2.1 Pan-cancer analysis of MCM6 expression

To investigate the discrepancy expression of MCM6 in tumour versus normal tissues, we analysed the physiological mRNA levels of MCM6 across various normal tissues using the GTEx data set (Figure S1A). As illustrated in Figure 1A, the expression level of the MCM6 gene was found to be significantly upregulated in numerous cancer types, including bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), oesophageal carcinoma (ESCA), glioblastoma multiforme (GBM), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD) and uterine corpus endometrial carcinoma (UCEC). Additionally, data from canSAR.ai indicated that MCM6 expression correlated with the pathological stages of most cancers (Figure S1B). Following this, we utilized canSAR.ai and DISGENET Plus to evaluate the relationship between elevated MCM6 expression and various cancers and diseases. The upregulation of MCM6 was found to have a strong correlation with leukaemia, breast cancer, male-specific cancers, skin cancer and head and neck cancer, while also demonstrating a significant association with lactose intolerance and some symptom (Figure S1C).

2.2 High expression of MCM6 has clinical value in BRCA

To explore the potential role of MCM6 in tumorigenesis and the unclear mechanisms underlying MCM6 dysregulation in the neoplastic process, we utilized the BioGRID online database to construct MCM6-binding protein–protein interaction (PPI) networks (Figure S2A). Subcluster analysis performed through Metascape indicated that MCM6-binding proteins were primarily clustered with proteins involved in cell cycle regulation, DNA replication, and DNA damage repair (Figure S2B). Additionally, further Kyoto Encyclopedia of Genes and Genomes and Genome Ontology (KEGG/GO) enrichment analysis, as conducted by Coexpedia and Metascape, revealed that MCM6 and its binding proteins were predominantly associated with DNA replication, DNA damage repair, and cell cycle regulation (Figure 1B, Figure S2C,D).

According to the Coexpedia database and the MeSH (Medical Subject Headings) database, we identified that the medical subject term most strongly correlated with MCM6 was breast cancer (Figure 1C). Consequently, we further investigated the specific association between MCM6 and breast cancer. Our findings demonstrated a significant increase in MCM6 expression in BRCA (Figure 1D). Additionally, data from the Human Protein Altas (HPA) indicated that the staining intensity of MCM6 was markedly elevated in BRCA tissue compared to normal tissues, where it was undetectable (Figure 1E).

Subsequently, we assessed the diagnostic potential of MCM6 in breast cancers through receiver operating characteristic (ROC) analysis. Our results, illustrated in Figure 1F and Figure S3A, indicated that MCM6 may serve as an excellent diagnostic marker across multiple cancers, including breast cancer (AUC >0.5). We further analysed the associations between the overall survival (OS) and progressive free interval (PFI) of patients and MCM6 expression levels using Kaplan–Meier survival analysis. The results demonstrated a significant negative correlation between increased MCM6 expression levels and both OS and PFI in BRCA (Figure 1F and Figure S3B,C). The officially recognized biomarkers for breast cancer include BRCA1, BRCA2, PIK3CA, MKI67, PALB2, ATM, CHEK2, RAD51C, BARD1 and TP53, as reported in the latest publication by The Lancet. To further elucidate the correlation between MCM6 and breast cancer, a relevance analysis revealed that MCM6 exhibits distinct and positive relevance with these biomarkers (Figure 1G).

Next, we conducted a comprehensive bibliometric analysis utilizing the Web of Science (WOS) database. Notably, MCM6 has emerged as a significant trend and focal point in recent years, particularly since 2021. Several countries and research institutions are concentrating on fundamental related to MCM6, while comparatively limited studied address its role in breast cancer (Figure S4A–C).

Subsequently, we employed the Linkedomics database to construct the co-expression network of MCM6 in BRCA, aiming to elucidate the primary pathways in which MCM6 is involved. The top five positively correlated genes identified are MCM3, MCM5, MCM2, MCM7 and MCM4 (Figure 1H). Furthermore, KEGG analysis revealed enrichment in pathways associated with DNA replication, chromosome segregation, cell cycle checkpoint regulation and DNA damage response (Figure S4D). Analysis of single-cell sequencing data obtained from cancerSEA, indicates that MCM6 predominantly participates in DNA replication, cell cycle regulation, DNA damage repair and the DNA damage response during the progression of breast cancer (Figure 1I). This evidence suggests that MCM6 is likely to play a critical role in the regulation of DNA replication and the DNA damage response in breast cancer progression.

