*2.3. Assays*

A Ras activity assay was performed according to the manufacturers instructions for the Active Ras Detection Kit (Cell Signaling Technology, Danvers, MA, USA). Briefly, cells were lysed with the Lysis/Binding/Wash bu ffer and pelleted, then the supernatant was used as the cell lysate. In the positive control, 5 μL of 10 mM GTP γS was added to 500 μL of lysates and incubated at 30 ◦C for 15 min. Cell lysates were incubated with glutathione resin, together with the purified GST-Raf1-RBD protein at 4 ◦C for 1 h in a spin cup. The resin was washed and the bound proteins were eluted by incubating with dithiothreitol (DTT)-containing sample bu ffer at RT for 2 min. Eluted samples were heated and analyzed by anti-Ras Western blotting.

A cell proliferation assay was performed using Cell Counting Kit-8 from Dojindo Molecular Technologies. Cells in 100 μL media in a 96-well plate were incubated with 10 μL of WST-8, a tetrazolium salt, at 37 ◦C in a tissue culture incubator. Absorbance was measured at 450 nm in a PerkinElmer Victor<sup>3</sup> plate reader. Half maximal inhibitory concentrations (IC50s) were determined by incubating cells at a range of concentrations for 72 h and were calculated by GraphPad Prism 5.0 using the log (inhibitor) vs. response function and non-linear fit.

#### *2.4. Chemoprevention in NPcis Mice*

NPcis (*cis Nf1*+/−*;Tp53*+/−) mice in C57BL/6 background (B6;129S2-Trp53tm1Tyj Nf1tm1Tyj/J, Stock No: 008191, Jackson Laboratory) were bred by pairing male heterozygous NPcis mice with the female wildtype mice to better generate MPNST animals [21,23]. Since homozygous *Nf1*/*Tp53* KO mice are embryonically lethal, only heterozygous and wildtype pups were born [21,25]. Mice were genotyped via qPCR by Transnetyx using the following primer pairs: Nf1 wildtype (WT) (5--GGTATTGAATTGAAGCACCTTTGTTTGG-3-, 5--CGTTTGGCATCATCATTATGCTTACA-3- , reporter: 5--AATATATGACCCCATGGCTGTC-3-), Nf1 KO (5--TGGAGAGGCTTTTTGCTTCCT-3- , 5--CGTTTGGCATCATCATTATGCTTACA-3-, reporter: 5--CTGCTCGACATGGCTG-3-), Tp53 WT (5--GTGAGGTAGGGAGCGACTTC-3-, 5--TTGTAGTGGATGGTGGTATACTCAGA-3-, Reporter: 5--CCTGGATCCTGTGTCTTC-3-) and Tp53 KO (5--TGTTTTGCCAAGTTCTAATTCCATCAGA-3- , 5--TTGTAGTGGATGGTGGTATACTCAGA-3-, reporter: 5--ACAGGATCCTCTAGAGTCAG-3-). At day 60 after birth, heterozygous mice were started on the medicated feed or water. The mouse diet consisting of 45 kcal% fat containing soybean oil and lard for fat (D12451, Research Diets) was used as the control feed. Diets with 175, 195, 215 or 250 mg/kg of MBZ polymorph C (Aurochem Laboratories Ltd., Mumbai, India) or 1000 ppm (mg/kg) celecoxib (Sigma) were manufactured with the D12451 formulation in color codes. Sulindac (Sigma) was added to drinking water at 160 ppm (0.5 mg/day) in 4 mM sodium phosphate bu ffer as previously described [20]. Animals were palpated weekly for tumors and survival and cause of death, as detailed in the Results section, were recorded. All animal experiments were performed under an approved protocol and in accordance with Johns Hopkins Animal Care and Use guidelines.
