PILS-Nir1 detects PA produced downstream of PLC. (A) Overexpression of the M3 receptor in HEK293A cells elevated PILS-Nir1 and NES-PABDx2-Spo20 PM localization even without agonist addition. The DAG biosensor C1ab-Prkd1 localization was unchanged. Data were taken from time point 0 of experiments in 1D, 1F, 5B, and 5C. 30–46 cells (small symbols) were analyzed from 3 to 4 individual experiments (large symbols). Statistics were calculated using an ordinary two-way ANOVA on the average of each experimental replicate (large symbols, n = 3–4). DF = 1, MS = 7.185, F(1, 14) = 30.37, P < 0.0001. (B) C1ab-Prkd1 showed the activation and subsequent attenuation of PLC signaling upon addition of 10 µM CCh for 2 min and then 5 µM atropine (Atro) for 15 min. Control cells were treated with cell media (veh) after CCh stimulation. (C) PILS-Nir1 response to CCh and then atropine treatment. Cells were treated as in B. The scatter plot shows the change in intensity ratio of PILS-Nir1 at the final time point. Small symbols represent individual cells (n = 38–46), which are color-coded according to their experimental replicate as shown by the large symbols (n = 3–4). Statistics were calculated using an unpaired t test on the average AUC for each replicate (n = 3–4). A two-tailed P value was used, t = 3.328, df = 4. (D) PILS-Nir1 translocation to the PM (data replicated from 5C) lagged behind C1ab-Prkd1 (data replicated from 5B) in response to CCh addition. The data have been normalized so that the maximum value is 1. All xy graphs show the grand means of each experimental replicate ± SEM. (E and F) 30 s after HEK293A cells were stimulated with 150 μM DiC8, 150 µM OAG, 5 µM PDBu, or 100 nM PMA, C1ab-Prkd1 translocated to the PM (E), but PILS-Nir1 did not bind the DAG analogs at the PM (F). The small circles indicate the change in the intensity ratio of individual cells (n = 30–46) 30 s after stimulation. The large circles show the average change in intensity ratio for each experimental replicate (n = 3–4). Cells in each replicate are color-coded accordingly. Statistics were calculated using a one-sample t and a Wilcoxon test with 0 as the hypothetical value. Statistics used the average change in ratio of each experimental replicate (n = 3–4). PILS-Nir1-DiC8: t = 26.25, df = 2; PILS-Nir1-OAG: t = 3.343, df = 2; PILS-Nir1-PDBu: t = 2.523, df = 2; PILS-Nir1-PMA: t = 0.2635, df = 3. C1ab-Prkd1-DiC8: t = 11.49, df = 2; C1ab-Prkd1-OAG: t = 6.916, df = 2; C1ab-Prkd1-PDBu: t = 7.118, df = 2; C1ab-Prkd1-PMA: t = 3.334, df = 3. PMA data are duplicated from Fig. 1 F.
Figure 5.

PILS-Nir1 detects PA produced downstream of PLC. (A) Overexpression of the M3 receptor in HEK293A cells elevated PILS-Nir1 and NES-PABDx2-Spo20 PM localization even without agonist addition. The DAG biosensor C1ab-Prkd1 localization was unchanged. Data were taken from time point 0 of experiments in 1D, 1F, 5B, and 5C. 30–46 cells (small symbols) were analyzed from 3 to 4 individual experiments (large symbols). Statistics were calculated using an ordinary two-way ANOVA on the average of each experimental replicate (large symbols, n = 3–4). DF = 1, MS = 7.185, F(1, 14) = 30.37, P < 0.0001. (B) C1ab-Prkd1 showed the activation and subsequent attenuation of PLC signaling upon addition of 10 µM CCh for 2 min and then 5 µM atropine (Atro) for 15 min. Control cells were treated with cell media (veh) after CCh stimulation. (C) PILS-Nir1 response to CCh and then atropine treatment. Cells were treated as in B. The scatter plot shows the change in intensity ratio of PILS-Nir1 at the final time point. Small symbols represent individual cells (n = 38–46), which are color-coded according to their experimental replicate as shown by the large symbols (n = 3–4). Statistics were calculated using an unpaired t test on the average AUC for each replicate (n = 3–4). A two-tailed P value was used, t = 3.328, df = 4. (D) PILS-Nir1 translocation to the PM (data replicated from 5C) lagged behind C1ab-Prkd1 (data replicated from 5B) in response to CCh addition. The data have been normalized so that the maximum value is 1. All xy graphs show the grand means of each experimental replicate ± SEM. (E and F) 30 s after HEK293A cells were stimulated with 150 μM DiC8, 150 µM OAG, 5 µM PDBu, or 100 nM PMA, C1ab-Prkd1 translocated to the PM (E), but PILS-Nir1 did not bind the DAG analogs at the PM (F). The small circles indicate the change in the intensity ratio of individual cells (n = 30–46) 30 s after stimulation. The large circles show the average change in intensity ratio for each experimental replicate (n = 3–4). Cells in each replicate are color-coded accordingly. Statistics were calculated using a one-sample t and a Wilcoxon test with 0 as the hypothetical value. Statistics used the average change in ratio of each experimental replicate (n = 3–4). PILS-Nir1-DiC8: t = 26.25, df = 2; PILS-Nir1-OAG: t = 3.343, df = 2; PILS-Nir1-PDBu: t = 2.523, df = 2; PILS-Nir1-PMA: t = 0.2635, df = 3. C1ab-Prkd1-DiC8: t = 11.49, df = 2; C1ab-Prkd1-OAG: t = 6.916, df = 2; C1ab-Prkd1-PDBu: t = 7.118, df = 2; C1ab-Prkd1-PMA: t = 3.334, df = 3. PMA data are duplicated from Fig. 1 F.

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