tp53 oe  (Santa Cruz Biotechnology)

Journal: International immunopharmacology

Article Title: Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

doi: 10.1016/j.intimp.2023.110574

Figure Lengend Snippet: Fig. 3. p53 was required for heat stress-induced necroptosis in the intestine of mice. A. Schematic of the in vitro experimental design for downregulation of p53 in IEC cells before they were subjected to heat stress. B. Effects of the treatment of siRNA-Tp53 on the expression of p53, p-p53, RIP3 and p-RIP3 in IEC cells following heat stress. C. Heat stress-induced upregulation of inflammatory cytokines in IEC cells were restored by knockdown of p53. D. Schematic of the in vivo experimental design for the treatment of Pifithrin-α. In brief, mice were received a daily intraperitoneal (i.p.) injection with Pifithrin-α (3 mg/kg body weight) for three days before they were subjected to heat stress. E and F. Effects of the treatment of Pifithrin-α on the expression of p53, p-p53, RIP3 and p-RIP3 in the intestine (E) and the levels of inflammatory cytokines in plasma of heatstroke mice following heat stress (F) (N = 6 for each group). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: siRNA-Tp53 (sc-29436), siRNA-Ripk3 (sc-61483), siRNA-FITC (sc36869), Tp53 OE (sc-423509-LAC), Ripk3 OE (sc-425224-LAC), Tlr3 OE (sc-431258-LAC), Control lentiviral activation particles (sc-437282) and copGFP control lentiviral particles (sc-108084) were purchased from Santa Cruz Biotechnology, Inc. (USA).

Techniques: In Vitro, Expressing, Knockdown, In Vivo, Injection, Clinical Proteomics

Journal: International immunopharmacology

Article Title: Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

doi: 10.1016/j.intimp.2023.110574

Figure Lengend Snippet: Fig. 4. Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF3-RIP3 pathway was dependent on p53.Tp53-/- mice. B. Effect of p53 deletion on the levels of inflammatory cytokine in the plasma of mice (N = 6 for each group). C. Heat stress-induced upregulation of RIP3, p-RIP3, p-Mlkl and reduction of p- Drp1 were normalized by p53 deletion (N = 6 for each group). D. Effect of p53 deletion on the expression of TLR3 and TRIF in the intestine of mice (N = 6 for each group). E. Schematic of the in vitro experimental design. IEC Tp53-/- cells were treated with Tp53 OE to re-express p53 or with Tlr3 OE to upregulate the expression of TLR3 before they were subjected to heat stress. F. Effect of re-expression of p53 and upregulation of TLR3 on IEC Tp53-/- cells to response to heat stress. G. Immunoblotting analysis showed that heat stress-induced activation of Mlkl was blocked by the deletion of p53, which was activated again by re-expression of p53. H. Flow cytometry with Annexin V-FITC/PI staining was used to evaluate the induction of HS-induced cell death. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: siRNA-Tp53 (sc-29436), siRNA-Ripk3 (sc-61483), siRNA-FITC (sc36869), Tp53 OE (sc-423509-LAC), Ripk3 OE (sc-425224-LAC), Tlr3 OE (sc-431258-LAC), Control lentiviral activation particles (sc-437282) and copGFP control lentiviral particles (sc-108084) were purchased from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Clinical Proteomics, Expressing, In Vitro, Western Blot, Activation Assay, Flow Cytometry, Staining

Journal: International immunopharmacology

Article Title: Heat stress-induced intestinal epithelial cells necroptosis via TLR3-TRIF-RIP3 pathway was dependent on p53.

doi: 10.1016/j.intimp.2023.110574

Figure Lengend Snippet: Fig. 5. Schematic diagram of p53 meditated in heat-induced intestinal epithelial cells. Heat stress promoted the phosphorylation of p53, which further upregulated TLR3 and enhanced the interaction of TRIF-RIP3, leading to the activation of RIP3-MLKL mediated necroptosis in intestinal epithelial cells. The necroptosis of intestinal epithelial cells amplificated the inflammatory of intestine. In turn, inflammation further aggravate the intestine injury.

Article Snippet: siRNA-Tp53 (sc-29436), siRNA-Ripk3 (sc-61483), siRNA-FITC (sc36869), Tp53 OE (sc-423509-LAC), Ripk3 OE (sc-425224-LAC), Tlr3 OE (sc-431258-LAC), Control lentiviral activation particles (sc-437282) and copGFP control lentiviral particles (sc-108084) were purchased from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Phospho-proteomics, Activation Assay

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 expression is elevated in MM patients with TP53 lesions. A) NEK2 mRNA levels in MM patients with ( n = 51) or without ( n = 497) TP53 deletion, and data presented as mean ± SD, p ‐values are calculated using unpaired t test, **** p < 0.0001. B) Kaplan‐Meier analyses of overall survival in MM patients with TP53 N & NEK2 N (normal), TP53 N & NEK2 Amp (Amplification), TP53 D (deletion) & NEK2 N , and TP53 D & NEK2 Amp ( n = 548). C) NEK2 mRNA levels in MM patients with ( n = 40) or without ( n = 703) TP53 mutation, and data presented as mean ± SD, p ‐values are calculated using unpaired t test, **** p < 0.0001. D) Kaplan‐Meier analyses of overall survival in MM patients with wild type TP53 ( TP53 N ) & low expression NEK2 ( NEK2 L ), TP53 N & high expression NEK2 ( NEK2 H ), mutant TP53 ( TP53 mut ) & NEK2 L and TP53 mut & NEK2 H ( n = 743). E) Correlation between NEK2 and TP53 mRNA expression in the TT2 and TT3 trial form GEP data (GSE2658, n = 559, p ‐values are calculated Pearson correlation coefficient ). F) Kaplan‐Meier analyses of overall survival in TT2 and TT3 MM patients ( n = 559) with high NEK2 ( NEK2 H ) & low TP53 expression ( TP53 L ), NEK2 H & high TP53 expression ( TP53 H ), low NEK2 ( NEK2 L ) & TP53 L and NEK2 L and TP53 H . G) Scatter plot showing the percentage of cells with amplified NEK2 copy number in MM patients with or without TP53 deletion. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, ** p < 0.01, *** p < 0.001. H) Scatter plot showing the percentage of cells with amplified NEK2 copy number in TP53 WT or TP53 −/− MM cells. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, * p < 0.05, ** p < 0.01. I,J) Representative immunofluorescence images and statistical analysis for p53 (Green) and NEK2 (Red) protein expression in MM newly diagnosed patients (AD, n = 51) and relapsed patients (RD, n = 16, p ‐values are calculated using one‐way ANOVA with Bonferroni correction).