In conclusion, MCM6 is correlated with an increased risk of breast cancer, indicating that it serves as a putative and vital regulator in breast cancer, especially in regulating its DNA replication and DNA damage response.

2.3 Knockdown of MCM6 inhibited proliferation of breast cancer cells

To confirm the tumour-promoting role of MCM6, we selected the high expression of the MCM6 breast cancer cell line MCF7 based on data from the Cancer Cell Line Encyclopedia (CCLE) and HPA data sets (Figure S4E,F). We subsequently knocked down MCM6 expression in breast cancer cells MCF7 and 4 T1 cells (The siMCM6#1 was utilized for subsequent research, hereinafter denoted as siMCM6) (Figure 2A), which resulted in suppression of cell growth in vitro (Figure 2B). This observation was corroborated by colony formation assays and BrdU staining, which demonstrated that knockdown of MCM6 inhibited colony formation and reduced the fraction of BrdU-positive cells (Figure 2C,D). Furthermore, MCM6-knockdown affected the cell cycle, by decreasing the S-phase fraction and increasing the G1-phase fraction (Figure 2E). Our analysis of DNA fibres revealed a substantial reduction in the length of ongoing DNA replication following MCM6 deficiency, indicating that MCM6-knockdown remarkably obstructed DNA replication and resulted in stalled DNA replication forks (Figure 2F). These findings suggest that aberrant expressed MCM6 regulates breast cancer cell growth and proliferation, while safeguarding DNA replication from DNA replication stress induced by MCM6 deficiency.

2.4 Knockdown of MCM6 DNA inducing replication stress caused cell apoptosis

The replication-coupled DNA damage repair mechanism effectively addresses the challenges posed by DNA replication stress, thereby facilitating the completion of DNA synthesis.³⁰ To investigate whether the replication stress induced by knockdown of MCM6 was a consequence of endogenous DNA damage, we assessed DNA damage following depletion of MCM6. As anticipated, MCM6-knockdown significantly increased DNA damage in MCF7 cells, as evidenced by the length and intensity of the comet tail signal. Furthermore, treatment with hydroxyurea (HU) exacerbated DNA replication stress, a finding corroborated by the DNA comet assay (Figure 2G). This result suggested that MCM6-knockdown led to the accumulation of DNA damage following HU treatment. Mechanistically, MCM6-knockdown was associated with elevated levels of γH2AX, a well-established marker of DNA damage (Figure 2H, Figure S5A). Additionally, the key factors involved in DNA damage repair, BRCA1 and Rad51, remained persistently increased in MCM6-deficient cells, implying that the absence of MCM6 compromises the efficiency of DNA damage repair (Figure 2I, Figure S5B,C).

Long-standing DNA damage triggers cell apoptosis, contributing to genomic instability. Flow cytometry analysis, utilizing Annexin V-PE and 7-AAD double staining, demonstrated that MCM6-knockdown induced apoptosis in MCF7 cells, as evidenced by an increase in the populations of 7-AAD and Annexin V positive cells (Figure 2J). Additionally, TUNEL-positive cells were identified through green fluorescence staining, revealing a substantial number of TUNEL-positive cells in MCM6-depleted breast cancer cells, consistent with the results from Annexin V staining (Figure S5D,E). Moreover, MCM6-knockdown led to the upregulation of the pro-apoptosis gene Bax expression while downregulating the anti-apoptosis gene Bcl-xl expression (Figure S5F), suggesting that MCM6 serves as a crucial regulatory factor in cell apoptosis.

It is reasonable to speculate that MCM6-knockdown induced DNA damage triggers cellular apoptosis, enhancing the sensitivity to chemotherapy. Furthermore, resistance to paclitaxel (PTX) contributes to poorer clinical outcomes for patients with breast cancer. MCM6 has been shown to increase the sensitivity of breast cancer cells to PTX in a dose-dependent manner (Figure 2K). Collectively, these findings suggest that MCM6 plays a crucial role in DNA damage repair and enhances sensitivity to chemotherapy to breast cancer.

