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. 2020 Sep 14;106(11):2927–2939. doi: 10.3324/haematol.2020.268235

Figure 4.

Figure 4.

TBL1 modulates MYC levels in a post-transcriptional/β-CATENIN independent manner. (A) Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with either dimethylsulphoxide (DMSO) control (NT) or tegavivint (T) for 12 hours. Lysates were immunoprecipitated with anti-TBL1 or anti-IgG and then immunoblotted with the indicated antibodies. Confocal images of proximity ligation assay (PLA) (60x) using anti-TBL1 and anti-β-CATENIN antibodies after treatment of DLBCL cells with either DMSO control (NT) or tegavivint (T) for 12 hours. Red indicates PLA signal, blue indicates cell nuclei (Dapi) (n=3). (B) Chromatin immunoprecipitation (ChIP) assay on crosslinked chromatin from DLBCL cells after treatment with either DMSO control (NT) or tegavivint (T) for 12 hours. Either preimmune (PI) or the indicated immune antibodies were used and the retained DNA was amplified using MYC or BIRC5 promoter-specific primers. Fold enrichment with each antibody was calculated relative to the PI. n=3; data represent means ± standard error of the mean (SEM). ns: P>0.05, *P<0.05, ***P<0.0005 by linear mixed effects models. (C) Immunoblot showing TBL1, β-CATENIN, MYC and SURVIVIN expression after treatment of DLBCL cells with either DMSO control (NT) or tegavivint (T) for 24 hours (n= 3). (D) Immunoblot showing MYC and SURVIVIN protein levels after TBL1 knock-down (KD) using two TBL1 specific shRNA constructs (n=3). Lysates are the same used in Figure 2A however probed for different proteins. (E) Quantitative real-time polymerase chain reaction (qRT-PCR) showing the mRNA fold changes of the indicated oncogenes after treating DLBCL cells with tegavivint (T) for 12 hours relative to the untreated control (NT). n=3, data represent means ± SEM. ns: P>0.05, *P<0.05, **P<0.005 by paired t-test. (F) Immunoblot showing MYC and SURVIVIN expressions after efficient β-CATENIN KD using two β-CATENIN specific short hairpin RNA (shRNA) constructs. Viability obtained by trypan blue exclusion 72 hours post-transduction. n=3, data represent means ± SEM. ns: P>0.05 by paired t-test (ns: not significant). Tegavivint (T): Riva, Pfeiffer: 70 nM; OCI-ly3: 50 nM; WSU-NHL: 15 nM (for all experiments).