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Amplification, Mutagenesis, Immunofluorescence

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 expression is reduced and correlates inversely with loss of wild type TP53 in MM cells. A) Relative mRNA levels of NEK2 and TP53 in the H929 TP53 WT MM cell line were detected with qPCR after treatment with nutlin‐3a for 48 h at concentrations of 0, 2, and 4 µM. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. B) Relative protein levels of p53, NEK2, and GAPDH in TP53 WT H929 cells after treatment with 0, 2, and 4 µM of nutlin‐3a for 48 h, as determined with immunoblotting. C) Relative mRNA levels of NEK2 and TP53 in the ARP1 TP53 −/− MM cell line were detected with qPCR after treatment with nutlin‐3a for 48 h at concentrations of 0, 2, and 4 µM. D) Relative protein levels of p53, NEK2, and GAPDH in TP53 −/− ARP1 cells after treatment with 0, 2, and 4 µM of nutlin‐3a for 48 h, as determined with immunoblotting. E) The mRNA levels of TP53 and NEK2 in H929 cells with or without TP53 deletion edited by CRISPR/Cas9, as determined with qPCR. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. F) p53 and NEK2 protein levels in H929 cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with immunoblotting. G) TP53 and NEK2 mRNA levels in MM.1s cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with qPCR. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, *** p < 0.001. H) The protein levels of p53 and NEK2 in MM.1s cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with immunoblotting. I) Relative mRNA levels of TP53 and NEK2 in ARP1 cells were detected with qPCR 72 h after infection with virus containing WT or mutant TP53 (E285K)‐expressing vectors. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05. J) Relative protein levels of p53, NEK2, and GAPDH in ARP1 cells were detected with immunoblotting 72 h after infection with virus containing WT or mutant TP53 (E285K)‐expressing vectors. K) The mRNA levels of TP53 and NEK2 in H929 cells with or without TP53 mutation, as determined with qPCR. L) The protein levels of p53 and NEK2 in H929 cells with or without TP53 mutation, as determined with immunoblotting. M) The mRNA levels of TP53 and NEK2 in TP53 ‐deleted H929 (H929‐TP53 KO ) cells with or without TP53 mutation, as determined with qPCR. N) The protein levels of p53 and NEK2 in TP53 ‐deleted H929 (H929‐TP53 KO ) cells with or without TP53 mutation, as determined with immunoblotting.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Western Blot, CRISPR, Infection, Mutagenesis

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Dual defects in NEK2 and p53 enhance mitotic abnormalities, cell proliferation, apoptosis, and tumorigenesis in MM. A) Representative images of clonogenic analysis in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells (images are shown in 4x magnification). B) Statistical analysis of clone numbers formed in soft agarose of the five cell groups shown in panel (A). Data presented as mean ± SD, p ‐values are calculated using unpaired one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. C) Representative flow cytometry dot plots for detection of BrdU‐positive cells among H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells. D) Statistical analysis of the number of BrdU‐positive cells among the five cell groups shown in panel (C). Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. E) Representative histograms for detection of apoptotic cells in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells treated with 4 nM BTZ for 48 h. F) Statistical analysis of the percentage of apoptotic cells among the five cell groups shown in panel (E) after treatment with 4 nM BTZ for 48 h. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. G) Representative images of tumor xenografts from B‐NDG mice with subcutaneous injections of H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells into the right abdomen (6 mice measured for each group). H) Statistical analysis of tumor volumes of xenografts from B‐NDG mice as shown in panel (G) (6 mice measured for each group). Data presented as mean ± SD, p ‐values are calculated using two‐way ANOVA with Dunnett post‐hoc test, n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001. I) Representative images for IHC detection of Ki‐67 protein in the tumor nodules derived from B‐NDG mice injected subcutaneously with H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Flow Cytometry, Derivative Assay, Injection

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: TP53 deletion upregulates NEK2 expression by inducing amplification and chromosomal instability in MM cells. A) Comparative analysis of HEK293‐TP53 KO versus HEK293‐Ctrl cells using CGH array. Red column represents gains of chromosomes. Blue column represents deletions. Chromosomal aberrations are across the whole genome. B) Schematic depiction of NEK2 location in chromosome 1q21.1‐q44. C) Comparative analysis of HEK293‐NEK2 OE versus HEK293‐Ctrl cells using CGH array. Red column represents gain. Blue column represents deletion. D) Left, representative images of FISH analysis using probes targeting TP53 (17P, Red) and NEK2 (1q32.2, Green) in HEK293‐Ctrl and HEK293‐TP53 KO cells. Right, scatter plot showing the percentage of cells from both groups with amplified NEK2 copy numbers. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. E) Scatter plot showing the percentage of H929‐Ctrl and H929‐TP53 KO cells with amplified NEK2 copy numbers as determined by FISH assay. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. F,G) GSEA of enrichment, including chromosome 1q32 and chromosomal instability, from differentially expressed genes between HEK293‐Ctrl and HEK293‐TP53 KO cells. H) Representative images of spindles (Red: Tubulin), Plasmid‐GFP (Green), and nuclei (Blue) in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells. I) Quantification showing the percentage of cells with abnormal monopolarity among each of the five groups shown in panel (H). All results are shown as means ± SD of three independent experiments, and at least 60 spindles per experiment were randomly chosen and counted in each experiment.Data presented as mean ± SD, p‐values are calculated usingone‐way ANOVA with Dunnett post‐hoc test, * p < 0.05.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Amplification, Plasmid Preparation