2.5 MCM6-Kcr is upregulated upon DNA replication stress

Since the advancement of modern technology, the characterisation of Kcr has been extensively detailed, however, the functional elucidation of non-histone modifications remains in its infancy. We employed an integrated approach that combined label-free quantification, high-performance liquid chromatography (HPLC) fractionation and high-resolution LC–MS/MS to investigate Kcr substrates in response to DNA replication stress stimuli (Figure 3A). The results indicated that 85 Kcr sites in 82 proteins were upregulated, while 33 sites in 32 proteins were downregulated in the IR-treated group compared with the control group (CTRL) (Figure S6A). Notably, crotonylation of MCM6 (MCM6-Kcr) was extremely upregulated in IR-treated cells (Figure 3B, Figure S6B). KEGG/GO enrichment analysis revealed that the upregulated Kcr proteins are predominantly involved in various cellular processes, particularly in response to stimuli (Figure 3C, Figure S6C). This finding provides new insight into the role of MCM6-Kcr, suggesting it may serve as a potential target for breast cancer treatment in regulating DNA damage response.

To further investigate whether MCM6 undergoes crotonylation in response to DNA replication stress, MCF7 cells were subjected to HU and IR, following by immunoprecipitation. The lysates were analysed using coomassie blue staining. The results indicated a significant upregulation of MCM6-Kcr levels following exposure to IR and HU (Figure 3D). Chromatin-binding extracts from both control or HU-treated cells were immunoprecipitated with anti-MCM6 antibodies and subsequently analysed via immunoblotting with specific antibodies. The findings demonstrated that MCM6-Kcr levels markedly increased upon HU treatment, suggesting that MCM6-Kcr significantly heightened the DNA damage (Figure 3E). Furthermore, sodium crotonate (NaCR) was utilized as an exogenous crotonyl donor to promote MCM6-Kcr, allowing for the assessment of its effects on the viability of breast cancer cells. An increase in NaCR treatment concentration and duration led to a progressive inhibition of the cell cycle due to enhanced MCM6-Kcr level, as evidenced by the phosphorylation of checkpoint kinase 2 (p-CHK2), a recognized marker of cell cycle arrest. This implies that MCM6-Kcr induced DNA replication stress, thereby resulting in cell cycle arrest (Figure 3F, Figure S6D). Additionally, cell proliferation and colony formation assays further confirmed that MCM6-Kcr suppresses breast cancer cell growth due to cell cycle blockage (Figure 3G–J).

2.6 MCM-K599cr induced DNA replication stress, resulting in cell apoptosis

Based on tandem MS, the putative MCM6-Kcr site lysine 599 (MCM6-K599cr) was measured to increase by a factor of 4.007 under HU-induced DNA replication stress, located at MCM6 AAA⁺ ATPase domain (Figure S7A,B). To demonstrate the evolutionary relationship and conservation of the site, we examined the identified MCM6-K599 using MEGA 7.0 and WebLogo software, which indicated that MCM6-K599 was conserved among multiple species (Figure S7C,D). A detailed analysis of the identified MCM6-K599 site via the PhosphoSitePlus website revealed that MCM6-K599 could also undergo ubiquitination and acetylation. This suggests that MCM6-K599 may serve as a hotspot for various PTMs, highlighting the vital functionalities associated with PTMs of MCM6-K599 (Figure S7E).

To investigate the specific Kcr sites, site-specific mutagenesis was employed to substitute K599 with glutamine (Q) and alanine (A) to mimic or inhibit crotonylation, respectively. MCF7 and 4 T1 cells transfected with HA-tagged wild type or point mutant MCM6 were validated by DNA sequencing (Figure S7F). To determine the mechanism underlying the role of MCM6-Kcr in HU-induced DNA replication stress and NaCR-induced cell cycle arrest, we assessed cell growth and proliferation using cell proliferation assays, colony formation assays, and BrdU assays. The results showed that breast cancer cells expressing the MCM6-K599Q mutant (referred to as K599Q) exhibited reduced cell growth and migration compared to the CTRL and cells expressing the MCM6-K599A mutant (referred to as CTRL and K599A, respectively) (Figure 4A–C, Figure S8A). Consistently, the K599Q mutant also resulted in cell cycle arrest in the G1 phase, mirroring the phenotype observed with MCM6-knockdown cells (Figure 4D). Additionally, we employed a DNA fibre assay to investigate whether MCM6-K599cr induced DNA replication stress. The findings confirmed that the K599Q mutant led to evident DNA replication stress characterised by numerous stalled DNA replication forks. In contrast, both the CTRL and K599A mutant exhibited comparable DNA replication lengths and spreads (Figure 4E).

