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10.1016/j.neo.2024.101033
101033
Original Research
WDR68 stimulates cellular proliferation via activating ribosome biogenesis in 293T cells
Wu Zhaoxia oasis_hn@163.com
ac1⁎
Xie Lanfeng b1
Yuan Ping c
Chu Yimin a
Peng Haixia phx1101@shtrhospital.com
a⁎⁎
a Digestive Endoscopy Center, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
b Department of Infectious Disease, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
c Hongqiao International Institute of Medicine, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China
⁎ Corresponding author at: Digestive Endoscopy Center, Key Laboratory for Translational Research and Innovative Therapeutics of Gastrointestinal Oncology, Hongqiao International Institute of Medicine, Shanghai Tongren Hospital, No.1111, Xianxia Rd, Changning District, Shanghai 200336, PR China. oasis_hn@163.com
⁎⁎ Corresponding author at: Digestive Endoscopy Center, Shanghai Tongren Hospital, No.1111, Xianxia Rd, Shanghai 200336, PR China. phx1101@shtrhospital.com
1 These authors contribute equally to this work.

26 7 2024
10 2024
26 7 2024
56 10103312 1 2024
17 7 2024
17 7 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
WDR68, a conserved WD40 repeat-containing protein, interacts with E1A and is involved in the E1A-induced cell proliferation and oncogenic transformation, but the intrinsic molecular mechanisms of this process remain to be elucidated. Here, we demonstrate that WDR68 promotes the proliferation of 293T cells by interacting with a series of ribosome biogenesis-regulating proteins. Gene Set Enrichment Analysis (GSEA) of RNA-seq data also revealed that the ribosome biogenesis-associated gene signatures could be the most significantly enriched in the WDR68 expression groups. In accordance, 293T cells are more sensitive to the ribosome biogenesis inhibitors than 293 cells. Taken together, our results indicated that WDR68 could promote cell proliferation through the activation of ribosome biogenesis in the 293T cell context. This provides new insights into the understanding of the function of WDR68 and the molecular characterisation of 293T tool cells.

Keywords

WDR68
E1A
Cellular proliferation
Ribosome biogenesis
==== Body
pmcIntroduction

WDR68 is a highly conserved WD40 repeat protein that acts as a scaffolding protein to assemble different components into multi-subunit complexes for various regulatory functions [1,2]. WD40 repeat proteins, also known as WD repeat (WDR) or WD40 proteins, are defined by the presence of a highly conserved WD40 motif across a wide range of species. This specific motif starts with a glycine-histidine (GH) pair from the N-terminus and ends with a tryptophan-aspartic acid (WD) pair at the C-terminus, also known as the Trp-Asp motif. In general, proteins containing WD40 repeats form a large family of proteins with diverse functions, including signal transduction, cell division, cytoskeleton assembly, chemotaxis, RNA processing and transcriptional regulation [1,3].

WDR68 was initially identified as a gene (AN11) that controls pigmentation of petunia flowers by activating the transcription of anthocyanin biosynthetic genes. AN11 and its orthologs all encode a protein with five WD40-repeats and are therefore termed collectively as WDR68. WDR68, also known as DCAF7 (DDB1 and CUL4 associated factor 7), is a substrate recognition factor for CUL4 E3 ubiquitin ligase complexes (CUL4–RBX1–DDB1). The substrate recruitment function of WDR68 that gathers DYRK1A to target RNA polymerase II and tethers several kinases together has been demonstrated [4,5]. Meanwhile, WDR68, has been reported to promote proteasomal degradation of targeted proteins [6,7].

Previously, WDR68 was identified as an essential gene required for cell viability in a large-scale CRISPR knockout screen [8]. Research in model organisms has shown that WDR68 is required for normal wing vein patterning and adult jumping muscle development in Drosophila [9] and for craniofacial development in zebrafish [10]. However, functional characterization of WDR68 remains to be unknown.

