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

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Copyright© 2021 Ferrata Storti Foundation

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Figure 5. — TBL1 is a critical component of a SCF complex in diffuse large B-cell lymphoma through which it modulates MYC stability. (A) The indicated diffuse large B-cell lymphoma (DLBCL) cell lines were treated with either dimethylsulfoxide (DMSO) control (NT) or tegavivint (T) for 12 hours, immunoprecipitated with either an anti-TBL1 or anti-SKP1, and then immunoblotted with the indicated antibodies. β-CATENIN and histone H3 were used as positive and negative controls, respectively. (B) Confocal images (60x) of proximity ligation assay (PLA) showing the CAND1-CUL1 interaction in the indicated DLBCL cells lines after treatment with either DMSO control (NT) or tegavivint (T) for 12 hours. Red indicates PLA signal, blue indicates cell nuclei (Dapi). Graph represents the mean value of PLA signal from no less than 300 cells (n=3). **P<0.005 by paired t-test. (C) Proposed model of the mechanism of tegavivint-induced activation of the SCF complex in DLBCL. (D) Immunoblot showing PLK1 and MYC expression levels after treatment with either DMSO control (NT) or tegavivint (T) at the indicated time points. (E) Immunoblot showing PLK1 level after treatment with either the translation inhibitor cycloheximide (CHX), tegavivint (T) or the combination (CHX+T) for the indicated time points. (F) Immunoblot showing PLK1 and MYC expression after treatment with either DMSO control (NT), tegavivint (T), the proteasome inhibitor MG132 or the combination (T+MG132) for 24 hours. (G) Cytotoxicity assay in DLBCL cell lines treated with either DMSO control (NT) or tegavivint (T), MG132 or the combination (T+MG132). Viability was determined by annexinV/propidium (Ann-/PI-) staining and flow cytometry at 24 hours. n= 3, data represent means ± SEM. *P<0.05, **P<0.005 by linear mixed effects models including interaction test between T and MG132. CHX: 70 mg/mL added for 1 hour and then washed before adding tegavivint treatment. MG132 (Riva, OCI-Ly3 and WSU-NHL: 0.5 uM and Pfeiffer: 0.3 uM) was added 6 hours after tegavivint treatment was started.