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: p53 deletion promotes aberrantly high NEK2 expression through indirect transcriptional regulation. A,B) Luciferase activity driven by NEK2 promoter with normal (full length) or mutant p53 binding site in HEK293‐TP53 KO and ARP1 cells transiently co‐transfected with p53 OE. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. C) ChIP confirming that the transcription factor p53 specifically binds to the NEK2 promoter region in H929 cells. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, **** p < 0.0001. D) Correlation of expression between NEK2 and E2F8 in MM patients based on GEP database (GSE2658, n = 559, p ‐values are calculated Spearman correlation coefficient ). E) The mRNA and protein levels of TP53 , CDKN1A ( p21 ), and E2F8 in H929 cells with or without p53 deletion. Here and in panels (F–J), mRNA and protein data were obtained using qPCR and immunoblotting, respectively. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. F) The mRNA and protein levels of TP53 , CDKN1A ( p21 ), and E2F8 in ARP1 cells with or without p53 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, *** p < 0.001. G,H) The mRNA and protein levels of NEK2 and E2F8 in H929‐Ctrl and H929‐TP53 KO cells with (H929‐TP53 KO /E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. I,J) The mRNA and protein levels of NEK2 and E2F8 in ARP1‐EV and ARP1‐TP53 OE cells with (ARP1‐TP53 OE/E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. K) ChIP confirmed that the transcription factor E2F8 specifically binds to the NEK2 promoter region. L) Schematic of p53 deletion enhancing NEK2 expression through E2F8 upregulation.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Binding Assay, Transfection, Western Blot, Over Expression

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: p53 suppresses NEK2 expression by regulating the expression of DNMTs. A) Detailed BGS analysis confirmed methylation status of the NEK2 promoter's distal CpG island in H929 cells with or without p53 deletion. B) Statistical analysis of methylated CpG dinucleotides in the NEK2 promoter's distal CpG nucleotides in H929 cells with or without p53 deletion. Data presented as mean ± SD, n = 11, p ‐values are calculated using one‐way ANOVA with Bonferroni correction, ** p < 0.01, **** p < 0.0001. C) Heatmap of the ratios of the signal intensities of differential methylation‐related genes in H929 cells with or without p53 deletion. D) The mRNA levels of histone methylation‐related genes ( NSD3 , PRMT1 , SETD5 , and SETD7 ) detected by qPCR in H929 cells with or without p53 deletion. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. E) H3K36me3 protein levels detected using immunoblotting in H929 cells with or without p53 deletion. F,G) The mRNA and protein levels of DNMT1 , DNMT3a , and DNMT3b in H929 cells with or without p53 deletion using qPCR and immunoblotting. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. H,I) The mRNA and protein levels of NEK2 after shRNA‐mediated DNMT3b knockdown in wild type p53 and p53‐deleted H929 cells, detected by qPCR and immunoblotting. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. J) Endogenous p53 was pulled down by p53 antibodies in H929 cells with or without p53 deletion, and the DNMT1, DNMT3a, and DNMT3b proteins were analyzed by western blotting. The lysates before IP were used as a positive control. K) Dysfunctional p53 elevates NEK2 expression in MM by upregulating DNMT expression, thereby increasing methylation of the NEK2 promoter.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Methylation, Western Blot, shRNA, Positive Control

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Downregulation of NEK2 in TP53 ‐deleted MM cells inhibits cell growth and decreases drug resistance. A,B) Representative images of spindles (Red, Tubulin), GFP (Green), and nuclei (Blue) in ARP1‐Ctrl, ARP1‐TP53 OE, and ARP1‐TP53 OE/shNEK2 cells. Quantifications show the percentage of abnormal monopolarity from three independent experiments. At least 60 spindles were randomly chosen and counted in each experiment. All results are shown as means ± SD of three independent experiments, Data presented as mean ± SD, p‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, * p < 0.05. Flow cytometric detection of BrdU‐positive cells in ARP1‐Ctrl, ARP1‐TP53 OE, and ARP1‐TP53 OE/shNEK2 cells. Representative C) dot plots and D) quantification. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01. E) Representative histograms for detection of apoptotic cells and F) statistical analysis of the percentage of apoptotic cells in ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TRP53 OE/NEK2 OE, and ARP1‐TP53 OE/shNEK2 cells treated with 4 nM BTZ for 48 h. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, ** p < 0.01. G) Representative images of tumor xenografts in B‐NDG mice after subcutaneous injection of ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells into the right abdomen. Cells were treated with BTZ or with PBS as a negative control. Tumor volumes of xenografts derived from B‐NDG mice injected with ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53OE/shNEK2 cells ( n = 4). Cells were treated I) with BTZ or H) with control. Data presented as mean ± SD, p ‐values are calculated using two‐way ANOVA with Dunnett post‐hoc test, n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001. J) Representative images for IHC detection of Ki‐67 protein in the tumor nodules derived from B‐NDG mice injected subcutaneously with ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells. Cells were treated with BTZ (right) or with control (left).

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Injection, Negative Control, Derivative Assay

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Working model depicting the regulation of NEK2 expression by TP53 .

