Putrescine d4

Total polyamine detection was conducted with a total polyamine assay kit (Fluorometric) (Cat# K475, BioVision) according to the manufacturer’s instructions. Individual polyamines were measured using LC-MS after dansylation in the CRI’s Metabolomics Facility at UT Southwestern. Briefly, tissues and cultured cells were extracted with 80% acetonitrile (20–30 mg tissue per 300 μL, 1 million cells per 100 μL), and 100 μL of the supernatant was dried in SpeedVac. The samples were reconstituted in 45 μL of water, then 10 μL of 10 × PBS (pH 11, adjusted by ammonium hydroxide), 5 μL of 10 μM polyamine internal standards including d8-spermine (Cat# 705330, Sigma), d4-putrescine (Cat# 491136, Sigma) and d6-spermidine (Cat# 709891, Sigma) prepared in water, and 45 μL of 1 mg/mL dansyl chloride (Cat# 03641, Sigma) prepared in acetonitrile were added into the same tube. The mixture was incubated at 55 °C for 10 min and then 400 μL of water was added. The samples were then extracted with Oasis HLB 96-well plate (Cat# 186000679, Waters). Samples were eluted with 98% acetonitrile and dried under SpeedVac. Finally, samples were reconstituted with 50 μL of 0.1% formic acid in water, and 20 μL was subjected to LC-MS analysis. Final concentration of polyamines was normalized to internal standards and total soluble protein content.