Fig. 1
Biochemical and genetic screens reveal ZEB1 interacts with NuRD complex members. a Fusion of an E. coli abortive biotin ligase mutant (BirA*) to ZEB1 allows for biotinylation of transient or stable ZEB1 interacting proteins. Biotinylated proteins are captured by streptavidin conjugated sepharose beads and identified by mass spectrometry. Human Zeb1 was cloned into the pcDNA5-FlagBirA*-FRT/TO vector and stably integrated in to HEK293 Flp-In cells. Subsequent to selection, cell lines were divided into two pools (denoted Pool A or B) and reflect biological replicates). Expression of the fusion protein repressed the established ZEB1 target, E-cadherin, as assessed by b qPCR and c immunoblot; all asterisks indicate statistical significance by t-test (n ≥ 3, *p ≤ 0.05); error bars represent standard error mean. d Depiction of the Epigenome short hairpin RNA (shRNA) dropout screen. Briefly, (1) an shRNA library consisting of 235 unique mouse or human epigenetic regulators was infected in to the murine Kras/p53 lung cancer cell lines, 393P and 344P. (2) Syngeneic 129/Sv mice were implanted with 400 cells/shRNA and monitored for four weeks; (3) Tumors (denoted In Vivo) and cell lines (denoted In Vitro) were sequenced to determine the barcoded shRNAs abundance and e rank was determined by differential analysis of 344P and 393P RSA score. Graphs represent top fifteenth percentile in in vitro and in vivo analyses and reveal hits with the most significant rank change between the mesenchymal 344P and the epithelial 393P cells