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 amplification in MM patients is associated with poor outcomes. A) Assessment of NEK2 CNV distribution in MM patients ( n = 575). B) The correlation between CNV and NEK2 mRNA expression, and data presented as mean ± SD, n = 573, p ‐values are calculated using unpaired t test, **** p < 0.0001. C) Kaplan‐Meier analysis of overall survival in MM patients with or without NEK2 CNV amplification ( n = 574). D) Left: Representative images of FISH analysis using probes specific for CEP1 (Green) and NEK2 (Red) in eight MM cell lines, healthy donors (HD, n = 4), and newly‐diagnosed (AD, n = 17) and relapsed (RD, n = 4) MM patients. Upper: normal diploid. Middle: three copies. Lower: four copies ( NEK2 amplification). Right: scatter plot showing statistical results of the proportion of cells with different NEK2 copy numbers in MM patients and MM cell lines. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, * p < 0.05, ** p < 0.01, *** p < 0.001. The MSP results of NEK2 proximal and distal CpG islands in E) seven MM cell lines and F) three MM patients. M, MSP products using methylation‐specific primers; U, MSP products using methylation‐unspecific primers.

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Amplification, Expressing, Methylation

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 expression is elevated in MM patients with TP53 lesions. A) NEK2 mRNA levels in MM patients with ( n = 51) or without ( n = 497) TP53 deletion, and data presented as mean ± SD, p ‐values are calculated using unpaired t test, **** p < 0.0001. B) Kaplan‐Meier analyses of overall survival in MM patients with TP53 N & NEK2 N (normal), TP53 N & NEK2 Amp (Amplification), TP53 D (deletion) & NEK2 N , and TP53 D & NEK2 Amp ( n = 548). C) NEK2 mRNA levels in MM patients with ( n = 40) or without ( n = 703) TP53 mutation, and data presented as mean ± SD, p ‐values are calculated using unpaired t test, **** p < 0.0001. D) Kaplan‐Meier analyses of overall survival in MM patients with wild type TP53 ( TP53 N ) & low expression NEK2 ( NEK2 L ), TP53 N & high expression NEK2 ( NEK2 H ), mutant TP53 ( TP53 mut ) & NEK2 L and TP53 mut & NEK2 H ( n = 743). E) Correlation between NEK2 and TP53 mRNA expression in the TT2 and TT3 trial form GEP data (GSE2658, n = 559, p ‐values are calculated Pearson correlation coefficient ). F) Kaplan‐Meier analyses of overall survival in TT2 and TT3 MM patients ( n = 559) with high NEK2 ( NEK2 H ) & low TP53 expression ( TP53 L ), NEK2 H & high TP53 expression ( TP53 H ), low NEK2 ( NEK2 L ) & TP53 L and NEK2 L and TP53 H . G) Scatter plot showing the percentage of cells with amplified NEK2 copy number in MM patients with or without TP53 deletion. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, ** p < 0.01, *** p < 0.001. H) Scatter plot showing the percentage of cells with amplified NEK2 copy number in TP53 WT or TP53 −/− MM cells. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, * p < 0.05, ** p < 0.01. I,J) Representative immunofluorescence images and statistical analysis for p53 (Green) and NEK2 (Red) protein expression in MM newly diagnosed patients (AD, n = 51) and relapsed patients (RD, n = 16, p ‐values are calculated using one‐way ANOVA with Bonferroni correction).

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Expressing, Amplification, Mutagenesis, Immunofluorescence

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 expression is reduced and correlates inversely with loss of wild type TP53 in MM cells. A) Relative mRNA levels of NEK2 and TP53 in the H929 TP53 WT MM cell line were detected with qPCR after treatment with nutlin‐3a for 48 h at concentrations of 0, 2, and 4 µM. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. B) Relative protein levels of p53, NEK2, and GAPDH in TP53 WT H929 cells after treatment with 0, 2, and 4 µM of nutlin‐3a for 48 h, as determined with immunoblotting. C) Relative mRNA levels of NEK2 and TP53 in the ARP1 TP53 −/− MM cell line were detected with qPCR after treatment with nutlin‐3a for 48 h at concentrations of 0, 2, and 4 µM. D) Relative protein levels of p53, NEK2, and GAPDH in TP53 −/− ARP1 cells after treatment with 0, 2, and 4 µM of nutlin‐3a for 48 h, as determined with immunoblotting. E) The mRNA levels of TP53 and NEK2 in H929 cells with or without TP53 deletion edited by CRISPR/Cas9, as determined with qPCR. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. F) p53 and NEK2 protein levels in H929 cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with immunoblotting. G) TP53 and NEK2 mRNA levels in MM.1s cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with qPCR. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, *** p < 0.001. H) The protein levels of p53 and NEK2 in MM.1s cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with immunoblotting. I) Relative mRNA levels of TP53 and NEK2 in ARP1 cells were detected with qPCR 72 h after infection with virus containing WT or mutant TP53 (E285K)‐expressing vectors. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05. J) Relative protein levels of p53, NEK2, and GAPDH in ARP1 cells were detected with immunoblotting 72 h after infection with virus containing WT or mutant TP53 (E285K)‐expressing vectors. K) The mRNA levels of TP53 and NEK2 in H929 cells with or without TP53 mutation, as determined with qPCR. L) The protein levels of p53 and NEK2 in H929 cells with or without TP53 mutation, as determined with immunoblotting. M) The mRNA levels of TP53 and NEK2 in TP53 ‐deleted H929 (H929‐TP53 KO ) cells with or without TP53 mutation, as determined with qPCR. N) The protein levels of p53 and NEK2 in TP53 ‐deleted H929 (H929‐TP53 KO ) cells with or without TP53 mutation, as determined with immunoblotting.

