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. Author manuscript; available in PMC: 2021 May 18.
Published in final edited form as: Nature. 2020 Nov 18;588(7836):164–168. doi: 10.1038/s41586-020-2925-1

Fig. 3. |. BCL6 polymerization enhances SIAH1 interaction and ubiquitination.

Fig. 3. |

a, Correlation of p-values for two genome-wide CRISPR-Cas9 knockout screens: x-axis - reporter screen for eGFPBCL6 stability in HEK293TCas9 cells upon BI-3802 treatment, and y-axis - BI-3802 resistance screen in SuDHL4Cas9 cells. Guides were collapsed to gene level (n = 3; 4 guides/gene; two-sided empirical rank-sum test-statistics). b, Flow cytometry analysis of HEK293TCas9 cells expressing the indicated BCL6-BTB domain fusion construct treated with DMSO or 1 μM BI-3802 for 7 hours (bars represent mean, n = 3). c, Immunoblots of eGFP immunoprecipitation in the presence of 2μM BI-3802 or DMSO from HEK293TCas9 cells transduced with indicated eGFPBCL6 constructs and V5SIAH144C>S (n = 2). d, Immunoblots of StrepBCL65−360 in vitro ubiquitination by SIAH1FL in the presence of DMSO or 1 μM BI-3802 (n = 2). e, BodipyBCL65−360 was titrated to 0.2 μM BiotinSIAH1SBD in DMSO, 2 μM BI-3812, or 2 μM BI-3802, and the signal was measured by TR-FRET. Lines represent standard four parameter log-logistic curve fit (n = 3). f, HEK293TCas9 cells expressing the eGFPBCL61−275 reporter and V5SIAH1 were treated with 0.5 μM MLN7243 for 2 hours, and 1 μM BI-3802 for 1 hour. Cells were imaged by indirect immunofluorescence as indicated (n = 2). Scale bar is 5 μm.