Intracellular flow cytometry and mass cytometry reveals loss of the LSC population in response to NRAS^G12V withdrawal. Primary AML cells were harvested from the spleens of leukemic mice at 0, 48, 72, and 96 hours after doxycycline treatment to abolish NRAS^G12V expression. Flow cytometry was used to assay for levels of (A) Mac1 (myeloid lineage–commitment surface marker). (B) Phospho-AKT, -ERK1/2, -MAPAPKII, and -mTOR. (C) Using mass cytometry, Mac1^High and Mac1^Low populations were gated separately, and the levels of pERK were assessed in each group in each sample. Because mass cytometry data lack autofluorescence (as seen with traditional flow cytometry), these histograms abut the y-axis. (D) SPADE trees reveal loss of Mac-1^Low cells in response to NRAS^G12V withdrawal. These cells were also submitted for mass cytometry to simultaneously evaluate the levels of 26 surface markers and intracellular signaling molecules. SPADE analysis displaying the patterns of Mac-1^Low (top 2 panels) and β-catenin are displayed. Expression values represent arcsinh difference: for Mac-1 the range is 1.15 to 6.13 and for β-catenin the range is −0.14 to 2.6. The smallest node corresponds to 1 cell and the largest node corresponds to 150 cells.