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Expressing, Western Blot, CRISPR, Infection, Mutagenesis

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Dual defects in NEK2 and p53 enhance mitotic abnormalities, cell proliferation, apoptosis, and tumorigenesis in MM. A) Representative images of clonogenic analysis in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells (images are shown in 4x magnification). B) Statistical analysis of clone numbers formed in soft agarose of the five cell groups shown in panel (A). Data presented as mean ± SD, p ‐values are calculated using unpaired one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. C) Representative flow cytometry dot plots for detection of BrdU‐positive cells among H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells. D) Statistical analysis of the number of BrdU‐positive cells among the five cell groups shown in panel (C). Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. E) Representative histograms for detection of apoptotic cells in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells treated with 4 nM BTZ for 48 h. F) Statistical analysis of the percentage of apoptotic cells among the five cell groups shown in panel (E) after treatment with 4 nM BTZ for 48 h. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. G) Representative images of tumor xenografts from B‐NDG mice with subcutaneous injections of H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells into the right abdomen (6 mice measured for each group). H) Statistical analysis of tumor volumes of xenografts from B‐NDG mice as shown in panel (G) (6 mice measured for each group). Data presented as mean ± SD, p ‐values are calculated using two‐way ANOVA with Dunnett post‐hoc test, n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001. I) Representative images for IHC detection of Ki‐67 protein in the tumor nodules derived from B‐NDG mice injected subcutaneously with H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells.

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Flow Cytometry, Derivative Assay, Injection

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: TP53 deletion upregulates NEK2 expression by inducing amplification and chromosomal instability in MM cells. A) Comparative analysis of HEK293‐TP53 KO versus HEK293‐Ctrl cells using CGH array. Red column represents gains of chromosomes. Blue column represents deletions. Chromosomal aberrations are across the whole genome. B) Schematic depiction of NEK2 location in chromosome 1q21.1‐q44. C) Comparative analysis of HEK293‐NEK2 OE versus HEK293‐Ctrl cells using CGH array. Red column represents gain. Blue column represents deletion. D) Left, representative images of FISH analysis using probes targeting TP53 (17P, Red) and NEK2 (1q32.2, Green) in HEK293‐Ctrl and HEK293‐TP53 KO cells. Right, scatter plot showing the percentage of cells from both groups with amplified NEK2 copy numbers. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. E) Scatter plot showing the percentage of H929‐Ctrl and H929‐TP53 KO cells with amplified NEK2 copy numbers as determined by FISH assay. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. F,G) GSEA of enrichment, including chromosome 1q32 and chromosomal instability, from differentially expressed genes between HEK293‐Ctrl and HEK293‐TP53 KO cells. H) Representative images of spindles (Red: Tubulin), Plasmid‐GFP (Green), and nuclei (Blue) in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells. I) Quantification showing the percentage of cells with abnormal monopolarity among each of the five groups shown in panel (H). All results are shown as means ± SD of three independent experiments, and at least 60 spindles per experiment were randomly chosen and counted in each experiment.Data presented as mean ± SD, p‐values are calculated usingone‐way ANOVA with Dunnett post‐hoc test, * p < 0.05.

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Expressing, Amplification, Plasmid Preparation

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: p53 deletion promotes aberrantly high NEK2 expression through indirect transcriptional regulation. A,B) Luciferase activity driven by NEK2 promoter with normal (full length) or mutant p53 binding site in HEK293‐TP53 KO and ARP1 cells transiently co‐transfected with p53 OE. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. C) ChIP confirming that the transcription factor p53 specifically binds to the NEK2 promoter region in H929 cells. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, **** p < 0.0001. D) Correlation of expression between NEK2 and E2F8 in MM patients based on GEP database (GSE2658, n = 559, p ‐values are calculated Spearman correlation coefficient ). E) The mRNA and protein levels of TP53 , CDKN1A ( p21 ), and E2F8 in H929 cells with or without p53 deletion. Here and in panels (F–J), mRNA and protein data were obtained using qPCR and immunoblotting, respectively. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. F) The mRNA and protein levels of TP53 , CDKN1A ( p21 ), and E2F8 in ARP1 cells with or without p53 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, *** p < 0.001. G,H) The mRNA and protein levels of NEK2 and E2F8 in H929‐Ctrl and H929‐TP53 KO cells with (H929‐TP53 KO /E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. I,J) The mRNA and protein levels of NEK2 and E2F8 in ARP1‐EV and ARP1‐TP53 OE cells with (ARP1‐TP53 OE/E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. K) ChIP confirmed that the transcription factor E2F8 specifically binds to the NEK2 promoter region. L) Schematic of p53 deletion enhancing NEK2 expression through E2F8 upregulation.

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Binding Assay, Transfection, Western Blot, Over Expression

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: p53 suppresses NEK2 expression by regulating the expression of DNMTs. A) Detailed BGS analysis confirmed methylation status of the NEK2 promoter's distal CpG island in H929 cells with or without p53 deletion. B) Statistical analysis of methylated CpG dinucleotides in the NEK2 promoter's distal CpG nucleotides in H929 cells with or without p53 deletion. Data presented as mean ± SD, n = 11, p ‐values are calculated using one‐way ANOVA with Bonferroni correction, ** p < 0.01, **** p < 0.0001. C) Heatmap of the ratios of the signal intensities of differential methylation‐related genes in H929 cells with or without p53 deletion. D) The mRNA levels of histone methylation‐related genes ( NSD3 , PRMT1 , SETD5 , and SETD7 ) detected by qPCR in H929 cells with or without p53 deletion. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. E) H3K36me3 protein levels detected using immunoblotting in H929 cells with or without p53 deletion. F,G) The mRNA and protein levels of DNMT1 , DNMT3a , and DNMT3b in H929 cells with or without p53 deletion using qPCR and immunoblotting. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. H,I) The mRNA and protein levels of NEK2 after shRNA‐mediated DNMT3b knockdown in wild type p53 and p53‐deleted H929 cells, detected by qPCR and immunoblotting. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. J) Endogenous p53 was pulled down by p53 antibodies in H929 cells with or without p53 deletion, and the DNMT1, DNMT3a, and DNMT3b proteins were analyzed by western blotting. The lysates before IP were used as a positive control. K) Dysfunctional p53 elevates NEK2 expression in MM by upregulating DNMT expression, thereby increasing methylation of the NEK2 promoter.

