p-luc/mdm2 utr  (Promega)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PSMB10 maintains the stemness of chemotherapeutic drug-resistant leukemia cells by inhibiting senescence and cytotoxic T lymphocyte-mediated killing in a ubiquitinated degradation manner

doi: 10.1186/s13046-025-03420-9

Figure Lengend Snippet: The increased PSMB10 impedes RPL6/RPS6-MDM2-P21-induced senescence initiation in AML cells. A Scattergram of upregulated pathways based on KEGG analysis of quantitative proteomics. B-C Changes in protein levels ( B ) and representative images of SA-β-Gal staining ( C ) in the indicated lentivirus-transfected THP-1 cells. Scale bar, 50 μm. D-G Immunoprecipitation assay between RPL6 or RPS6 and PSMB10 ( D ), RPS6 (top) or RPL6 (bottom) and MDM2 or PSMB10 ( E ), MDM2 and RPL6 or RPS6 or P21 ( F ), and MDM2 and P21 ( G ) in THP-1 cells. H shPSMB10-transfected THP-1 cells were treated with CHX for the indicated times, and RPS6 and RPL6 protein levels were detected via WB analysis. I Ubiquitination assay of (left) RPL6 and (right) RPS6 in shPSMB10- or shCTRL-transfected THP-1 cells. J-K Changes in the protein levels of RPL6, MDM2, and P21 ( J ), and representative images of SA-β-Gal staining ( K ) in shCTRL- and shRPL6-transduced THP-1 cells with shPSMB10. Scale bar, 50 μm. L Polysome profiling of shRPL6- or shCTRL-transduced THP-1 cells. M Polysome profiling coupled with qRT‒PCR analysis of shCTRL- and shRPL6-transduced THP-1 cells: MDM2 mRNA distribution in different ribosome fractions (left), and statistical histogram of MDM2 mRNA in the nonribosome portion and polysomes (right). N–O Changes in the protein levels of RPS6, MDM2, and P21 ( N ), and representative images of SA-β-Gal staining ( O ) in shCTRL- and shRPS6-transduced THP-1 cells with shPSMB10. Scale bar, 50 μm. P Statistical histogram of MDM2 translation initiation efficacy, defined as the quotient of reporter protein production (F-luc/R-luc). Q Immunoprecipitation assay between RPS6 and MDM2 in control shRNA- or RPL6 shRNA-transfected THP-1 cells transfected with shPSMB10. R Immunoprecipitation assay between RPL6 and MDM2 in control shRNA- or RPS6 shRNA-transfected THP-1 cells transfected with shPSMB10. OE: overexpression; SA-β-Gal: senescence-associated β-galactosidase; WT: wild-type; IP: immunoprecipitation; CHX: cycloheximide; Fract: fraction; IgG: Immunoglobulin G; NC: negative control; Ub: ubiquitination. **** p < 0.0001 (t test). ns, not significant. The error bars denote the means ± SDs

Article Snippet: The RPS6 overexpression plasmid and its control plasmid, the MDM2 luc-5' UTR plasmid and its control plasmid were constructed by Genechem Company (Shanghai, China).

Techniques: Quantitative Proteomics, Staining, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Control, shRNA, Over Expression, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PSMB10 maintains the stemness of chemotherapeutic drug-resistant leukemia cells by inhibiting senescence and cytotoxic T lymphocyte-mediated killing in a ubiquitinated degradation manner