Our observation of significantly increased comet tail formation in the K599Q mutant indicates DNA damage, accompanied by an extended accumulation of fragmented DNA (Figure 4F). HU further exacerbated DNA damage in the K599Q mutant, as confirmed by γH2AX staining assay. This suggests that MCM6-Kcr enhances DNA replication stress and aggravates DNA damage (Figure S8B). Further supportive evidence was obtained through western blot analysis of chromatin-binding proteins, which revealed increased expression of γH2AX, BRCA1, and Rad51 in K599Q mutant cells, particularly following HU-induced DNA replication stress (Figure 4G). Moreover, BRCA1 and Rad51 were also detected in these cells. Consistent with the γH2AX staining results, the K599Q mutant continued to display numerous foci even after a 12 h repair interval (Figure S8C,D). Collectively, these findings indicate that MCM6-Kcr induces DNA replication stress and compromises that ability to repair long-standing DNA damage.

The Annexin V-PE/7-AAD flow cytometry assay demonstrated a significant increase in Annexin V-positive cells (Figure 4H). The TUNEL assay revealed that the K599Q mutant markedly enhanced TUNEL-positive signals, which were further corroborated by Annexin V staining (Figure S8E,F). In alignment with these results, western blot analysis indicated an increase in Bax expression accompanied by a decrease in Bcl-xl expression (Figure S8G).

Subsequently, MTT assay results indicated that K599Q mutant cells exhibited a significant reduction in cell viability in response to PTX (Figure 4I). Collectively, these results strongly suggest that MCM6-K599cr may be implicated in the chemotherapeutic resistance associated with cell apoptosis.

2.7 MCM6-K599cr blocked MCM2-7 complex assembly, which may be regulated by its ubiquitination

MCM6 is a critical component of the MCM2-7 complex, closely linked with MCM2 and MCM4, respectively. Structural analysis using date from the Protein Data Bank (PDB) revealed that MCM6-K599 exhibits a strong interaction with MCM2-E673 and MCM2-F904 (Figure 5A), implying MCM6-K599cr may have potential roles to regulate the MCM2-7 complex assembly. To this end, co-immunoprecipitation assay indicated that the K599Q mutant disrupts its interaction with MCM2 in chromatin lysates (Figure 5B). Additionally, an exogenous HA-tag immunoprecipitationassay and reciprocal co-immunoprecipitation using Flag-MCM2 confirmed that MCM6-K599cr inhibited its interaction with MCM2 (Figure 5C). These findings indicate that MCM6-Kcr is a primary target influencing the formation of the MCM2-7 complex formation, which subsequently induces replication stress.

As shown in Figure S7E, the MCM6-K599 site may also be subject to ubiquitination. Our work team has previously investigated RNF8-mediated protein lysine ubiquitination (Kub) in regulating multiple cellular processes.^31-33 Based on our identical label-free proteomics analysis of Kub, 351 Kub sites in 272 proteins were downregulated in RNF8-knockdown cells (siRNF8), including MCM6 (Figure 5D), which are primarily involved in metabolic processes, responses to stimuli, developmental processes and immune system processes (data not show). The comparison of the Kcr and Kub results revealed 22 overlapping proteins. Notably, MCM6 could also undergo ubiquination in an RNF8-dependent manner (Figure 5E).

To investigate the mechanism of RNF8-dependent MCM6-Kub in MCF7 cells, we first generated RNF8-knockdown and RNF8-overexpressing mutants (referred to as siRNF8 and oeRNF8, respectively), which were then transfected into MCF7 cells. The co-immunoprecipitation assay results showed that depletion of RNF8 remarkably inhibited MCM6-Kub, a process that be rescued by overexpressing of RNF8 (Figure 5F,G). Given that both MCM6-Kcr and MCM6-Kub are present concurrently in the cells, we next sought to determine whether the dynamic transformation between MCM6-Kcr and MCM6-Kub is regulated by RNF8. Co-immunoprecipitation assays demonstrated an increase in the MCM6-Kcr level. Conversely, MCM6-Kub decreased following treatment with 5 mM NaCR (Figure 5H). This observation aligns with the finding that RNF8 deficiency leads to reduced MCM6-Kub levels alongside elevated MCM6-Kcr levels (Figure 5I). These results further support the idea that MCM6-Kcr and MCM6-Kub may influence one another through RNF8, suggesting that RNF8 could represent a novel target for therapeutic intervention in breast cancer.