Human embryonic kidney (HEK) 293 cell line and its derivatives are the most frequently used tool cells for studying the function of proteins. Conventionally, we constructed stable pool cells with WDR68 knockout using CRISPR−CAS9 technology in 293T cell lines and serendipitously found that WDR68 knockout significantly inhibited cell proliferation, and WDR68 rescue promoted cell growth in 293T cell clones without WDR68. Immunoprecipitation combined with liquid chromatography-mass spectrometry showed that WDR68 interacts with many cell-cycle related proteins such as RB family proteins and AMBRA1(activating molecule in BECN1-regulated autophagy protein 1). Subsequently, co-immunoprecipitation assays confirmed that WDR68 binds to the small pocket domain of RB in 293T cells. Transcriptome sequencing data showed that WDR68 is closely linked to the transcription of a large number of crucial genes in 293T cells. However, the proliferation inhibition effect is irreproducible in other cell lines including 293 cells. We thus performed the GSEA and found that ribosome biogenesis-associated gene signature could be significantly enriched in WDR68 expression groups. Secondly, 293 T cells are more sensitive to ribosome biogenesis inhibitors CX-5461 or oxaliplatin than 293 cells. Thirdly, ribosome biogenesis regulating factors such as DYRK1B, CCT2, RING1, XPO1 and TRMT112 are found in the list of WDR68-interacting proteins. All these results supported that WDR68 probably promote cell proliferation via activating ribosome biogenesis in 293T cells.

In conclusion, we have demonstrated the essential function of WDR68 for cell proliferation in 293T cells and WDR68 probably stimulates ribosome biogenesis via interacting with ribosome biogenesis regulating factors. These findings not only expand the understanding of function of WDR68 gene, but also provide a deeper recognition of the molecular properties of 293T tool cells.

Results

WDR68 depletion inhibited cell proliferation in 293T cells

Although the role of WDR68 in cell proliferation has been reported [11], the limitations of RNA interference (RNAi) technology with imperfect mRNA knockdown and confounding off-target effects should be considered [8]. To characterise the function of WDR68, we constructed stable WDR68 knockout pool cells by infecting 293T cell lines with lentiviruses encoding CAS9 and two guide RNAs targeting the WDR68 locus (gWDR68) with gNS as a nonspecific control. Unexpectedly, depletion of WDR68 by CRISPR-CAS9 (Fig. 1A) significantly inhibited cell growth (Fig. 1B). Next, we generated 293T∆WDR68 clones by using CRISPR/Cas9 gene editing, and these clones exhibited significant inhibition of cell growth. Stable expression of WDR68 in 293T∆WDR68 5# cells, a representative clone, significantly promotes cell proliferation (Fig. 1C & D).Fig. 1 WDR68 deletion inhibits cell proliferation in 293T cell lines. (A)Cell lysates were prepared from 293T cells infected with gNS, gWDR68#1 or gWDR68#3 and detected blot with WDR68 and β-actin antibodies respectively. (B)Number of cells 2⁓10 days after seeding. Cells were grown in a 6-well cell culture plate, trypsinized and counted every two days. (C)Western blot analysis was carried out to identify stable expression of WDR68 in 293T∆WDR68 5# cells with WDR68 and β-actin antibodies respectively. (D)Cell number on 2⁓10 days post cell seeding. Cells were grown in a 6-well cell culture plate, trypsinized and counted every two days. (E)The KEGG enrichment of the top 50 WDR68-interacting proteins. (F)The validation of marked WDR68-interacting proteins. The experiments in B and D were independently repeated three times for 3 replicates with similar results, and the results of one representative experiment are shown. For B and D, data represent means ± s.d. Statistical significance was determined by two-way ANOVA. NS, non-specific; EV,empty vector.

Fig 1

To investigate the underlying mechanism of WDR68, 293T cells were instantaneously transfected with Flag-tagged WDR68 plasmids, whole-cell lysates were subjected to affinity purification using Flag-M2 beads, and the bound proteins were analyzed by liquid chromatography–tandem mass spectrometry(LC−MS/MS). 668 unique proteins interacting with WDR68 were identified(Supplementary Table A-1). The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that top 50 WDR68-interacting proteins were involved in cell senenscence, human papillomavirus infection, cell cycle, viral carcinogenesis (Fig. 1E)(Supplementary Table A-2&3). To confirm the interaction between WDR68 and these proteins, total cell proteins were extracted from 293T cells with ectopic expression of WDR68, and co-immunoprecipitations (co-IPs) were performed with anti-Flag antibody, followed by immunoblotting with antibodies shown (Fig. 1F). The results indicated that the interaction between WDR68 and a few representative proteins could be validated. Among these proteins, CCNA2, ATM and RB are in the top 50 WDR68-interacting protein list.

