40, ZT44 and ZT48. Statistical analysis was cosine fitted using OriginPro 8.0 (https://www.originlab.com/).

For cell cycle analysis, cells were fixed in 70% ethanol overnight at 4°C and then were washed twice with ice-cold PBS. Cells were stained with PI staining solution (50 µg/ml PI, 100 µg/ml RNase, 0.05% Triton X-100 in ddH2O) at room temperature for 20–30 min. PI-stained cells were analyzed for their DNA content using FACSCalibur flow cytometer (BD Biosciences) and the data analyzed using FlowJo 7.6 (www.flowjo.com).

Cellular apoptosis was measured using the Annexin V-FITC Apoptosis Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.). Briefly, 1×10⁵ cells were resuspended in Annexin V binding buffer and stained with Annexin V-FITC and PI (1 µg/ml) at room temperature for 15 min. The apoptotic rate was quantified via FACSCalibur flow cytometer (BD Biosciences) and the data analyzed using FlowJo 7.6 (www.flowjo.com).

To assess cell viability, the cells were seeded (5×10³ cells/well) into 96-well plates and incubated at 37°C with 5% CO₂ for 24 h. Then, the cells were treated with increasing concentration of cisplatin (0–40 µg/ml) or PTX (0-1,000 nM) for 48 h at 37°C. After incubation, 20 µl MTS solution was added into each well and incubated for 2 h at 37°C. The number of viable cells was evaluated by measuring the absorbance at 490 nm with a microplate reader (Thermo Fisher Scientific, Inc.). In total ≥3 independent experiments were conducted.

Total RNA was extracted from cells with TRIzol^® reagent (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. cDNA was synthesized with PrimeScript™ RT Master mix (Takara Biotechnology Co., Ltd.) for 15 min at 37°C, and then the reverse transcriptase was inactivated for 5 sec at 85°C. qPCR was performed with SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus; Takara Biotechnology Co., Ltd.) with the following conditions in an Applied Biosystems instrument: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 5 sec and 72°C for 15 sec, 95°C for 15 sec, 60°C for 60 sec. The samples were quantified with 2^−ΔΔCq method and β-actin was used as internal reference (24). The following primers were used: BMAL1 forward, 5′-TGCAACGCAATGTCCAGGAA-3′ and reverse, 5′-GGTGGCACCTCTTAATGTTTTCA-3′; CLOCK forward, 5′-TGCGAGGAACAATAGACCCAA−3′ and reverse, 5′-ATGGCCTATGTGTGCGTTGTA−3′; CRY1 forward, 5′-TTGGAAAGGAACGAGACGCAG−3′ and reverse, 5′-CGGTTGTCCACCATTGAGTT-3′; PER2 forward, 5′-GACATGAGACCAACGAAAACTGC-3′ and reverse, 5′-AGGCTAAAGGTATCTGGACTCTG-3′; β-Actin forward, 5′-CATGTACGTTGCTATCCAGGC−3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT−3′; and p53 forward, 5′-CAGCACATGACGGAGGTTGT-3′ and reverse 5′-TCATCCAAATACTCCACACGC−3′.

Total proteins from cells were extracted with RIPA lysis buffer containing 1 mM PMSF (Fude: http://www.fdbio.net) and phosphatase inhibitor (Fude). Protein concentration was measured using a BCA Protein Assay kit (CWBio: http://www.cwbiotech.bioon.com.cn). The proteins (50 µg/lane) were loaded on 10% SDS-PAGE and separated by electrophoresis, followed by blotting on a PVDF membrane (EMD Millipore). The membrane was blocked in Tris-buffered saline (pH 8.0)+0.1% Tween-20 and 5% skim milk at room temperature for 2 h. The corresponding primary antibody (1:1,000) was incubated overnight at 4°C and then incubated with the horseradish peroxide-conjugated secondary antibody (Fude; 1:10,000) at room temperature for 1 h. Immunological signals were detected using an electrochemical luminescence kit (Yeasen; www.yeasen.com). The band intensities were quantified using Image-Pro-Plus 6.0 software (MediaCybernetics, Inc.). The primary antibodies are as follows: CRY1 (cat. no. ab245564; Abcam), PER2 (cat. no. ab179813; Abcam), CLOCK (cat. no. 18094-1-AP; ProteinTech Group, Inc.), BMAL1 (cat. no. 14268-1-AP; ProteinTech Group, Inc.), p53 (cat. no. 2524; Cell Signaling Technology, Inc.), p21 (cat. no. 10355-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. AP0063; Biogot Technology Co., Ltd.).

