Unlike hSARM1, the NADase activity of ceTIR-1 is not inhibited in cultured cells. NAD+consumption in HEK293F cells was measured by the fluorescence resazurin assay (top) and by LC-MS/MS (middle). Cellular toxicity was measured by live count (bottom). ceTIR-1 activity in cultured cells is significant in both the resazurin and cell death assays but is the most conspicuous in the LC-MS/MS measurements, where NAD+levels drop by 75% relative to NADase-negative control (hSARM1E642Q) or to hSARM1. Point mutations designed to disturb the ARM-TIR interaction ceFF407-8RR do not lead to further significant NAD+consumption (resazurin) or cell death, demonstrating that ARM-TIR interactions do not play a significant role in ceTIR-1 inhibition. Gray and black-striped bars show values gained 48 and 72 h post transfection, respectively. Three biological repeats, Student’s t test;***p < 0.001;**p < 0.01;*p < 0.05; n.s., no significance.
