flat-bottom tissue culture plates the day before treatment at a density of 12,000 cells per well. Cells were washed with serum-free media before treatment and pre-purchase CSP-1103 incubated with inhibitors at the indicated concentrations for 1 hour prior to the addition of chlorambucil or chlorambucil conjugates. The plates were incubated for 18 hrs at 37 uC in a humidified incubator and then 10 mL of CCK-8 viability dye was added to each well and the plates were further incubated till color development occurred. Absorbance at 450 nm was then measured 18362028 and non-linear curve fitting and statistical analysis were performed using the GraphPad Prism software. Annexin-V apoptosis assay. HeLa cells were seeded at 100,000 cells/well the day prior to an experiment in a 12-well flatbottom tissue culture plate. Chlorambucil and chlorambucil-conjugate at indicated concentrations were added to the cells in triplicate in serum-free cell-appropriate media. Cells were incubated for 90 minutes with mt-Cbl at 37uC with 5% CO2. Cells were later harvested and washed with ice cold PBS, then washed again with annexin V binding buffer. Cells were resuspended in annexin V binding buffer plus annexin V-FITC and incubated for 15 min. at room temperature. Later, cell suspensions were diluted with more annexin V binding buffer plus SYTOX Red and incubated for a further 15 min. Flow cytometry was then performed on a FACSCanto flow cytometer. A minimum of 10,000 cells were analyzed for every sample. 3 Effects of Shifting the Site of Alkylation Damage Assessment of caspase 3/7 activity, protease activity, and ATP cellular levels. HeLa cells were seeded at 7500 cells/well a day prior to experiments in white flat clear-bottom 96-well plates. Cells were incubated with Cbl, Cbl conjugates, staurosporin or digitonin as appropriate at indicated concentrations. Cellular ATP levels and protease activity were measured after a 90 minute incubation using the Mitochondrial ToxGlo assay as per manufacturer’s instructions. To assess caspase 3/7 activity, cells were incubated with compound for 6 hours prior to measurement with the Caspase-GloH 3/7 Assay as per manufacturer’s instructions. Western blots. After treatment, cells were harvested and washed with ice cold 22286128 PBS prior to lysis with RIPA buffer plus protease inhibitor cocktail at 4uC for 20 min. Cells were then centrifuged at 16,000 g at 4uC for 10 minutes and the supernatant was collected. Total protein concentrations were quantified using the BCA assay and 40 mg of total protein in each sample was run on a 10% or 15% SDS-PAGE gels. Gels were 
transferred onto nitrocellulose or PVDF membranes and blocked with 5% BSA-TBST. Membranes were probed with primary antibody, 1:1000 PARP-1 antibody, 1:1000 cleaved Caspase-3 antibody, 1:5000 Biotin antibody, 1:1000 TBP antibody and 1:1000 Histone H3 antibody ). Membranes were then washed with TBST and incubated with 1:5000 donkey anti-mouse or goat anti-rabbit IgG-HRP secondary Ab for 1 hour at room temperature in 2% milk-TBST prior to chemiluminescence detection. Cbl-conjugate Labeling of mtDNA. HL-60 cells were incubated with Cbl-TPP for 30 min, after which the cells were collected. The cells were then washed with ice-cold PBS, and their mitochondria were isolated using Mitochondrial Isolation Kit for Mammalian Cells. The mtDNA was extracted from the mitochondrial pellets using AllPrep DNA/RNA Mini Kit. DNA concentration was measured using a NanoDrop 1000 Spectrophotometer and then normalized. After th