The small pocket domain of RB is indispensable for binding to WDR68 in 293T cells

Next, we found that WDR68 binds equally to three RB family proteins (The abundance of proteins pulled down is similar) (Fig. 2A & B). To map the domains of RB involved in its interaction with WDR68, the HA-tagged RB and its truncations were transiently transfected into 293T cells respectively, followed by co-IP with anti-HA antibody. As depicted in Fig 2C and D, RB ΔN and RB ΔC pulled down WDR68, but not RB ΔP1 and RB N642, indicating that the small pocket domain of RB is required for its interaction with WDR68. Next, plasmids encoding flag-tagged full-length WDR68 (WDR68-FL) and its four deletion mutants, including the WDR68-ΔN, WDR68-ΔC, WDR68-ΔWD1&2 and WDR68-ΔWD3&4&5 fragments(Fig 2E) were transfected into 293T∆WDR68 clone cells, and co-IP assays were performed with Flag antibody. Notably, WDR68-FL pulled down RB, but deletion of either the N-terminal or the C-terminal domain completely abolished the interaction of WDR68 with RB, DYRK1A (a positive control) [12] (Fig 2F). These data showed that a complete molecular architecture of WDR68 is necessary for its interaction with RB in 293T cells.Fig. 2 The small pocket domain of RB is indispensable for binding to WDR68. (A) Flag-WDR68 plasmids were transiently transfected into 293T cells and co-IP assays were performed with Flag antibody. Co-eluted proteins were detected by western blot assays using Flag and RB antibodies respectively. (B)HA-tagged RB, RBL1 and RBL2 were transiently transfected into 293T cells and co-IP assays were performed with HA antibody. The co-eluted proteins were detected by western blot assays with HA and WDR68 antibodies respectively. (C) A diagram showing a series of HA-tagged truncation mutants of RB. (D) The small pocket domain, amino acid residues between No.380 and No.787 of RB is critical for binding to WDR68. (E)A schematic representation of the structure of flag-tagged WDR68 deletion mutants is indicated. (F) The binding of WDR68 mutants to RB is shown.The experiments shown in A, B, D and F were repeated three times with similar results, and the results of one representative experiment are shown.

Fig 2

Transcriptome and DEGs analysis with or without WDR68 in 293 T cells

To elucidate the molecular basis of WDR68 in 293T cells, we carried out RNA-sequencing assays with the pooled WDR68-knockout or WDR68-rescue cell lines. For the transcriptomic analysis, we selected the sgRNA number 3 pool cell line, which is more efficient. In total, about 45.47–52.59 Mb and 42.68–48.44 Mb raw reads for each sample were sequenced and uniquely mapped to the human genome in two indicated groups individually. Using |Log2FC| ≧1 and p < 0.05 as cutoffs to filtrate the differentially expressed genes (DEGs), we found that 243 genes were upregulated and 117 were downregulated in the WDR68 knockout group, and 542 were upregulated and 219 were downregulated in the WDR68 rescue group (Fig. 3A and B; Supplementary Table B 1-4).Fig. 3 Differentially expressed genes profiling of rescue of the WDR68 protein in 293T∆WDR68 cells or 293T knockout pool cells. (A)Volcano plot showing the profile of differentially expressed genes (DEGs) between WDR68 rescue or WDR68 deletion and the control group. (B)The heat map of differentially expressed genes (DEGs) with a cutoff of log2FC >1 and of p<0.05(n=3). (C) The top 20 enrichment of GO or KEGG analysis(D). "Number"represents the number of differential genes, the larger the number the more.

