Lineage tracing of colon cancer cells after MAPK or NOTCH inhibition. (A) Lentiviral vectors for expression of rtTA (pLenti rtTA3G), doxycycline dependent CreERT2 (pLenti TetO-CreERT2), and the Cre-responsive color transgene (pLenti Trace). Upon Cre-recombination, the RFP transgene element flanked by loxN will be removed, causing an irreversible switch from expression of RFP to YFP fluorescence. BlastR/PuroR, blasticid and puromycin resistance genes; LTR, long terminal repeat; PRE posttranscriptional regulatory element; TRE, tetracycline response element. (B) Triple transduced colon cancer cells were xenografted into NOD/SCID mice. Experimental schedule for Cre-recombination by doxycycline (DOX) and tamoxifen (TAM) in AZD- or DBZ-treated xenografts. (C) Representative double immune fluorescence images for YFP, FRA1, and NICD at 2 and 15 d after recombination in AZD- and DBZ-treated SW480 xenografts, as indicated. Narrow panels are higher magnifications of areas boxed in squared panels. Arrowheads point to FRA1- and NICD-positive tumor cells within single YFP-positive clones at 15 d after recombination. Bars, 25 µm. (D and E) Quantification of FRA1–/YFP– and NICD–/YFP–double positive tumor cells in vehicle- (Ctrl), AZD-, and DBZ-treated SW480 xenografts at 2 d (D) and 15 d (E) after recombination. Error bars are mean ± SD. *, P < 0.05; ***, P < 0.001 by t test; n.s., not significant. n ≥ 3 independent biological replicates.