CBD induces apoptosis in activated HSCs. (a) Cell viability of activated rat HSCs was determined using an acid phosphatase assay following serum starvation and 8-h treatment with indicated concentrations of cannabinoid ligands. Cell survival is presented as percent of acid phosphatase activity in vehicle-treated cells, expressed as mean±S.E.M. of triplicate experiments. AM630, CB₂R antagonist; JWH (JWH133), CB₂R agonist; PF (PF514273), CB₁R antagonist; AM251, CB₁R antagonist; ACEA, CB₁R agonist; CP55940, CB₁R/CB₂R agonist; AEA, endogenous CB₁R/CB₂R agonist; 2-AG, endogenous CB₁R/CB₂R agonist. (b) Cell death was determined by western blot analysis of cleaved PARP in activated-rat HSCs, following 12-h incubation in serum-free media in the presence of either vehicle (DMSO) or 5 μM of indicated cannabinoid ligands. PARP cleavage is expressed as cleaved PARP over total PARP. (c) PARP cleavage was examined as in (b) in activated rat HSCs treated with CBD for the indicated concentrations and time periods. Western blots are representative of n=three experiments. (d) Activated rat HSCs were incubated in serum-free media in the absence or presence of 5 μM CBD for 2, 4, and 8 h, then stained for FITC-Annexin V and propidium iodide (PI). Cells were sorted and analyzed by flow cytometry. The percentage of cells that are either negative or positive for Annexin V and/or PI is indicated in the appropriate quadrant of each panel

CBD induces ER dilation and expression of ER stress markers. (a) Activated rat HSCs were incubated for 6 h in serum-free media with 5 μM CBD in the presence or absence of either 1 μM AM251 or 1 μM AM630. Cell lysates were subjected to western blot analysis and probed for cleaved PARP to detect cell death. ^**P<0.01 versus control (Student's t-test). Representative of n=three independent experiments. (b) Cell viability of activated rat HSCs treated with 5 μM CBD in the presence or absence of 1 μM AM251 or 1 μM AM630 was determined using the acid phosphatase assay as described in Materials and Methods. (c) Vehicle- or 5 μM CBD-treated activated rat HSCs were visualized by light microscopy at 20 × magnification after 4-h treatment. (d) Immunofluorescent staining of calnexin (green) in activated rat HSCs. Activated HSCs were incubated in serum-free media with either vehicle or 5 μM CBD for the indicated time periods. The cells were fixed and processed for immunostaining as described in Materials and Methods. Nuclei were stained with DAPI (blue). Cells were visualized at 63 × magnification by confocal microscopy. (e) Activated rat HSCs were incubated in serum-free media in the presence or absence of 5 μM CBD for 4 h, then fixed and processed for electron microscopy. Cells were imaged at 12 000 × magnification. White arrows indicate the ER. (f) Activated rat HSCs were incubated in serum-free media for 0–24 h with indicated concentrations of CBD. Cell lysates were subjected to western blot analysis and probed for CHOP and calnexin. Representative of n=three independent experiments

Pro-apoptotic effect of CBD is specific to activated HSCs. (a) HSCs from control or ethanol-fed rats, (b) primary mouse in vivo-activated HSCs, (c) LX-2 cells (human HSC line derived from cirrhotic patient), and (d) primary rat hepatocytes were incubated in serum-free media with 5 μM CBD for 0–8 h. Cell lysates were subjected to western blot analysis and probed for PARP, calnexin, and CHOP to detect cell death and ER stress. (e) Acid phosphatase activity was measured in order to determine cell viability following treatment with indicated concentrations of CBD for 8 h in HSCs from control rats, HSCs from ethanol-fed rats, and primary rat hepatocytes. Inset, western blot analysis of α-SMA expression in HSCs from either control or ethanol-fed rats. Western blots and cell survival assays are representative of n=three independent experiments

