Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Association between LEF1 mRNA expression with survival status of MM patients. A, The overall survival curve exhibited the overall survival of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software ( www.genomicscape.com ). B, The progression-free survival (Left) and overall survival (Right) status of MM patients with low or high expression levels of LEF1 mRNA using GenomicScape software ( www.genomicscape.com ). Red line: high expression group, blue line: low expression group. P values were denoted on the plot.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Expressing, Software

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Knockdown of LEF1 suppressed growth of U266 and RPMI8226 myeloma cells. A, The mRNA expression level of LEF1 in U266 and RPMI8226 cells infected with LEF1 shRNA (shLEF1) or scramble shRNA as the control (shCon) for 48 h was assessed by quantitative real-time RT-PCR (qRT-PCR). B, The protein expression level of LEF1 in U266 and RPMI8226 cells infected with shLEF1 or shCon for 72 h was detected by Westernblot analysis. C, The cell viability of U266 and RPMI8226 cells following transduction with shLEF1 or shCon was determined by CCK-8 assay. D, Brdu incorporation was determined in U266 and RPMI8226 cells transduced with shLEF1 or shCon. * P < 0.05 and ** P < 0.01 versus shcon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Knockdown, Expressing, Infection, shRNA, Control, Quantitative RT-PCR, Transduction, CCK-8 Assay, BrdU Incorporation Assay

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Depletion of LEF1 induced apoptosis of U266 and RPMI8226 myeloma cells. A-B, The cellular apoptosis was detected in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 5 days by flow cytometry analysis following Annexin V/7AAD staining. C, The protein levels of PARP and cleaved caspase 3 was detected in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 5 days by Westernblot analysis. D, Intracellular ROS production was determined by CM-H2DCFDA staining in U266 and RPMI8226 cells following transduction with shLEF1 or shCon for 72 h. * P < 0.05 and ** P < 0.01 versus shCon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Transduction, Flow Cytometry, Staining

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: LEF1 inhibited transcription of CYLD gene through promoter binding. A, The protein levels of CYLD in U266 and RPMI8226 cells following transduction with shLEF1 or shCon for 72 h was measured by Westernblot analysis. B, The CYLD mRNA expression level in U266 and RPMI8226 cells transduced with shLEF1 or shCon for 72 h was determined using qRT-PCR. C, Schematic representation of potential LEF1-binding sites in CYLD promoter and the location of the primers used for ChIP assay. D, U266 cells transduced with shLEF1 or shCon were co-transfected with either wild-type CYLD luciferase reporter construct (pGL3-CYLD-wt) or the plasmid containing the mutation in the putative LEF1 binding site (pGL3-CYLD-mt) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined with dual-luciferase reporter assay kit. E, ChIP assay was performed to show binding of LEF1 on CYLD promoter in U266 cells. Anti-LEF1 antibody or control IgG was used to precipitate sonicated chromatin from U266 cells. The purified DNA was analyzed by real-time PCR with primers flanking potential LEF1 binding site. Data showed fold enrichment relative to control IgG. * P < 0.05 and ** P < 0.01 versus shCon group.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Binding Assay, Transduction, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Construct, Plasmid Preparation, Mutagenesis, Activity Assay, Reporter Assay, Control, Sonication, Purification, Real-time Polymerase Chain Reaction

Journal: Technology in Cancer Research & Treatment

Article Title: Downregulation of LEF1 Impairs Myeloma Cell Growth Through Modulating CYLD/NF-κB Signaling

doi: 10.1177/15330338211034270

Figure Lengend Snippet: Depletion of LEF1 repressed NF-κB signaling pathway through modulation of CYLD in U266 cells. A, The protein levels of p65 and p-p65 in U266 cells following LEF1 knockdown for 72 h was measured by Westernblot analysis. B, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. C, The mRNA expression levels of Cyclin D1, Bcl-2 and Bcl-xL in U266 cells transduced with shLEF1 or shCon for 72 h were determined using qRT-PCR. * P < 0.05 and ** P < 0.01 versus shCon group. D, U266 cells transduced with shLEF1 or shCon were co-transfected with NF-κB reporter construct (p-NF-κB-luc) in the presence of CYLD siRNA or the control siRNA together with the pRL-CMV plasmid for normalization of transfection efficacy for 24 h, and relative luciferase activity was determined using dual-luciferase reporter assay kit. * P < 0.05 and ** P < 0.01.

Article Snippet: When indicated, cells were co-transfected with the siRNA specific for human LEF1 (sc-35804, Santa Cruz, USA) or the control siRNA (sc-37007, Santa Cruz, USA) at the final concentration of 20 nM.

Techniques: Knockdown, Transduction, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Control