Cell proliferation was detected with a EdU labeling/detection kit (Guangzhou RiboBio Co., Ltd.). Cells were plated in 24-well plates (1×10⁵ cells/well). EdU labeling medium (1:1,000) was added into wells and incubated for 2 h at 37°C according to the manufacturer's instructions. After EdU staining, cells were counterstained with Hoechst 33342 for 30 min at room temperature. The percentage of EdU⁺ cells was calculated from five random fields each in three wells via fluorescence microscopy (magnification, ×400; Nikon Corporation).

Cry1-targeting siRNAs (si-Cry1-1, 5′-GATGCAGATTGGAGCATAA-3′; and si-Cry1-2, 5′-GGATGAAACAGATCTATCA-3′) were designed by Guangzhou RiboBio Co., Ltd. Cells (5×10⁵) were seeded into 6-well plates and then were transfected with 100 nM Cry1 siRNA at 37°C for 24 or 48 h using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Non-targeting siRNA was used as control.

Senescent cells were detected using the SA-β-Gal staining kit according to the manufacturer's instruction (Cell Signaling Technology, Inc.). Cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. Each well (6-well plates) was filled with 1 ml β-Gal staining solution. The plate was sealed with a parafilm and incubated at 37°C overnight in a dry incubator (no CO₂). The percentage of SA-β-Gal-positive cells were identified as bluish green-stained cells under a fluorescent microscope (magnification, ×400; Nikon Corporation).

After washing in cold PBS, cells were lysed with RIPA lysis buffer containing 1 mM PMSF (Fude) and phosphatase inhibitor (Fude). Total cell lysates (1.0 ml) were incubated with 1 µg anti-p53 (cat. no. 2524; Cell Signaling Technology, Inc.) overnight at 4°C, and then mixed with 40 µl protein-A + G conjugated beads (Beyotime Institute of Biotechnology) to each sample. Centrifuged at 4°C for 14,000 × g for 15 min. Beads were then washed with lysis buffer and centrifugation was repeated three times. The immunoprecipitated protein complexes were analyzed via western blotting.

Statistical analysis was performed using SPSS 20.0 software (IBM Corp.). Differences between groups were compared using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. In total, three duplicated experiment were performed.

To study drug resistance, Res UMUC3 and EJ cells were established via prolonged culture in the presence of cisplatin. As presented in Fig. S1, the selected cells were resistant to cisplatin, as well as PTX. Res cells exhibited normal proliferative profile as demonstrated by results of DNA replication (Fig. 1A), cell cycle distribution (Fig. 1B) and the expression of cell cycle-related proteins, including Cyclin D1, Cyclin E1 and minichromosome maintenance complex component 7 (MCM7) (Fig. 1C). Upon acute PTX treatment, Res cells presented with significantly decreased DNA replication (Fig. 1A) and G₁/G₀ accumulation (Fig. 1B). Moreover, the proteins expression levels of Cyclin D1, Cyclin E1 and MCM7 were significantly decreased (Fig. 1C). These data indicated that the Res cells entered a non-proliferative quiescent state upon PTX stress.

It has been reported that prolonged proliferation arrest could lead to cell senescence (25) and that PTX can induce cell senescence (16,17). In UMUC3 and EJ cells with a 2-day PTX exposure, it was observed that the staining of SA-β-Gal was 20–50 times higher compared with that in cells without PTX treatment (Fig. 1D). Accordingly, the expression levels of the canonical senescence-associated proteins, p53 and p21, were increased after PTX treatment; however, p53 expression demonstrated no statistical difference after PTX treatment in EJ cells (Fig. 1E). While UMUC3 cells carry a homozygous tumor protein p53 mutation, it has been revealed that p53 function still remains in UMUC3 cells (26). However, in the UMUC3 Res and EJ Res cells, nearly no SA-β-Gal positive signals were detected after PTX treatment (Fig. 1D). Consistently, p53 protein expression was decreased in PTX-treated Res cells (Fig. 1E). p21 expression was also decreased in EJ Res cells, but increased in the UMUC3 Res cells, indicating the discrepancy between the different cell types (Fig. 1E). According to the ATCC and a previous report, the cyclin dependent kinase inhibitor 2A (cdkn2a), a gene encoding p16 protein, is mutated or deleted in UMUC3 and EJ cells (27,28). Consistent with this, no p16 protein was detected in both cell lines (data not shown). These results demonstrated that the Res cells appeared to escape from senescence and remained in a quiescent state under PTX stress.