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Expressing, Methylation, Western Blot, shRNA, Positive Control

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Downregulation of NEK2 in TP53 ‐deleted MM cells inhibits cell growth and decreases drug resistance. A,B) Representative images of spindles (Red, Tubulin), GFP (Green), and nuclei (Blue) in ARP1‐Ctrl, ARP1‐TP53 OE, and ARP1‐TP53 OE/shNEK2 cells. Quantifications show the percentage of abnormal monopolarity from three independent experiments. At least 60 spindles were randomly chosen and counted in each experiment. All results are shown as means ± SD of three independent experiments, Data presented as mean ± SD, p‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, * p < 0.05. Flow cytometric detection of BrdU‐positive cells in ARP1‐Ctrl, ARP1‐TP53 OE, and ARP1‐TP53 OE/shNEK2 cells. Representative C) dot plots and D) quantification. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01. E) Representative histograms for detection of apoptotic cells and F) statistical analysis of the percentage of apoptotic cells in ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TRP53 OE/NEK2 OE, and ARP1‐TP53 OE/shNEK2 cells treated with 4 nM BTZ for 48 h. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, ** p < 0.01. G) Representative images of tumor xenografts in B‐NDG mice after subcutaneous injection of ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells into the right abdomen. Cells were treated with BTZ or with PBS as a negative control. Tumor volumes of xenografts derived from B‐NDG mice injected with ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53OE/shNEK2 cells ( n = 4). Cells were treated I) with BTZ or H) with control. Data presented as mean ± SD, p ‐values are calculated using two‐way ANOVA with Dunnett post‐hoc test, n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001. J) Representative images for IHC detection of Ki‐67 protein in the tumor nodules derived from B‐NDG mice injected subcutaneously with ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells. Cells were treated with BTZ (right) or with control (left).

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Injection, Negative Control, Derivative Assay

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Working model depicting the regulation of NEK2 expression by TP53 .

Article Snippet: For Figures and , 1 × 10 6 H929‐Ctrl, H929‐NEK2 OE, H929‐TP53 KO , H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of immunodeficient female B‐NDG (6–8 weeks old) mice without mature T cells, B cells, and NK cells [ ] (Biocytogen Co, Beijing, China).

Techniques: Expressing

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 expression is elevated in MM patients with TP53 lesions. A) NEK2 mRNA levels in MM patients with ( n = 51) or without ( n = 497) TP53 deletion, and data presented as mean ± SD, p ‐values are calculated using unpaired t test, **** p < 0.0001. B) Kaplan‐Meier analyses of overall survival in MM patients with TP53 N & NEK2 N (normal), TP53 N & NEK2 Amp (Amplification), TP53 D (deletion) & NEK2 N , and TP53 D & NEK2 Amp ( n = 548). C) NEK2 mRNA levels in MM patients with ( n = 40) or without ( n = 703) TP53 mutation, and data presented as mean ± SD, p ‐values are calculated using unpaired t test, **** p < 0.0001. D) Kaplan‐Meier analyses of overall survival in MM patients with wild type TP53 ( TP53 N ) & low expression NEK2 ( NEK2 L ), TP53 N & high expression NEK2 ( NEK2 H ), mutant TP53 ( TP53 mut ) & NEK2 L and TP53 mut & NEK2 H ( n = 743). E) Correlation between NEK2 and TP53 mRNA expression in the TT2 and TT3 trial form GEP data (GSE2658, n = 559, p ‐values are calculated Pearson correlation coefficient ). F) Kaplan‐Meier analyses of overall survival in TT2 and TT3 MM patients ( n = 559) with high NEK2 ( NEK2 H ) & low TP53 expression ( TP53 L ), NEK2 H & high TP53 expression ( TP53 H ), low NEK2 ( NEK2 L ) & TP53 L and NEK2 L and TP53 H . G) Scatter plot showing the percentage of cells with amplified NEK2 copy number in MM patients with or without TP53 deletion. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, ** p < 0.01, *** p < 0.001. H) Scatter plot showing the percentage of cells with amplified NEK2 copy number in TP53 WT or TP53 −/− MM cells. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, * p < 0.05, ** p < 0.01. I,J) Representative immunofluorescence images and statistical analysis for p53 (Green) and NEK2 (Red) protein expression in MM newly diagnosed patients (AD, n = 51) and relapsed patients (RD, n = 16, p ‐values are calculated using one‐way ANOVA with Bonferroni correction).

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Amplification, Mutagenesis, Immunofluorescence

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: NEK2 expression is reduced and correlates inversely with loss of wild type TP53 in MM cells. A) Relative mRNA levels of NEK2 and TP53 in the H929 TP53 WT MM cell line were detected with qPCR after treatment with nutlin‐3a for 48 h at concentrations of 0, 2, and 4 µM. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. B) Relative protein levels of p53, NEK2, and GAPDH in TP53 WT H929 cells after treatment with 0, 2, and 4 µM of nutlin‐3a for 48 h, as determined with immunoblotting. C) Relative mRNA levels of NEK2 and TP53 in the ARP1 TP53 −/− MM cell line were detected with qPCR after treatment with nutlin‐3a for 48 h at concentrations of 0, 2, and 4 µM. D) Relative protein levels of p53, NEK2, and GAPDH in TP53 −/− ARP1 cells after treatment with 0, 2, and 4 µM of nutlin‐3a for 48 h, as determined with immunoblotting. E) The mRNA levels of TP53 and NEK2 in H929 cells with or without TP53 deletion edited by CRISPR/Cas9, as determined with qPCR. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. F) p53 and NEK2 protein levels in H929 cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with immunoblotting. G) TP53 and NEK2 mRNA levels in MM.1s cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with qPCR. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, *** p < 0.001. H) The protein levels of p53 and NEK2 in MM.1s cells with or without CRISPR‐Cas9‐mediated TP53 deletion, as determined with immunoblotting. I) Relative mRNA levels of TP53 and NEK2 in ARP1 cells were detected with qPCR 72 h after infection with virus containing WT or mutant TP53 (E285K)‐expressing vectors. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05. J) Relative protein levels of p53, NEK2, and GAPDH in ARP1 cells were detected with immunoblotting 72 h after infection with virus containing WT or mutant TP53 (E285K)‐expressing vectors. K) The mRNA levels of TP53 and NEK2 in H929 cells with or without TP53 mutation, as determined with qPCR. L) The protein levels of p53 and NEK2 in H929 cells with or without TP53 mutation, as determined with immunoblotting. M) The mRNA levels of TP53 and NEK2 in TP53 ‐deleted H929 (H929‐TP53 KO ) cells with or without TP53 mutation, as determined with qPCR. N) The protein levels of p53 and NEK2 in TP53 ‐deleted H929 (H929‐TP53 KO ) cells with or without TP53 mutation, as determined with immunoblotting.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Western Blot, CRISPR, Infection, Mutagenesis