Fig 3

GO enrichment analysis of 360 DEGs in WDR68 knockout cell lines using MetaScape online tool revealed that they gathered into 20 significant top-ranked GO terms (p < 8.75E-11, Fig. 3C; Supplementary Table B-7). Notably, the DEGs mainly correlated with cellular differentiation, development and morphogenesis, including tissue development, blood vessel development, system development, animal organ development, multicellular organism development and animal organ morphogenesis, animal organ morphogenesis. Similarly, GO enrichment analysis of 761 DEGs in WDR68-rescue group presented that the top 20 significant clusters also mainly include these pathways (p < 4.24E-20, Fig. 3C; Supplementary Table B-8). By KEGG enrichment analysis we obtained the top 20 significant pathway terms with the up- or down-regulated DEGs in WDR68-knockout group. The KEGG term included most of the PI3K-AKT, hippo, TGF-β signaling pathways and cancers (Fig. 3D; Supplementary Table B-9). Similar results were also obtained in the WDR68-rescue group(Supplementary Table B-10). These data indicated that WDR68 exerted a key role on cell proliferation and differentiation, organ development and cancer development in 293T cells.

Based on the RNA-sequencing results, we have screened 82 genes that are positively regulated and 93 genes that are negatively regulated by WDR68 (Fig.s 4A)(Supplementary Table B-5&6). Among them, we selected 24 genes with significant difference (Table 1) to validate the RNA-sequencing results by Quantitative Real-time PCR(qRT-PCR). As a result, deregulation of 24 genes was completely reproduced in 293T cells (Fig. 4B & C).Fig. 4 Validation of differentially expressed genes in RNA-sequencing analysis. (A)Venn diagram showing the number of genes positively and negatively regulated by WDR68 by RNA-sequencing analysis. (B)qRT-PCR analysis of 24 genes with large multiple differences and important functions in 293T cells with WDR68 deletion or (C) in 293T∆WDR68 with WDR68 rescue. In B and C, data represent means ± s.d. (n= 3 independent experiments; two-tailed unpaired t-test). All other experiments were independently repeated three times with similar results, and the results of one representative experiment are shown.

Fig 4

Table 1 The information of 24 validated genes.

Table 1gene ID	gene Name	baseMean_EV	baseMean_D7	log2FoldChange	pval	Regulation	
595	CCND1	14.50073568	1679.48242	6.855746788	1.08E-88	Up Regulation	
650	BMP2	6.477242139	335.3235655	5.694030387	3.96E-61	Up Regulation	
64641	EBF2	3.086836744	91.14349937	4.883938673	1.69E-25	Up Regulation	
7547	ZIC3	25.97146925	638.3193924	4.619278941	3.44E-34	Up Regulation	
2034	EPAS1	55.45719467	1147.355163	4.370793597	1.91E-68	Up Regulation	
9421	HAND1	18.64835968	304.9174922	4.031298275	8.71E-42	Up Regulation	
147372	CCBE1	3.123207608	35.86967976	3.521664488	3.02E-06	Up Regulation	
5154	PDGFA	13.04652085	135.4346057	3.375859382	5.76E-23	Up Regulation	
1305	COL13A1	29.83598835	238.6871338	2.999995328	5.17E-26	Up Regulation	
55273	TMEM100	4.870708867	38.05444814	2.96586144	1.34E-08	Up Regulation	
1846	DUSP4	29.28457626	222.3218332	2.924436712	3.09E-24	Up Regulation	
654	BMP6	56.65681346	398.5952748	2.81460324	2.24E-28	Up Regulation	
760	CA2	1401.008005	8433.193562	2.589613869	4.06E-35	Up Regulation	
7481	WNT11	52.33174992	279.8891541	2.419097175	2.12E-20	Up Regulation	
23194	FBXL7	5.924661134	26.73198254	2.173762292	0.003222873	Up Regulation	
389692	MAFA	11.87662101	50.59659121	2.090915754	5.11E-07	Up Regulation	
653	BMP5	218.2989358	9.129045645	-4.579697239	4.19137E-13	Down Regulation	
29103	DNAJC15	59.95741942	4.758710485	-3.655295701	5.49915E-12	Down Regulation	
10512	SEMA3C	1051.904861	105.5490726	-3.31701842	9.41147E-09	Down Regulation	
1950	EGF	45.21785291	4.994690238	-3.178425378	1.54637E-10	Down Regulation	
84432	PROK1	16.23460817	3.236158882	-2.32671822	0.000881198	Down Regulation	
54541	DDIT4	4743.600287	1101.267546	-2.106817443	0.002932055	Down Regulation	
1490	CCN2	483.5444435	128.4348358	-1.912611934	1.4205E-16	Down Regulation	
3491	CCN1	1449.119179	450.3087468	-1.686189844	1.22538E-15	Down Regulation	

WDR68 promotes cell proliferation probably through activating ribosome biogenesis in 293T cells

To confirm the inhibitory function on cell proliferation, we used CRISPR/Cas9 to generate several WDR68 knockout cell lines such as NCM460, SW620, MCF-7 and Hela cells. However, WDR68 depletion did not significantly inhibit cell proliferation (data not shown). These results indicated that the regulation of cell proliferation by WDR68 is limited in 293 T cell lines.