2.8 The crotonylation eraser SIRT7 decreased MCM6-Kcr during DNA replication stress

Previous studies have demonstrated that certain histone deacetylases exhibit de-crotonylation activity.³⁴ To clarify the decrotonylase of MCM6, we treated cells with the specific HDAC family inhibitor Trichostatin A (TSA) and the SIRT family inhibitor Nicotinamide (NAM) to identify the decrotonylase responsible for MCM6. Co-immunoprecipitation experiments involving chromatin-binding proteins revealed that treatment with 10 mM NAM resulted in an increased MCM6-Kcr level, whereas treatment with 1 μM TSA did not (Figure 6A,B). This suggests that the SIRT family may function as a potential decrotonylase for MCM6.

Currently, seven mammalian SIRT members named SIRT1 to SIRT7 have been identified. As far as we know, SIRT1, SIRT6 and SIRT7 are mostly located in the chromatin, whereas SIRT3-5 localizes primarily to mitochondria and SIRT2 is mostly found in the cytoplasm. Meanwhile, some reports also indicated that SIRT1,³⁵ SIRT6^{36, 37} and SIRT7^38-40 were considered as the central regulator of DNA damage repair and were usually caused by the overproduction of reactive oxygen species (ROS).⁴¹ Thus, we designed siRNA to target knockdown SIRT1, SIRT6 and SIRT7. Knockdown of SIRT7 but not SIRT1 or SIRT6 significantly rescued the level of MCM6-Kcr (Figure 6C). MCM6-Kcr levels were significantly elevated in the chromatin segment of cells with SIRT7 depletion (Figure 6D). To validate this observation, an in vitro decrotonylase assay was conducted using a dual transfection model, which further corroborated SIRT7 inhibiting MCM6-Kcr (Figure 6E). Consequently, we concluded that SIRT7 functions as a specific decrotonylase for MCM6, thereby modulating MCM6-Kcr and inducing replication stress (Figure 6F).

2.9 Kaempferol targeted SIRT7 to modulate MCM6 crotonylation

Several natural products have been reported to reverse chemoresistance by inhibiting proliferation, regulating DDR-related proteins and inducing apoptosis in cancer cells, such as flavonoids compounds.^{42, 43} Natural products have unique advantages with low toxicity and multi-target features, making them widely used against chemoresistance. Therefore, we aimed to identify a small molecule compound targeting SIRT7 to modulate the MCM6-Kcr, thereby reversing chemotherapeutic resistance. Utilizing the STITCH, SymMap V2 and CTD database, we identified kaempferol as a potential ingredient targeting SIRT7 (Figure 6G, Figure S9A). Molecular docking analysis using CB-dock 2.0 revealed that kaempferol binds to SIRT7 through two binding pockets with a strong binding affinity, as indicated by an absolute vina score less than −7.0 (vina score = −8.4; vina score = −7.6) (Figure S9B).

As illustrated in Figure 6H, MCM6-Kcr exhibited a clear dose-dependent response to kaempferol, while no significant difference in MCM6 level was observed. A similar result was obtained through chromatin protein detection (Figure 6I). As excepted, kaempferol upregulated MCM6-Kcr by inhibiting SIRT7 expression (Figure 6J).

2.10 Kaempferol suppressed proliferation and induced DNA replication stress in breast cancer cells

Kaempferol, a flavonoid compounds, has been shown to inhibit the proliferation of oral cancer cells. It also increased the expression of γH2AX and caspase-3, leading to DNA damage and inducing cell apoptosis.⁴⁴ However, the precise mechanism by which kaempferol exerts therapeutic effects in breast cancer, potentially through the modulation of SIRT7-mediated MCM6-Kcr, remains to be fully elucidated.

