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. Author manuscript; available in PMC: 2020 Apr 1.
Published in final edited form as: Free Radic Biol Med. 2018 Dec 26;134:1–8. doi: 10.1016/j.freeradbiomed.2018.12.029

Fig. 4.

Fig. 4.

Brain phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) activity assays were performed at room temperature in the dark according to the manufacturers’ instructions. The data are presented as percent saline control. (a) PC-PLC activity was measured after 22.5h of incubation of assay reagents and samples, the time at which the fluorescence of the corresponding positive control was maximum. Cleavage of the assay substrate by PC-PLC yields a dye-labeled diacylglycerol (DAG) that fluoresces using excitation and emission wavelength maxima of 509 and 516nm, respectively. Dox administration to WT mice caused a significant decrease in PC-PLC activity compared to saline treated WT mice (****p<0.0001). Dox treatment of TNFKO mice resulted in similar decrease in PC-PLC activity compared to saline treated TNFKO mice (****p<0.0001). (b) Brain PLD activity at 1h of incubation of assay reagents and samples, the time of maximal fluorescence of the corresponding positive control in these trials, was performed according to manufacturer’s instructions at 37°C protected from light. PLD cleaves the alcohol component of the head group of phospholipids thereby releasing the choline from PtdCho. Assay reactions involving the choline produce a product that fluoresces using excitation and emission wavelength maxima of 571 and 585nm, respectively. Dox treatment resulted in significantly decreased PLD activity in WT mice brain compared to that of saline-treated WT mice (****p<0.0001). Dox treatment also resulted in significantly decreased PLD activity in TNFKO mice brain compared to that of saline-treated TNFKO mice (***P=0.0005). However, PLD activity was preserved in Dox-treated TNFKO mice, significantly higher than PLD activity in brain of Dox-treated WT mice (***p=0.0008). (For determination of PC-PLC activity, n=6 for all groups except for the saline-treated TNFKO group n=7; for determination of PLD activity, n=9, 10, 11 and 7 for WT saline, WT Dox, TNFKO Dox and TNFKO saline groups, respectively).