293 T cell lines, a derivative of 293 cells, harbor a temperature-sensitive allele of the simian virus 40 (SV40) large T antigen [13]. The SV40 T antigen binds to and inhibits p53 activity to further compromises genome integrity [14]. HEK293 was generated from an aborted human embryonic kidney cell by transformation with sheared fragments of human adenovirus type 5(ADV5) DNA [15]. It has been reported that the 293 cell line contains a single, collinear insertion of ADV5 DNA fragments encoding the early region 1 (E1A/E1B) proteins with no rearrangements [16]. Expression of E1A promotes the entry of quiescent, growth-arrested rodent cells into cell cycle to stimulate cell proliferation [17].

To investigate whether SV40 T antigen and E1A contribute to the proliferation-promoting effect of WDR68, two stable pool cells of either E1A or WDR68 knockout were generated using CRISPR-CAS9 in 293T cells and 293 cells, respectively. The effect of knockout was analyzed by western blot on day 5 after lentiviruses infection(Fig. 5A&C). Depletion of E1A by CRISPR-CAS9 in 293T and 293 cells significantly inhibited cell proliferation (Fig. 5B&D). However, WDR68 deficiency had no effect on cell growth in 293 cells (Fig. 5D). These results suggested that proliferation-promoting effect of WDR68 is confined in 293T cell context. To trace the regulatory mechanism of WDR68, RNA-sequencing data was analyzed by Gene set enrichment analysis (GSEA). GSEA demonstrated that ribosome biogenesis-associated gene signatures were enriched in the up-regulated genes when WDR68 was expressed compared to the empty vector, with an enrichment ranking in the top 3(Fig. 5E)(Supplementary Table C-1). Correspondingly, the depletion of WDR68 resulted in the enrichment of ribosome biogenesis-associated gene signatures in the down-regulated genes, with this enrichment being ranked in the top 1 (Fig. 5F)(Supplementary Table C-2). These results showed that WDR68 could activate ribosome biogenesis. The ribosome is the “protein synthesis factory”. Ribosome biogenesis is the process by which ribosomes are made and plays an essential role in cell proliferation, differentiation, apoptosis, development and transformation [18]. Dysregulation of ribosome biogenesis can lead to cellular dysfunction and ultimately cause various diseases, while hyperactivated ribosome biogenesis likely drives cancer initiation and progression [19,20]. Therefore, ribosome biogenesis and ribosome function have become targets for cancer therapies [21,22]. A number of new inhibitors of rDNA transcription are in early clinical trials, such as CX-3543, BMH-21, and CX-5461 [[23], [24], [25]]. CX-5461, a small molecule inhibitor of RNA Pol I, activates P53-dependent anti-cancer signalling by decreasing the binding affinity of the RNA-Pol I complex to rDNA promoters [26]. 293T and 293 cells were treated individually with CX-5461 for 48 hours at concentrations ranging from 0 to 25 μM. A significant dose-dependent growth inhibition was observed in response to CX-5461 and 293T was more sensitive than 293 cells to CX-5461(Fig. 5G). Oxaliplatin, a cancer chemotherapeutic agent, kills cells by inducing ribosome biogenesis stress, not through the DNA-damage response [27]. 293T had higher sensitivity than 293 cells to the inhibitory effects of oxaliplatin (Fig. 5H). These results showed that WDR68 promotes cell proliferation in 293T cells probably through activating ribosome biogenesis.Fig. 5 WDR68 promotes cell proliferation probably through activating ribosome biogenesis in 293T cells. 293T (A) and 293 cells(C) were transduced by lentiviruses with gNS, gE1As and gWDR68s infection, followed by Western blot analysis of the indicated proteins. (B&D)Cell growth on 1~7 days after seeding. Cells were grown in a 96-well cell culture plate, detected every two days by the CCK8 assay. (E&F) GSEA of RNA-seq data from 293T∆WDR68 cells with and without WDR68(E) and 293T cells transduced by lentiviruses encoding Cas9 and gRNAs designed to target WDR68 (gWDR68) (F) with the indicated ribosome biogenesis genesets. (G&H)Inhibition rate of cell proliferation was detected when 0.04–25 μM CX-5461(G) or 0.08-50μg/mL oxaliplatin(H) was used in 293T and 293 cells. Experiments in B, D, G and H were independently repeated three times for 3 repetitions with similar results, and the results of one representative experiment are shown, data represent means± s.d. Statistical significance was determined by two-way ANOVA.