Based on BATMAN database and DO analysis (Figure S10A,B), a total of 52 genes were recognized as candidate targets, while 64 disease targets were found in CTD database and 59 disease targets in OMIM database, all of which are closely related to breast cancer. Pathway enrichment analysis from KEGG/GO enrichment indicated that kaempferol is primarily involved in pathways in cancer, response to stimuli, cellular process and cell growth (Figure S10C,D). These findings suggest that kaempferol may regulate DNA replication stress, hereby modulating the progression of breast cancer.

Further validation of the effect of kaempferol was conducted through proliferation and cell cycle assays. The results showed that kaempferol inhibits cell growth and blocks cell cycle (Figure 7A–D). We subsequently assessed DNA replication stress and DNA damage. As illustrated in Figure 7E,F, kaempferol suppressed DNA synthesis and induced a significant accumulation of stalled DNA forks, leading to replication-stress-induced DNA damage in a dose-dependent manner (Figure 7G). We also investigated whether kaempferol induced cell apoptosis similar to MCM6-Kcr. As anticipated, kaempferol resulted in cell apoptosis (Figure 7H).

2.11 Kaempferol inhibited tumour growth via enhancing PTX sensitivity

Under identical experimental conditions, strong enhanced antiproliferative activity of kaempferol (KPL) on PTX regulation of cell proliferation was demonstrated in MCF7 and 4 T1 cells (Figure 8A). Encouraged by these results, we subsequently assessed the antitumor effects of paclitaxel-kaempferol (PTX-KPL) treatment in vivo. 4 T1 cells were subcutaneously injected into the flank of BALB/c mice to establish the tumour model. The tumour-bearing mice were randomly divided into four groups and subjected to the injection schedule outline in Figure 8B. Tumour measurements were taken after 21 days post-injection (Figure 8C). Compared to the CTRL, both tumour weight and volume exhibited a gradually decrease (p < 0.05) following treatment with either PTX or KPL alone, while the PTX-KPL combination therapy demonstrated significant anti-tumour effects (p < 0.01) (Figure 8D,E).

Additionally, we evaluated the potential toxicity of the different formulations. Body weight did not significantly differ among the groups, indicating that all treatments were devoid of evident toxicity (Figure 8F). Furthermore, the IHC and co-immunoprecipitation assay corroborated previous findings, showing that kaempferol enhanced potency of PTX by reducing SIRT7 expression, thereby inducing MCM6-Kcr-mediated DNA replication stress (Figure 8G,H).

4.1 Gene expression level analysis

The mRNA levels of MCM6 in a variety of cancers were analysed by Tumour Immune Estimation Resource, version 2 (TIMER2.0, TIMER2.0 (cistrome.org)).⁵⁶ Differential expression levels of MCM6 were observed between the cancerous region and normal tissue for various types of tumours. The range was set as follows:log2-fold change (log2 FC) ≥ 1 or ≤ −1 and p value ≤0.05. Gene Expression Profiling Interactive Analysis, version 2 (GEPIA2, GEPIA 2 (cancer-pku.cn))⁵⁷ was employed to analyse the MCM6 mRNA levels in certain tumours with no corresponding normal tissues. Additionally, violin plots were applied to reveal the relationship between MCM6 and pathological stages of cancers by using the “Stage Plot” and “PR status” of GEPIA. In addition, canSAR.ai (canSAR.ai | the Cancer Drug Discovery Platform)⁵⁸ and DisGeNET (DisGeNET—a database of gene-disease associations)⁵⁹ were utilized to analyse the correlations of MCM6 with cancers and diseases.

4.2 Survival prognosis analysis and gene enrichment analysis

The OS and DFS of all tumour patients in the GEO cohorts were analysed using the Kaplan–Meier plotter (Kaplan–Meierplotter[Breast](kmplot.com)) and PrognoScan tools (PrognoScan: A new database for meta-analysis of the prognostic value of genes. (kyutech.ac.jp)). Furthermore, we visualized the protein–protein interaction network using Coexpedia software (Coexpedia)⁶⁰ and clarified the potential pathways of the top 100 MCM6-binding proteins using Metascape (Metascape)⁶¹ and GPS Heml2.0 (HemI 2.0—Heatmap Illustrator(biocuckoo.cn)).⁶²

4.3 Cell lines, culture method, and reagents

The human embryonic kidney cell line HEK293 and the human breast cancer cell lines MCF7 and MDAMB231 were purchased from ATCC (Manassas, VA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Servicebio) containing 10% fetal bovine serum (FBS, HyClone) supplemented with 5 μL penicillin/streptomycin (Servicebio) in a 5% CO2 atmosphere at 37°C.