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Dual defects in NEK2 and p53 enhance mitotic abnormalities, cell proliferation, apoptosis, and tumorigenesis in MM. A) Representative images of clonogenic analysis in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells (images are shown in 4x magnification). B) Statistical analysis of clone numbers formed in soft agarose of the five cell groups shown in panel (A). Data presented as mean ± SD, p ‐values are calculated using unpaired one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. C) Representative flow cytometry dot plots for detection of BrdU‐positive cells among H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells. D) Statistical analysis of the number of BrdU‐positive cells among the five cell groups shown in panel (C). Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. E) Representative histograms for detection of apoptotic cells in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells treated with 4 nM BTZ for 48 h. F) Statistical analysis of the percentage of apoptotic cells among the five cell groups shown in panel (E) after treatment with 4 nM BTZ for 48 h. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. G) Representative images of tumor xenografts from B‐NDG mice with subcutaneous injections of H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells into the right abdomen (6 mice measured for each group). H) Statistical analysis of tumor volumes of xenografts from B‐NDG mice as shown in panel (G) (6 mice measured for each group). Data presented as mean ± SD, p ‐values are calculated using two‐way ANOVA with Dunnett post‐hoc test, n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001. I) Representative images for IHC detection of Ki‐67 protein in the tumor nodules derived from B‐NDG mice injected subcutaneously with H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, or H929‐TP53 KO /shNEK2 cells.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Flow Cytometry, Derivative Assay, Injection

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: TP53 deletion upregulates NEK2 expression by inducing amplification and chromosomal instability in MM cells. A) Comparative analysis of HEK293‐TP53 KO versus HEK293‐Ctrl cells using CGH array. Red column represents gains of chromosomes. Blue column represents deletions. Chromosomal aberrations are across the whole genome. B) Schematic depiction of NEK2 location in chromosome 1q21.1‐q44. C) Comparative analysis of HEK293‐NEK2 OE versus HEK293‐Ctrl cells using CGH array. Red column represents gain. Blue column represents deletion. D) Left, representative images of FISH analysis using probes targeting TP53 (17P, Red) and NEK2 (1q32.2, Green) in HEK293‐Ctrl and HEK293‐TP53 KO cells. Right, scatter plot showing the percentage of cells from both groups with amplified NEK2 copy numbers. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. E) Scatter plot showing the percentage of H929‐Ctrl and H929‐TP53 KO cells with amplified NEK2 copy numbers as determined by FISH assay. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. F,G) GSEA of enrichment, including chromosome 1q32 and chromosomal instability, from differentially expressed genes between HEK293‐Ctrl and HEK293‐TP53 KO cells. H) Representative images of spindles (Red: Tubulin), Plasmid‐GFP (Green), and nuclei (Blue) in H929‐Ctrl, H929‐TP53 KO , H929‐NEK2 OE, H929‐TP53 KO /NEK2 OE, and H929‐TP53 KO /shNEK2 cells. I) Quantification showing the percentage of cells with abnormal monopolarity among each of the five groups shown in panel (H). All results are shown as means ± SD of three independent experiments, and at least 60 spindles per experiment were randomly chosen and counted in each experiment.Data presented as mean ± SD, p‐values are calculated usingone‐way ANOVA with Dunnett post‐hoc test, * p < 0.05.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Amplification, Plasmid Preparation

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: p53 deletion promotes aberrantly high NEK2 expression through indirect transcriptional regulation. A,B) Luciferase activity driven by NEK2 promoter with normal (full length) or mutant p53 binding site in HEK293‐TP53 KO and ARP1 cells transiently co‐transfected with p53 OE. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. C) ChIP confirming that the transcription factor p53 specifically binds to the NEK2 promoter region in H929 cells. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, **** p < 0.0001. D) Correlation of expression between NEK2 and E2F8 in MM patients based on GEP database (GSE2658, n = 559, p ‐values are calculated Spearman correlation coefficient ). E) The mRNA and protein levels of TP53 , CDKN1A ( p21 ), and E2F8 in H929 cells with or without p53 deletion. Here and in panels (F–J), mRNA and protein data were obtained using qPCR and immunoblotting, respectively. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. F) The mRNA and protein levels of TP53 , CDKN1A ( p21 ), and E2F8 in ARP1 cells with or without p53 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, *** p < 0.001. G,H) The mRNA and protein levels of NEK2 and E2F8 in H929‐Ctrl and H929‐TP53 KO cells with (H929‐TP53 KO /E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01. I,J) The mRNA and protein levels of NEK2 and E2F8 in ARP1‐EV and ARP1‐TP53 OE cells with (ARP1‐TP53 OE/E2F8 OE) or without E2F8 overexpression. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. K) ChIP confirmed that the transcription factor E2F8 specifically binds to the NEK2 promoter region. L) Schematic of p53 deletion enhancing NEK2 expression through E2F8 upregulation.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Binding Assay, Transfection, Western Blot, Over Expression