The circadian rhythm is closely associated with cell proliferation (29), and also regulates cell senescence (30). The expression levels of the core circadian genes, BMAL1, CLOCK, PER2 and CRY1, were detected in the Res cells. The mRNA expression levels of the four core circadian genes fluctuated over a period of 24 h, similar with those in the parental cells (Fig. 2A). However, the circadian oscillation was disturbed after PTX treatment demonstrated by an increased amplitude and a prolonged period in the Res cells. In the UMUC3 Res cells, the circadian period was ~48 h, and in the EJ Res cells circadian oscillation was not noticed in a period of 48 h (Fig. 2A). Moreover, the expression of the circadian genes at the protein level showed similar oscillation before and after PTX treatment. However, the amplitude of CRY1 oscillation was reduced by PTX in both UMUC3 Res (Fig. 2B) and EJ Res cells (Fig. S2).

To further clarify the relationship between the circadian rhythm and cell proliferation, the expression levels of circadian gene were examined in serum-starved cells. As expected, serum starvation induced a prolonged circadian oscillation (Fig. S3). These results indicated that the quiescent status of PTX-treated Res cells was accompanied with a prolonged circadian rhythm.

Next, the expression levels of the four circadian proteins were compared in the parental, Res and Res + PTX cells. In UMUC3 cells, BMAL1, CLOCK and PER2 expression levels were decreased in the Res and Res + PTX cells compared with parental UMUC3 cells, but there was no significant difference between Res and Res + PTX cells. Noticeably, CRY1 protein was significantly increased in the Res + PTX cells (Fig. 3A). In EJ cells, CRY1 and BMAL1 demonstrated similar trends as in UMUC3 cells. PER2 expression increased in the Res cells compared with control and Res + PTX cells. CLOCK expression was significantly increased in the Res + PTX group vs. Control and Res groups (Fig. 3A). The circadian proteins were further compared in the serum-starvation cells. After starvation for 72 h, CRY1 and PER2 accumulated significantly in both cell lines. Moreover, CLOCK and BMAL1 expression levels decreased in UMUC-3 cells, but there were no significant changes in EJ cells after serum-starvation for 72 h (Fig. 3B).

It was further examined whether CRY1 functioned as an inhibitor in PTX-induced senescence. To test this, siRNA was used to knockdown CRY1 expression in the PTX-treated Res cells. (Fig. 4A and B). After CRY1 knockdown, p21 mRNA expression was significantly downregulated (Fig. 4A), but its protein expression was not significantly changed in both Res cells (Fig. 4B). The p53 mRNA expression did not change significantly in EJ Res cells, but was significantly increased in UMUC3 Res cells (Fig. 4A). Moreover, its protein expression was significantly enhanced after CRY1 knockdown in both cell lines (Fig. 4B). The si-CRY1-1 demonstrated a higher efficiency compared with the si-CRY1-2 to inhibit CRY1 expression. Therefore, the si-CRY1-1 was used in subsequent experiments. It was identified that CRY1 knockdown induced apparent senescence in the PTX-treated Res cells (Fig. 4C). Furthermore, apoptosis was significantly enhanced in the PTX-treated Res cells by CRY1 knockdown (Fig. 4D). These results indicated that CRY1 contributed to resistance of senescence by decreasing p53 protein expression.

The E3 ubiquitin ligase MDM2 mediates p53 ubiquitination, leading to p53 degradation, which is the major mechanism for p53 protein turnover (31) Since CRY1 knockdown increased the expression of p53 protein, but not p53 mRNA, it was investigated whether CRY1 participated in MDM2-mediated p53 degradation in the Res cells. The Co-IP assay using anti-p53 antibody successfully detected an interaction between endogenous MDM2 and p53 proteins. It was found that CRY1 knockdown significantly decreased MDM2 expression in the immunoprecipitation complex (Fig. 5), suggesting that the MDM2/p53 interaction was attenuated by CRY1 knockdown. This indicated that CRY1 could facilitate the interaction of p53 with MDM2 and potentiate p53 degradation.