Sodium crotonate (NaCR, Absin, abs42074236), hycamptothecin (HCPT, Solarbio, IH2120), hydroxycarbamide (HU, Solarbio, H8420) and paclitaxel (PTX, Solarbio, YZ-100382) were dissolved in DMSO and used at the specified concentrations and times as indicated. The following primary antibodies were used for western blotting: MCM6 polyclonal antibody (Proteintech, 13347-2-AP), Anti-Crotonyllysine Mouse mAb (panKcr, ptm-biolab, PTM-502), RAD51 Polyclonal antibody (proteintech, 14961-1-AP), Anti-BRCA1 (Abcam, ab238983), Anti-53BP1 (Abcam, ab175933), anti-gamma H2A.X (phospho S139) (γH2AX, Abcam, ab81299), Anti-Chk2 (phospho T383) (p-CHK2, Abcam, ab59408), Beta Actin recombinant antibody (β-actin, Proteintech, 81115-1-RR), Histone-H3 Polyclonal antibody (proteintech, 17168-1-AP) and GAPDH Monoclonal antibody (Proteintech, 60004-1-lg). The following second antibodies were used for western blotting: HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Proteintech, SA00001-1) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00001-2).

4.4 Transfection with siRNA and plasmid DNA

The complementary DNA (cDNA) for control MCM6 was amplified by polymerase chain reaction and ligated into pcDNA plasmid containing HA-tag. For construction of the HA-MCM6-K599A and HA-MCM6-599Q plasmids, site-directed mutagenesis (Sino Biological) was applied and verified by sequencing. For generation of stable MCF7 RNF8 knockout cells, sgRNF8 were subcloned and inserted into the lentiCRISPRv2 vector. For transient transfection with plasmid DNA, MCF7 (K599A and K599Q) and MCF7 (siRNF8 and oeRNF8) cells were seeded in 6-well plates at a confluency of 70%–80% and transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium according to the manufacturer's protocol. After 48 h, MCM6 and RNF8 expression was evaluated by western blotting, and cells were used for downstream applications. For transfection with siRNAs, multiple predesigned siRNAs per target gene as well as scrambled control siRNA were obtained from Genepharma. MCF7 (siMCM6) cells were transfected at 70%–80% confluency in six-well plates with 50 nM of siRNA using GP-transfect-Mate (Genepharma) in Opti-MEM medium following the manufacture's protocol. Downregulation of target genes was analysed by western blotting and immunofluorescence microscopy 48 h after transfection.

4.5 Extraction of chromatin-bound proteins by biochemical fractionation

Cell fractionation was performed as described in the Bioprotocol.⁶³ Briefly, cells were harvested, washed with PBS and resuspended in E1 buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol, 0.5%NP-40, 0.25% Trixon X-100, 1 mM DTT, protease inhibitor cocktail) on ice for 10 min, and soluble proteins were separated by centrifuge at 1100 g. The nucleocyte proteins were extracted with E2 buffer (10 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitor cocktail) with the same procedure as before. The chromatin-bound pellet was isolated by ice E3 buffer (50 mM Tris–HCl pH 7.5, 20 mM NaCl, 1 mM MgCl₂,1% NP-40, protease inhibitor cocktail, 1:1000 bezonase) followed by sonication and centrifugation at 16000 × g.

4.6 Immunoprecipitation (IP) and immunoblotting (IB)

Cells were washed with ice-cold PBS, lysed in RIPA buffer (50 mM Tris–HCl pH 7.4, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA and 50 mM NaF) supplemented with protease inhibitor cocktail and MG132 (MCE, HY-13259), scraped and incubated in cold tubes for 30 min on ice. Cell lysates were centrifuged at 12000 rpm at 4°C for 20 min, and the supernatant was used to determine the protein concentration by spectrophotometer (DeNovix, DS-11). SDS sample buffer was added to the lysates, and standard immunoblotting procedures were followed. For IP, cells were lysed and quantified for protein concentration. The primary antibody was added and incubated in a 4°C rotation rack overnight. Protein G beads were then added for an additional 3 h at 4°C with rotation. The resin was then centrifuged for 4 min at 550 × g, washed three times with lysis buffer and analysed by immunoblotting.