doi: 10.1186/s13046-025-03420-9

Figure Lengend Snippet: A proposed mechanism for the increased PSMB10 to maintain the stemness of drug-resistant leukemia cells. Created with figdraw.com. PSMB10 is significantly upregulated in chemotherapeutic drug-resistant LSCs, leading to the downregulation of both RPL6 and RPS6 proteins through ubiquitination-mediated degradation. Then the decreased RPL6 and RPS6 proteins, on the one hand, result in an increased MDM2 protein via the upregulation of translation activity, on the other hand, lead to a decreased RPs complex binding-induced conformational change of MDM2, which further promotes the MDM2-mediated ubiquitin-independent degradation of P21 Waf1 protein and resistance to senescence in AML cells. Besides, the increased PSMB10 also induces leukemia cell resistance to CTL-mediated killing by a direct binding and the ubiquitinated degradation of MHC-I proteins. AML: acute myeloid leukemia; Ub: ubiquitination; SASP: senescence-associated secretory phenotype; CTL: cytotoxic T lymphocyte; TCR: T-cell Receptor; β2m: β2-microglobulin; MHC-I: major histocompatibility complex class I

Article Snippet: The RPS6 overexpression plasmid and its control plasmid, the MDM2 luc-5' UTR plasmid and its control plasmid were constructed by Genechem Company (Shanghai, China).

Techniques: Ubiquitin Proteomics, Activity Assay, Binding Assay, Immunopeptidomics

Journal: bioRxiv

Article Title: Δ133p53 and Δ160p53 isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.1101/2024.07.23.604790

Figure Lengend Snippet: (A) Schematic representation of the promoters of p53 target genes (p21, MDM2, BAX, and PUMA). An arrow denotes the transcription start site (TSS). The DNA sequences of p53 response elements (REs) are shown, with uppercase and lowercase letters indicating matched and mismatched bases, respectively, in relation to the canonical p53 RE sequence (RRRCWWGYYY). Paired arrows highlight the regions subjected to quantitative PCR (qPCR) amplification. Specifically, the distal (5’) p53 RE within the p21 gene promoter was analyzed. (B) Relative DNA-binding of the FLp53-FLAG, Δ133p53-FLAG, and Δ160p53-FLAG proteins to p53 target genes (p21, MDM2, BAX, and PUMA) in H1299 cells. (C) Relative DNA-binding of the FLp53-FLAG protein to the p53-target gene promoters in the presence of the V5-tagged protein Δ133p53 or Δ160p53. ChIP-qPCR assay data are shown as relative enrichment of promoter sequences of the target genes after normalization to the control, pcDNA3.1 plasmid transfected cells. Data are represented as the mean of technical triplicates ± standard deviation (SD), ** P < 0.01 (Student’s t-test).

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (Plasmid #16451) , pGL3-MDM2-Luc (Plasmid #32365) , and PUMA Frag1-Luc (Plasmid #16591) were obtained from Addgene.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Amplification, Binding Assay, ChIP-qPCR, Control, Plasmid Preparation, Transfection, Standard Deviation

Journal: bioRxiv

Article Title: Δ133p53 and Δ160p53 isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.1101/2024.07.23.604790

Figure Lengend Snippet: (A-D) H1299 cells were transfected with luciferase reporter plasmids driven by p21 (A), MDM2 (B), BAX (C), and PUMA (D) promoters, along with vectors expressing FLp53, Δ133p53, or Δ160p53. To evaluate the influence of the isoforms on FLp53’s transactivation capability, co-expression was performed at ratios of 1:1, 1:5, and 1:10 relative to FLp53. Basal promoter activity was determined by transfecting cells with the empty vector, pcDNA3.1. Relative promoter activity is shown after normalization to the pcDNA3.1-treated sample activity. Data represent mean values ± standard deviation (SD) (n=3). * P < 0.05; ** P < 0.01 (Student’s t-test). (E-H) The transcriptional activity of FLp53 on the p21 (E), MDM2 (F), BAX (G), and PUMA (H) promoters was inhibited by Δ133p53 and Δ160p53. Inhibition curve fitting was performed using the exponential function ExpDec1 in Origin 2018 software.

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (Plasmid #16451) , pGL3-MDM2-Luc (Plasmid #32365) , and PUMA Frag1-Luc (Plasmid #16591) were obtained from Addgene.

Techniques: Transfection, Luciferase, Expressing, Activity Assay, Plasmid Preparation, Standard Deviation, Inhibition, Software