Supplemental Figure 1: AEA induced ER stress in NMSCs
(A-B) JWF2 cells were pretreated with CM-H
2
DCFDA and then treated with vehicle (ETOH) or
20µM AEA for 1 hour. DCF fluorescence (A, upper panel) and phosphorylated eIF2α (B, upper panel) were measured using flow cytometry and analyzed with a BD FACSCanto II flow cytometer with BD FACSdiva software (BD Biosciences). Mean fluorescence intensities in AEA and vehicle treated cells were represented as mean ± SEM (A-B, lower panels). Data were analyzed using the Student’s t-test (* indicates the statistically significant difference between
AEA and vehicle treated cells, P<0.05).
(C-D) JWF2 cells were treated with AEA (20µM) or vehicle (ETOH) for 1 and 2 hours (C) or 4 and 6 hours (D). Phosphorylated eIF2α (P-eIF2α), total eIF2α (T-eIF2α) (E) and CHOP10 expression (F) were examined by Western blot analysis.
GAPDH expression was measured as loading control.
Band intensities were quantified using
ImageJ software. Fold induction was determined by comparing the band intensities of samples to vehicle-treated cells after normalizing GAPDH levels.
Supplemental Figure 2: ER stress is required for AEA-induced apoptosis
JWF2 cells were pretreated with 2.5mM 4-Phenylbutyric acid (PBA) for 1 hour and then treated with AEA. (A) Phosphorylated eIF2α (P-eIF2α) and total eIF2α (T-eIF2α) (2 hours), (B)
CHOP10 expression (4 hours) and GAPDH expression (loading control) were analyzed by
Western blot analysis. Band intensities were quantified using ImageJ software. Fold induction in protein expression was determined by comparing the band intensities of samples to vehicletreated cells after normalizing GAPDH levels.
(C) Caspase 3/7 activity was measured using
Caspase-Glo 3/7 Assay. Luminescence was measured and is presented as the percent increase

from untreated cells. (D) Cell viability was measured after 8 hours by using the MTS assay. Data were analyzed using one way ANOVA followed by Tukey’s multiple comparison test and are represented as mean ± SEM (* indicates the statistically significant difference between the samples and vehicle-treated cells. # indicates the statistically significant difference between the samples and AEA-treated cells. P<0.05).
Supplemental Figure 3: Selective CB1 (AM251) and CB2 (AM630) receptor antagonists inhibited the AEA-mediated reduction in cAMP
(A) JWF2 cells were pre-treated with the indicated concentrations of AM251 or AM630 for 30 minutes and then treated with 20µM AEA for 15 minutes. cAMP was measured using the cAMP-Glo ELISA kit. Data were analyzed using one way ANOVA followed by Tukey’s multiple comparison test and are represented as mean ± SEM (* indicates the statistically significant difference between the samples and vehicle-treated cells. # indicates the statistically significant difference between the samples and AEA-treated cells. P<0.05). (B) JWF2 and HCA-
7 cells were treated with vehicle (ETOH) or AEA and then COX-2, CB1, CB2, GAPDH (loading control) expression were analyzed using Western blot analysis.
Supplemental Figure 4: Selective TRPV1 receptor antagonist inhibited the AEA-mediated elevation of intracellular calcium
(A) AEA caused a dose-dependent increase in intracellular calcium.
JWF2 cells were treated with various concentrations of AEA for 1 minute.
The intracellular calcium Ca i
2+ was measured using Fluo-4 NW calcium assay kit. (B) JWF2 cells were incubated with 20µM AEA or vehicle and intracellular calcium Ca i
2+
was measured in one minute intervals for 10 minutes. (C) AEAinduced elevation of Ca i
2+ was mediated by the TRPV1 receptor. JWF2 cells were pretreated

with different concentrations of the TRPV1 receptor antagonist (AMG9810) for 30 minutes, treated with AEA for one minute, and then the intracellular calcium Ca i
2+ was measured. Data were analyzed using one way ANOVA followed by Tukey’s multiple comparison test and are represented as mean ± SEM (* indicates the statistically significant difference between the samples and vehicle-treated cells. # indicates the statistically significant difference between the samples and AEA-treated cells. P<0.05).