Journal: Clinical and Translational Medicine

Article Title: The positive regulatory loop of TCF4N/p65 promotes glioblastoma tumourigenesis and chemosensitivity

doi: 10.1002/ctm2.1042

Figure Lengend Snippet: TCF4N promotes NF‐κB activation by regulating p65 S536 phosphorylation, nuclear translocation and stability. (A) Western blot analysis indicating the activation of p65 and Stat3 in U87 and U251 cells expressing ectopic TCF4N. GAPDH served as a loading control. The right panel shows the statistical result of p65 protein level. n = 3, ** p < 0.01. (B) RT‐qPCR analysis showing ectopic TCF4N had no effect on RELA (p65) mRNA expression. (C) Western blot analysis of p65 expression in U87 cells with ectopic TCF4N. Cells were treated with cycloheximide (CHX, 100 μg/ml) with or without MG132 (20 μM) for the indicated hours before harvest (left panel). The alteration of relative p65 protein level was shown in the right panel. (D) Ubiquitination analysis of p65. The indicated cells were incubated with MG132 (20 μM) for 6 h before harvest. Ubiquitin (Ub)‐binding p65 was immunoprecipitated (IP) by p65 antibody and detected by Ub antibody. Target proteins in the total lysate were also detected by the indicated antibodies. (mean ± SD, n = 3, ** p < 0.01). (E) Ubiquitination analysis of indicated ectopic p65 in p65‐KO cells. Ub‐binding p65 was immunoprecipitated by Flag antibody and detected by Ub antibody. Target proteins in the total lysate were also detected by the indicated antibodies. (F) Western blot analysis of indicated proteins in p65‐KO cells expressing ectopic TCF4N. GAPDH served as a loading control. (G) Transcriptional activity analysis of NFκB using luciferase reporter system. n = 3, ** p < 0.01. (H) Diagram of indicated TCF4 constructs. (I) IP analysis indicating TCF4N binding of p65. U87 cells expressing TCF4N were transfected with an empty vector or a p65 vector (Flag‐p65) and followed with an IP analysis using HA antibody. IgG antibody was used as a negative control. (J) Analysis of the interaction between the indicated constructs and p65. U87 cells co‐expressing of p65 and TCF4 or TCF4∆N vectors and were subjected to IP analysis using HA antibody. (K) Western blot analysis of p65 level in U87 cells expressing ectopic TCF4 or TCF4∆N. (L) Double IF staining showing the distribution of p65 in U87 cells upon expressing the indicated TCF4 constructs. Bars, 20 μM. (M) IF staining showing the cellular localization of endogenous or ectopic p65 in U87 cells. Scale bars, 10 μm. (N) IF staining showing the cellular expression of ectopic p65 in p65‐KO cells with or without TCF4N expression. Scale bars, 10 μm

Article Snippet: For p65 stability assay, cells were treated with cycloheximide (CHX, 100 μg/ml; Selleck, Houston, TX) with or without MG132(20 μM; Selleck) for the indicated hours before harvest. p65 expression was determined by Western blot.

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay, Western Blot, Expressing, Control, Quantitative RT-PCR, Ubiquitin Proteomics, Incubation, Binding Assay, Immunoprecipitation, Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation, Negative Control, Staining