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. 2023 Dec 30;25(1):531. doi: 10.3390/ijms25010531

Figure 1.

Figure 1

The pharmacological or genetic manipulation of DAGLα decreases the level of 2-AG in bEnD.3 endothelial cells. bEnD.3 endothelial cells were treated with LEI-106 (1.3 mM) or vehicle (0.9% DMSO in media) for 15 min, then subjected to LC-MS/MS to measure 2-AG and AEA levels. In a separate set, the level of DAG was quantified by ELISA. In the siRNA transfection experiments, bEnD.3 cells were transiently transfected with siRNA targeting DAGLα or a scrambled, non-targeting control, then harvested 72 h post-transfection, followed by a Western blot to detect DAGLα expression. In a separate set of experiments, the cell pellet was lysed and subjected to LC-MS to measure 2-AG and AEA levels. (A) The schema of the experimental setting. (B) The pharmacological blockade of DAGLα with LEI-106 significantly reduced the 2-AG level in endothelial cells (LEI-106 vs. vehicle, p = 0.001, t(12) = 5.447 as assessed by unpaired t-test). Data are shown as mean ± SEM in nmol/g pellet (n = 4-10 in each group). *** denotes significantly different (p < 0.001). (C) After LEI-106 treatment, the level of AEA was unchanged (LEI-106 vs. vehicle, p = 0.7578, t(13) = 0.3149 as assessed by unpaired t-test). Data are shown as mean ± SEM in pmol/g pellet (n = 5–10 in each group). ns = non-significant. (D) The LEI-106 treatment did not significantly change the level of DAG compared to vehicle control (LEI-106, 650 µM vs. vehicle: p = 0.4364, t(14) = 0.8011; LEI-106, 1.3 mM vs. vehicle: p = 0.4626, t(14) = 0.7553 as assessed by unpaired t-test). Data are presented as % of media-treated ± SEM (n = 8/group). ns = non-significant. (E) Representative image showing the expression of DAGLα with α-tubulin as loading control in bEnd.3 cell lysate harvested 72 h after siRNA transfection. The transfection of DAGLα-targeting siRNA significantly reduced the detection of DAGLα protein compared to the non-targeting control (DAGLα siRNA vs. non-targeting control siRNA: p < 0.0001, t(14) = 7.227, as assessed by unpaired t-test). Data are shown as % of non-targeting control ± SEM (n = 8/group). **** denotes significantly different (p < 0.0001). (F) siRNA knockdown of DAGLα in bEnD.3 cells significantly reduced 2-AG levels compared to non-targeting control (DAGLα siRNA vs. non-targeting control siRNA: p = 0.0266, t(6) = 2.919, as assessed by unpaired t-test). Data are shown as mean ± SEM in nmol/g pellet (n = 4/group). * denotes significantly different (p < 0.05). (G) The AEA level of bEnd.3 cells was significantly increased after DAGLα siRNA transfection compared to non-targeting control, suggesting the presence of a compensatory mechanism after the loss of 2-AG (DAGLα siRNA vs. non-targeting control siRNA: p = 0.031, t(6) = 2.804, as assessed by unpaired t-test). Data are shown as mean ± SEM in pmol/g pellet (n = 4/group). * denotes significantly different (p < 0.05).