Loss of TSC1 or TSC2 attenuates the response to CC-90009. A-C) Assessment of the effect of TSC1 or TSC2 knockout on CC-90009 response by a flow cytometry based CRISPR competition assay. U937 cells stably expressing Cas9 were infected with a lentiviral vector constitutively co-expressing GFP and sgNT-1, or with lentiviral vectors constitutively co-expressing RFP and sgNT-1, sgNC-8, or 1 of the 3 sgRNAs targeting TSC1 or TSC2 as indicated. Three days after infection, RFP and GFP cells were mixed at a 1:1 ratio and treated with DMSO or 10 μM CC-90009. The change of RFP+/GFP+ ratio was monitored by flow cytometry every 2 days thereafter. A) Left, schematic design of the flow cytometry based CRISPR competition assay. Right, confirmation of TSC1 or TSC2 knockout by immunoblot analysis. B) and C) The RFP+/GFP+ ratios of U937-Cas9 cells co-expressing RFP and sgNT-1, sgNC-8, or 1 of 3 sgRNAs targeting TSC1 (B) or TSC2 (C) mixed with cells co-expressing GFP and sgNT-1 at each indicated timepoint were normalized to the RFP+/GFP+ ratio of the cell mixtures on “Day 0”. D) and E) Immunoblot analysis of U937-Cas9 parental cells or cells stably expressing sgRNAs as indicated. Cells were treated with DMSO or CC-90009 in the absence (D) or presence (E) of cycloheximide. (F) Immunoblot analysis of anti-HA immunoprecipitates (top) or whole cell extracts (bottom) of U937-Cas9 parental cells or cells stably expressing the indicated sgRNAs. Cells were treated with MLN4924 and DMSO or CC-90009. Result shown in all figure panels is representative of 3 biological replicates.