4.7 Immunofluorescence microscopy

Immunofluorescence analyses were performed as previously described.⁶⁴ Briefly, the cells were cultured on coverslips at a concentration of 2 × 10⁵ cells/well in a 6-well plate, and the cells were washed with PBS. Following the removal of PBS, the cells were fixed in 1 mL of 100% ice-cold methanol at −20°C for 7 min and then blocked with 2% BSA solution in PBS containing 0.1% Tween-20 at 4°C overnight. The cells were then incubated with the appropriate antibodies at 4°C overnight. After washing three times with blocking buffer, the cells were incubated with appropriate secondary antibodies for 1 h at 37°C in the dark and then incubated in DAPI solution for 2 min. Cells were subjected to immunofluorescence analyses using a fluorescence microscope.

4.8 DNA fibre assay

DNA fibre analyses were performed as previously described.⁶⁵ Briefly, cells were transfected with target siRNA or plasmid, pulse-labelled with CldU, and then pulse-labelled with IdU in fresh medium. After trypsinization, the cells were washed and resuspended in PBS. Then, 2 μL of cell suspension was placed on a glass slide, which was angled to allow DNA to spread, and lysis buffer. DNA was denatured with HCl and blocked with 5% bovine serum albumin (BSA) in PBS for 1 h after washing. Slides were stained with antibodies and sealed with coverslips. Slides were imaged using a microscope and analysed using ImageJ software. At least 200 replication forks were analysed per experimental condition, while the analysis shows the mean of three independent experiments.

4.9 Neutral comet assay

Neutral comet assays were carried out as previously described.⁶⁶ Briefly, 2 × 10⁵ cells were suspended in PBS, mixed with 1% low-melting-point agarose, pipetted onto a slide precoated with 1% agarose, and then covered with 0.5% low-melting-point agarose. The cells were lysed with lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 10% DMSO, 1% Triton-X100) at 4°C for 2 h. Excess ions were washed off with double-distilled water, the slides were placed in a horizontal electrophoresis tank containing electrophoresis solution (1 mM Na₂EDTA, 300 mM NaOH) to soak for 20 min, and electrophoresis was run for 40 min. Following electrophoresis, the slides were rinsed with neutralization buffer (0.4 M Tris, pH 7.5) for 15 min. The slides were the washed in distilled water for 5 min and stained with propidium iodide (PI, 5 μg/mL) for 20 min, and then excess PI was washed off with distilled water. The slides were observed under 590 nm excitation light using a fluorescence microscope. At least 100 comet images from each slide were analysed using comet image analysis software.

4.10 Apoptosis assay

Apoptosis was detected by a TUNEL (terminal deoxynucleotidyl transferase nick-end labeling) fluorescein kit (ZIKER, ZK-PL4019) and confirmed with the Annexin V-PE/7-AAD detection kit (Sino Biological, APK10448-P) using a fluorescence microscope and flow cytometer.

4.11 Tumorigenesis and treatment

All mice were kept for 1 week for environment adaptation before starting the injections. After adaptation, Baclb/c mice were inoculated into their left fat pad with 4 T1 cells (1 × 10⁶ in 100 μL PBS). Regular checks were performed to assess tumour growth until the palpable tumour was detected. After 5 days of injections, all the mice were randomly divided into 4 groups, tumour size were measured every three days. Then, 5 mice in the CTRL were injected with 0.9% normal saline every day; 5 mice in the kaempferol single group (KPL) were injected with 20 mg/kg kaempferol every day; 6 mice in the paclitaxel single group (PTX) were injected with 15 mg/kg every three days; 6 mice in kaempferol and PTX combination group (KPL + PTX). In the third week, mice were killed after anaesthesia, and then the tumour tissues were collected for further analysis.

4.12 Statistical analysis

Quantified results are presented as the mean ± SEM of at least three duplicates. Comparisons between groups were performed with an unpaired Student's t-test. The results were considered statistically significant only if the p value was less than 0.05, and the significance level is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.