Fig 5

Discussion

In this study, 293T cells, a widely used human renal epithelial line as an expression tool for recombinant proteins, were used to dissect the function of WDR68. We found that WDR68 deletion remarkably inhibits cell proliferation and WDR68 interacts with a series of key proteins and induces a number of fundamental gene deregulations at the transcriptional level in 293T cells. 293T cell lines, derived from 293 cells, are characterized with SV40 T antigen and one copy of AD5 E1A/E1B DNA fragment. In both 293T and 293 cells, E1A deficiency significantly inhibited cell growth. However, WDR68 depletion by CRISPR-CAS9 showed no impact on cell proliferation in 293 cells. These data indicated that proliferation-promoting effect of WDR68 is limited in 293 T cells. GSEA analysis revealed that ribosome biogenesis-associated gene signature could be significantly enriched in WDR68 expression group, suggesting that WDR68 expression could stimulate ribosome biogenesis. Ribosome biogenesis is closely linked to protein synthesis, cell proliferation, differentiation and apoptosis [28]. In addition, 293T cells are more sensitive to inhibitors of ribosome biogenesis than 293 cells. The results indicated that hyperactivated ribosome biogenesis might be indispensable for maintaining cell proliferation capacity of 293T cells.

WD40-repeat proteins are a large, rapidly expanding conservative family. WD40-repeat sequence forms the propeller structure and provides a extensive surface for protein and protein interactions [3]. WDR68 is also one of substrate recognition factors for CUL4 E3 ubiquitin ligase complex. Interactomics revealed that WDR68 interacts with a number of key proteins. Among them, several proteins are known ribosome biogenesis regulating factors including DYRK1B, CCT2, RING1, XPO1 and TRMT112 [29]. Ribosome biogenesis is an energetically demanding process involving three RNA polymerases, numerous RNA processing factors, chaperones and the coordinated expression of 79-80 ribosomal proteins [18]. Especiallly, the biogenesis of ribosome has become more complex in multicellular eukaryotes and the mechanism by which human ribosomes are made remains to be elucidated. Besides the above 5 proteins, other WDR68-interacting proteins perhaps also regulate ribosome biogenesis.

293T cells have a very complicated context containing Ad5 genomic DNA sequences from nucleotides (nt) 1 to 4344 and SV40 early gene and grow at a much faster rate than the parental line 293. We hypothesize that WDR68 mediates the pro-proliferative effect of T antigen or synergistic effects of T antigen and E1A. The complexity of 293T cell context determines that it is difficult to gain insight into the deeper molecular mechanisms.

Taken together, these data suggest that WDR68 promotes cell proliferation in the context of 293T cells; mechanistically, WDR68 binds to ribosome biogenesis regulatory factors such as DYRK1B, CCT2, RING1, XPO1 and TRMT112 or other unproved proteins. We will then focus on in vivo experiments to extend the recognition of WDR68. In the future, we believe that these findings might provide new insights into understanding the function of WDR68. In the meantime, it also reminds us to be careful with the 293T tool cells for functional studies of proteins.

Materials and methods

Cell lines and cell culture

The human embryonic kidney (HEK) 293T and 293 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). 293 and 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and all other cell lines were maintained in RPMI 1640. All media were supplemented with 10 % fetal bovine serum (FBS). All the cells were tested negative for mycoplasma contamination and authenticated by short tandem repeat (STR) fingerprinting prior to use.

