Design and selection ARTi-shRNAmirs.

A, Schematic outline of the ARTi approach. B, Sequence logo (https://weblogo.berkeley.edu) displaying nucleotide position biases of 2161 shRNAs with exceptionally high DSIR scores (>105). Inlay depicts miRNA seed sequence biases. C, Reporter assay comparing gene-specific shRNAs to ARTi-shRNAmirs. D, Competitive proliferation assays in human cell lines after transduction with ARTi-shRNAmirs, neutral (shRen.713) and essential control shRNAs (shRPL9.324 or shPSMA3.164). E, Principal component (PC) analysis of gene expression profiling upon stable expression of indicated shRNAs or treatment with MEK inhibitor (trametinib) in RKO cells. F, Volcano plots visualizing de-regulated genes upon expression of indicated shRNAs and trametinib treatment in RKO cellsk, compared to empty vector control.

Experimental validation of the ARTi approach.

A, Schematic of EGFRdel19-ARTi engineering in PC-9 cells. Blue color denotes overexpressed ARTi variant. Green denotes endogenous gene. B, Western Blot demonstrating knock-down of EGFRdel19-ARTi. Western Blot is a representative example of three independent biological repeat experiments. C, Proliferation assay and crystal violet staining of parental and engineered PC-9 cells in absence or presence of Dox. Crystal violet staining is a representative example of two independent biological repeat experiments. D, In vivo experiment comparing Dox-induced EGFRdel19-ARTi knockdown to pharmacological EGFRdel19 inhibition. Mean tumor volume and +/- s.e.m. is plotted for all in vivo experiments. E, Schematic of MIA PaCa-2 engineering. Blue color denotes overexpressed ARTi variant. Green denotes endogenous gene. F, Western blot for KRAS and Actin in indicated engineered MIA-PaCa-2 cells in the presence and absence of Dox. Western Blot is a representative example of two independent biological repeat experiments. G, Proliferation assay and crystal violet staining of parental and engineered MIA PaCa-2 cells in absence or presence of Dox. Crystal violet staining is a representative example of two independent biological repeat experiments. H, Growth curves of tumors implanted with engineered MIA PaCa-2 cells in absence and presence of Dox. In vivo I, Schematic of C-terminal endogenous tagging of STAG1. Green color denotes endogenous genes. J, Western Blot demonstrating knock-down of STAG1-ARTi. Western Blot is a representative example of three independent biological repeat experiments. K, Immunohistochemistry staining of STAG1 in engineered HCT-116 cells in absence and presence of Dox. Asterisk marks an area of murine fibroblasts that serve as an internal positive control. I, Growth curves of tumors implanted with engineered HCT 116 cells in the absence and presence of Dox.