PILS-Nir1 reveals that PLD contributes to PA production downstream of PLC. (A and B) HEK293A cells expressing either PILS-Nir1 (A) or NES-PABDx2-Spo20 (B) were treated with 750 nM FIPI for 5 min to inhibit PLD activity or cell media as a control, and then, 5 µM CCh was added for 15 min to induce M3 receptor signaling. The red insets show the biosensors at a region of the PM before stimulation, while the blue insets show the same region after stimulation. The xy graphs show the grand means of five to six experiments ± SEM. The scatter plots show the AUC of individual cells’ responses as the small symbols (n = 48–60) and average AUC of the experimental replicates as the large symbols (n = 5–6). Cells are color-coded according to their experimental replicate. Statistics were calculated using Student’s t test on the average replicate AUC (n = 5–6). For PILS-Nir1, t = 2.139, df = 8. For NES-PABDx2-Spo20, t = 0.8288, df = 9. In both tests, a two-tailed P value was used.