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: p53 suppresses NEK2 expression by regulating the expression of DNMTs. A) Detailed BGS analysis confirmed methylation status of the NEK2 promoter's distal CpG island in H929 cells with or without p53 deletion. B) Statistical analysis of methylated CpG dinucleotides in the NEK2 promoter's distal CpG nucleotides in H929 cells with or without p53 deletion. Data presented as mean ± SD, n = 11, p ‐values are calculated using one‐way ANOVA with Bonferroni correction, ** p < 0.01, **** p < 0.0001. C) Heatmap of the ratios of the signal intensities of differential methylation‐related genes in H929 cells with or without p53 deletion. D) The mRNA levels of histone methylation‐related genes ( NSD3 , PRMT1 , SETD5 , and SETD7 ) detected by qPCR in H929 cells with or without p53 deletion. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. E) H3K36me3 protein levels detected using immunoblotting in H929 cells with or without p53 deletion. F,G) The mRNA and protein levels of DNMT1 , DNMT3a , and DNMT3b in H929 cells with or without p53 deletion using qPCR and immunoblotting. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, *** p < 0.001. H,I) The mRNA and protein levels of NEK2 after shRNA‐mediated DNMT3b knockdown in wild type p53 and p53‐deleted H929 cells, detected by qPCR and immunoblotting. Data presented as mean ± SD, p ‐values are calculated using unpaired t test, n = 3, ** p < 0.01. J) Endogenous p53 was pulled down by p53 antibodies in H929 cells with or without p53 deletion, and the DNMT1, DNMT3a, and DNMT3b proteins were analyzed by western blotting. The lysates before IP were used as a positive control. K) Dysfunctional p53 elevates NEK2 expression in MM by upregulating DNMT expression, thereby increasing methylation of the NEK2 promoter.

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing, Methylation, Western Blot, shRNA, Positive Control

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Downregulation of NEK2 in TP53 ‐deleted MM cells inhibits cell growth and decreases drug resistance. A,B) Representative images of spindles (Red, Tubulin), GFP (Green), and nuclei (Blue) in ARP1‐Ctrl, ARP1‐TP53 OE, and ARP1‐TP53 OE/shNEK2 cells. Quantifications show the percentage of abnormal monopolarity from three independent experiments. At least 60 spindles were randomly chosen and counted in each experiment. All results are shown as means ± SD of three independent experiments, Data presented as mean ± SD, p‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, * p < 0.05. Flow cytometric detection of BrdU‐positive cells in ARP1‐Ctrl, ARP1‐TP53 OE, and ARP1‐TP53 OE/shNEK2 cells. Representative C) dot plots and D) quantification. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, * p < 0.05, ** p < 0.01. E) Representative histograms for detection of apoptotic cells and F) statistical analysis of the percentage of apoptotic cells in ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TRP53 OE/NEK2 OE, and ARP1‐TP53 OE/shNEK2 cells treated with 4 nM BTZ for 48 h. Data presented as mean ± SD, p ‐values are calculated using one‐way ANOVA with Dunnett post‐hoc test, n = 3, ** p < 0.01. G) Representative images of tumor xenografts in B‐NDG mice after subcutaneous injection of ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells into the right abdomen. Cells were treated with BTZ or with PBS as a negative control. Tumor volumes of xenografts derived from B‐NDG mice injected with ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53OE/shNEK2 cells ( n = 4). Cells were treated I) with BTZ or H) with control. Data presented as mean ± SD, p ‐values are calculated using two‐way ANOVA with Dunnett post‐hoc test, n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001. J) Representative images for IHC detection of Ki‐67 protein in the tumor nodules derived from B‐NDG mice injected subcutaneously with ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53 OE/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells. Cells were treated with BTZ (right) or with control (left).

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Injection, Negative Control, Derivative Assay

Journal: Advanced Science

Article Title: Genetic Aberrations and Interaction of NEK2 and TP53 Accelerate Aggressiveness of Multiple Myeloma

doi: 10.1002/advs.202104491

Figure Lengend Snippet: Working model depicting the regulation of NEK2 expression by TP53 .

Article Snippet: 1 × 10 6 ARP1‐Ctrl, ARP1‐TP53 OE, ARP1‐TP53/NEK2 OE, or ARP1‐TP53 OE/shNEK2 cells (in 150 µL PBS) were injected subcutaneously into the abdomen of 6–8 weeks old B‐NDG mice (Biocytogen Co, Beijing, China).

Techniques: Expressing

Journal: International Journal of Nanomedicine

Article Title: p53 Promoted Ferroptosis in Ovarian Cancer Cells Treated with Human Serum Incubated-Superparamagnetic Iron Oxides

doi: 10.2147/IJN.S282489

Figure Lengend Snippet: p53 promoted SPIO-Serum-induced ferroptosis. ( A ) Western blot detection of p53. ( B ) Quantification of p53 levels in SKOV3 and A2780 cells. ( C ) Light microscopy assessment of ovarian cancer cells survival (Scale bar: 20 μm). ( D ) SKOV3 and A2780 cells were treated with p53 overexpression, 100 μg Fe/mL SPIO-Serum, p53 overexpression combined with 100 μg Fe/mL SPIO-Serum for 24 h, MTT assay was used to determine the cell viability. ( E ) SKOV3 and A2780 cells treated as indicated, Western blot was used to determine the ferroptosis-associated proteins xCT and GPX4. ( F ) p53, GPX4 and xCT levels. ( G ) xCT protein expression and mitochondrial ROS production were observed by fluorescence microscopy including control group, 100 μg Fe/mL SPIO-Serum group, p53 overexpression group, and p53 overexpression combined with 100 μg Fe/mL SPIO-Serum group (Scale bar: 10 μm). ( H ) Prussian blue staining (Scale bar: 20 μm). All data presented the mean ± SD (n=3). * P <0.05 vs control group. ** P <0.01 vs control group.

Article Snippet: Trans-OE TM DNA TP53 was purchased from Genechem (Shanghai, China).

Techniques: Western Blot, Light Microscopy, Over Expression, MTT Assay, Expressing, Fluorescence, Microscopy, Control, Staining