Plasmids

The WDR68 and ADV5E1A were PCR amplified from HEK293T cell cDNA and inserted into pLVX-puro or PLVX-IRES-ZsGREEN1 plasmids. Full-length and truncated WDR68 or RB, RBL1, RBL2 were produced by PCR and cloned into pQCXIN or pcDNA3.1(+) expression vectors.

CRISPR/Cas9

Information on the sgRNAs is shown in Supplementary Table A-4. Individual two guide sgRNA sequences #1 and #3 targeting the WDR68 locus (gWDR68) or #2 and #4 targeting ADV5E1A were ordered as complementary primers, mixed in a 1:1 ratio, annealed and were cloned into lenti-CRISPR-v2 at the BsmBI site as described [30]. Using X-tremeGENE 9 DNA Transfection Reagent (Roche), lentivirus was produced by co-transfecting the lentiviral construct pCMV-dR8.91 (Δ8.9) plasmid, containing the genes gag, pol and rev, and the pMD2.G envelope expressing plasmid into 293T cells. At 24∼48h post-transfection, the viral supernatant was harvested and filtered through a 0.45 μm filter for infecting the target cells at 60 % confluence. Cells were infected with virus containing the sgRNA and Cas9 for 48h. Puromycin was used to select positive cells. HEK293T-ΔWDR68 clones were obtained by transiently transfecting lenti-CRISPR-sgWDR68-3# plasmids into 293T cells for 48h and screening puromycin-positive cells. The cells were then diluted and single cells were seeded into 96 well culture plates. The effect of sgRNA was analyzed by western blot. PCR and sequencing were used to confirm homozygous editing of the gene loci.

Immunoprecipitation

Cells were harvested and lysed with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, 1 mM PMSF, and 1× protease inhibitor cocktail (Calbiochem)). After brief sonication, the supernatants (whole cell lysates) were collected by centrifugation at 12,000 × g for 10 min at 4 °C. For immunoprecipitation of Flag or HA-labelled proteins, whole cell lysates were incubated with anti-HA or anti-Flag M2 affinity gel at 4 °C for 4 hours. Precipitates were washed three times with immunoprecipitation buffer, boiled in sample buffer and subjected to immunoblotting.

In-gel digestion and Mass spectrometry

Immunoprecipitation samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and visualized with colloidal Coomassie blue. The lane from the gels was cut into 1 mm slices and each slice was washed twice with 50 mM NH4HCO3, 50 % acetonitrile (ACN) and dehydrated with 10 mM DTT and 55 mM iodoacetamide were used for protein reduction and alkylation. The proteins were then washed with 50 mM NH4HCO3 and ACN, subjected to gel with trypsin (Promega) overnight at 37°C, followed by extraction of tryptic peptides. Extraction of tryptic peptides from the gel pieces with 0.1 % trifluoroacetic acid and 60 % ACN. The extracted peptides were dried by vacuum centrifugation and dissolved in 10 μl of 2 % ACN and 0.1 % trifluoroacetic acid.

The nano-HPLC liquid phase system UltiMate 3000 RSLCnano (ThermoFisher Scientific) was used to identify the interacting proteins as described [31]. Finally, the raw data were analyzed by ProteomeDiscoverer 2.5 software and matched to the Uniprot Homo sapiens database. Proteins with unique peptides identified in the Experimental group but not in the control group were considered as WDR68-interacting proteins.

Coimmunoprecipitation, Western blotting and antibodies

Cells were harvested, washed in cold 1×PBS and lysed after transfection using RIPA (Millipore) with 1 mM PMSF and 1×protease inhibitor cocktail (Calbiochem) according to the manufacturer's protocol. Whole cell lysates were incubated for 4 h at 4°C with anti-HA or anti-Flag M2 affinity gel as recommended by the manufacturer for immunoprecipitation. For the detection of most proteins, protein extracts were separated by SurePAGE, Bis-Tris, 4∼12 % gel (GenScript). After separation, proteins were transferred to a nitrocellulose membrane (Bio-Rad, Richmond, CA), blocked with 5 % non-fat milk for 1 h at room temperature and sequentially incubated with primary antibodies in 2 % BSA overnight at 4 °C. The next day, the blots were washed in PBS and incubated in horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling, Beverly, MA) in 2 % BSA for 1 hour at room temperature. Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore) was used for detection. Information on all antibodies is given in Supplementary Table A-5. Complete and untrimmed western blots are provided in the original data file.

