Figure 7.

Quantitative phospho-proteomics after DAGLα inhibition in bEnD.3 cells. bEnd.3 cells cultured on 150 mm plates were treated with vehicle (0.9% DMSO in media) or LEI-106 (650 µM) for 15 min, then subjected to quantitative phospho-proteomics. (A) Schema of the experiment. (B) Volcano plot of bEnd.3 of detected phospho-peptides showing a greater than 2-fold change after LEI-106 compared to vehicle treatment. (C) Principal Component Analysis (PCA) of cells treated with vehicle or LEI106 (650 µM). (D) Heatmap of phospho-peptides increased by LEI-106 treatment compared to vehicle or decreased compared to vehicle. (E) GO Biological process functional analysis showing the top 10 processes altered in each condition. (F) GO cellular compartment functional analysis showing the top 10 compartments in which phospho-peptides were impacted in each condition. (G) GO molecular function functional analysis shows the top 10 in which phospho-peptides were impacted in each condition. (H) Top 10 Reactome Pathways associated with changes in phospho-peptides in each outcome. n = 4 biological replicates per condition. (I) bEnd.3 cells were treated with LEI-106 (650 µM or 1.03 mM) or vehicle for 15 min, then subjected to Western to detect ZO-1. Representative image showing the expression of ZO-1 along with α-tubulin as a loading control. The blockade of DAGLα significantly decreased the detection of ZO-1, compared to vehicle control (vehicle vs. LEI-106-650 µM: p = 0.034, vehicle vs. LEI-106-1.3 mM: p = 0.0004, LEI-106-650 µM vs. LEI-106-1.3 mM: p = 0.0142 as assessed by one-way ANOVA with Tukey post-test, F(2,7) = 27.41). All data presented as % of vehicle-treated ± SEM (n = 3–4/condition). * p < 0.05, *** p < 0.001.