Skip to main content
. 2023 Dec 30;25(1):531. doi: 10.3390/ijms25010531

Figure 4.

Figure 4

The inhibition of DAGLα induced morphological changes in bEnD.3 cells accompanied with reduced expression of VE-cadherin. Cell viability assay on bEnD.3 cells was performed using increasing doses of LEI-106 (100 nM–1.3 mM). For ICC experiments, the bEnD.3 cells were treated with two doses of LEI-106 (650 uM and 1.3 mM) for 15 min, followed by 2-AG or vehicle application. ICC was performed to detect possible changes in the expression of two tight junction proteins, claudin 5 and VE-cadherin. (A) The panel represents the setting of the experiments. (B) LEI-106 did not cause significant changes in the cell viability at any doses applied in the study. (LEI-106 at any doses vs. vehicle: p > 0.05, as assessed by one-way ANOVA with Bartlett post-test, F(8,26) = 0.7738). All data are expressed as the mean of optical density ± SEM (n = 4 in each group). ns = non-significant. (C) Representative ICC images of bEnd.3 cells treated with LEI-106 ±2-AG application. (D) Significant changes in the nuclear: cytoplasmic ratio were observed following LEI-106 treatment, suggesting possible morphological alteration caused by 2-AG depletion (LEI-106-650 µM vs. vehicle: p < 0.0001, LEI-106-1.3 mM vs. vehicle: p < 0.0001, as assessed by one-way ANOVA with Tukey post-test, F(8,85) = 30.50). 2-AG treatment following LEI-106 application significantly reversed these effects (LEI-106-650 µM vs. LEI-106-650 µM + 2-AG: p < 0.0001, LEI-106-1.3 mM vs. LEI-106-1.3 mM + 2-AG: p < 0.0001, as assessed by one-way ANOVA with Tukey post-test, F(8,85) = 30.50). All data are shown as the mean % of cellular area ± SEM (n = 9–12/condition). **** p < 0.0001, compared to corresponding controls. (E) The higher dose of LEI-106 (1.3 mM) significantly reduced VE-cadherin CTCF; however, no significant change was detected at the lower dose (650 µM) compared to vehicle control (LEI-106-650 µM vs. vehicle: p = 0.9052, LEI-106-1.3 mM vs. vehicle: p = 0.0233, as assessed by one-way ANOVA with Bartlett post-test, F(2,33) = 4.100). The administration of 2-AG increased the VE-cadherin CTCF, moderating the loss of VE-cadherin expression after LEI-106 treatment (LEI-106-1.3 mM vs. LEI-106-1.3 mM + 2-AG: p = 0.005, as assessed by one-way ANOVA with Tukey post-test, F(4,49) = 5.568). All values are expressed as the mean of corrected total cell fluorescence (CTCF) ± SEM (n = 9–12/condition). * p < 0.05, ** p < 0.01, **** p < 0.0001, compared to the corresponding controls. ns = non-significant. (F) Neither dose of LEI-106 treatment significantly influenced the CTCF of claudin-5 compared to vehicle control (LEI-106-650 µM vs. vehicle: p = 0.8982, LEI-106-1.3 mM vs. vehicle: p = 0.6161, as assessed by one-way ANOVA with Tukey post-test, F(5,59) = 2.276). All values are expressed as the mean of corrected total cell fluorescence (CTCF) ± SEM (n = 9–12/condition). ns = non-significant.