RNA-Seq and differential expression analysis

Cells were harvested and RNA-Sequencing services were provided by Personal Biotechnology Co., Ltd. (Shanghai, China). Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), and then the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA was used as input material for the RNA sample preparations. The libraries were sequenced on an Illumina NovaSeq 6000 platform, and 150 bp paired-end reads were generated. In total, 44.88~52.6 Mb of raw reads were generated for each sample. The raw data (raw reads) in fastq format were first processed using fastp (Chen et al., 2018), and the low-quality reads were removed to obtain the clean reads. Then, approximately 42.05 to 49.42 Mb of clean reads for each sample were retained for subsequent analyses. The clean reads were mapped to the human genome assembly GRC38 by STAR version 2.5.

We used HTSeq [32] statistics to compare the read count value of each gene as the original expression of the gene, and then used FPKM to standardise the expression. Then, we used DESeq [33] to analyse the genes of differential expression with the following screened conditions: expression difference multiple |log2FoldChange| > 1, significant P-value < 0.05. At the same time, we used R language Pheatmap software package to perform bi-directional clustering analysis of all different genes of samples. We get heat map according to the expression level of the same gene in different samples and expression patterns of different genes in the same sample using Euclidean method to calculate the distance and complete linkage method to cluster. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) [34] pathway enrichment analysis of DEGs were then performed using R based on the hypergeometric distribution. Bioinformatic analysis was performed using the free online platform Personalbio GenesCloud (https://www.genescloud.cn).

Additionally, Gene Set Enrichment Analysis (GSEA) [35] was employed to rank all signature genes and RNA-seq data defined by the t-statistic of the differential expression analysis according to their association with the high or low expression of the core genes. We then selected the top 20 pathways associated with high expression of each core gene. Gene sets that exhibited enrichment with a nominal p-value of less than 0.05, an absolute normalized enrichment score (NES) greater than 1, and a false positive rate (FDR) q-value less than 0.25 were classified as statistically significant.

qRT-PCR

Total RNA was extracted from the indicated cells using TRIzol reagent (Life Sciences, Carlsbad, CA, USA) according to the manufacturer's instructions. Complementary DNAs were synthesised from 2 micrograms of total RNA using random primers (Takara) and M-MLV reverse transcriptase (Promega, Fitchburg, WI), followed by qRT-PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The expression levels of each gene were normalised to GAPDH. Primers used for quantitative real-time PCR are listed in Supplementary Table A-6.

Cell viability assay

The cytotoxicity of CX-5461(Topscience, Shanghai, China) and Oxaliplatin (Selleck, Shanghai, China) to 293T or 293 cells was determined by the CCK8 assay. 293T and 293 cells were seeded in 96-well plate and exposed to CX-5461 at concentration of 0, 0.04, 0.2, 1, 5, 25 μM or Oxaliplatin at concentration of 0, 0.08, 0.4, 2,10, 50 μg/mL for 48 h. 10 μl CCK8(Meilun, Dalian, China) was added to each well and incubated for 2 h. Then, the plate was shaken for 1 min. The absorbance was measured at 450 nm wavelength using a Microplate Reader (Bio-Rad, Hercules, CA, USA).

Statistical analysis

All in vitro experiments were repeated at least three times with comparable results. Results are expressed as mean ± s.d. Student's exact t-test was used to compare P values between two groups. All statistical analyses were two-tailed and P values < 0.05 were considered statistically significant.

Data availability

Datasets are available on request: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

CRediT authorship contribution statement

Zhaoxia Wu: Writing – original draft, Methodology, Conceptualization. Lanfeng Xie: Validation, Investigation. Ping Yuan: Data curation. Yimin Chu: Formal analysis. Haixia Peng: Writing – review & editing, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Acknowledgements

This work is supported by 10.13039/501100001809 National Natural Science Foundation of China (No. 82272653 ); 10.13039/100007219 Shanghai Municipal Natural Science Foundation (No. 21ZR1458600 ); Interdisciplinary Program of Shanghai Jiao Tong University(YG2022ZD031 ); Scientific Research Foundation for the introduction of talent of Shanghai Tongren Hospital (TR2019rc01 ).

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101033.
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