CBD induces ER stress response. (a) Schematic representation of the signaling pathways characterizing the ER stress response. In (b) through (e), activated rat HSCs were incubated in serum-free media with 5 μM CBD, unless indicated otherwise, for the indicated time periods and processed for either (b and c) western blot, (d) RT-PCR, or (e) qRT-PCR analysis of ER stress mediators. (b) Whole cell lysates were subjected to western blot analysis and probed for phospho-PERK, phospho-eIF2α, phospho-IRE1, and P58^IPK to detect the PERK- and IRE1-mediated arms of the ER stress response. (c) Cell lysates and nuclear extracts were subjected to western blot analysis and probed for ATF4, cleaved ATF6α, and XBP1s to detect the presence of ER stress transcription factors in the nucleus. (d) Total RNA was extracted from cells and reverse transcribed to obtain cDNA used for PCR with primers specific for XBP1. Spliced XBP1 was detected by agarose gel electrophoresis. (e) Real-time RT-PCR was carried out with gene-specific primers for ATF4, ATF6, CHOP, and XBP1 to detect relative mRNA levels at the indicated time points. Results are normalized to GAPDH, hypoxanthine-guanine phosphoribosyltransferase, and RNA polymerase II, and expressed as fold change over control±S.E.M. (n=three independent experiments in triplicate). ^*P<0.05; ^**P<0.01; ^***P<0.001 versus control (Student's t-test)

CBD-induced cell death is mediated by the IRE1/ASK1/JNK pathway. Expression levels of phospho-ASK1, phospho-JNK, and JNK were determined by western blot analysis in (a) LX-2 cells, (b) HSCs from ethanol-fed rats, and (c) primary mouse in vivo-activated HSCs, following incubation in serum-free media with 5 μM CBD for 0–8 h. (d) Expression of DN IRE1α in JS1 cells (mouse activated HSCs) was assessed by RT-PCR using primers specific for the mouse and human IRE1α. Wild-type JS1 and LX-2 cells were used as controls. Gel-purified PCR products were sequenced to confirm presence of the mutated IRE1α (K599A DN IRE1α). (e) JS1 cells expressing DN IRE1α were treated with 5 μM CBD in serum-free media for 0–4 h. Expression of PARP and phospho-JNK was determined by western blot analysis and is presented as fold over vehicle±S.E.M. for either control or DN IRE1α-expressing JS-1 cells, n=three independent experiments. (f) JS1 cells expressing DN IRE1α were treated with CBD as in (e), and then harvested for total RNA isolation and RT-PCR with XBP1-specific primers. Spliced XBP1 was detected by agarose gel electrophoresis. (g) LX-2 cells were treated with CBD as in (a) in the presence or absence of 10 μM JNK inhibitor for 4 h. Western blot analysis was used to determine PARP and phospho-c-Jun expression. Cleaved PARP is expressed as fold over control±S.E.M. for n=four independent experiments. (h) LX-2 cells were treated with CBD in the presence or absence of JNK inhibitor as in (g) for 8 h. Cell viability was determined using an acid phosphatase assay and is expressed as mean±S.E.M. of triplicate experiments. ^***P<0.001; ^**P<0.01; ^*P<0.05

Inhibition of ER stress response protects against CBD-induced cell death. (a) JS1 cells were transfected with siRNA against ATF4. After 16 h, cells were incubated with 5 μM CBD in serum-free media for 8 h. Western blot analysis was used to determine PARP, ATF4, and CHOP expression, presented as fold over control±S.E.M. for n=four independent experiments. (b) LX-2 cells were treated with 5 μM CBD in serum-free media for 8 h in the presence or absence of 10 μM imatinib to inhibit ER stress. Expression of PARP, CHOP, and phospho-JNK was determined by western blot analysis and is presented as fold over vehicle±S.E.M. for n=four independent experiments. (c) Acid phosphatase activity was used to measure LX-2 cell viability following treatment with CBD and imatinib as in (b). Cell viability is expressed as mean percent of control acid phosphatase activity±S.E.M. of triplicate experiments. ^***P<0.001; ^**P<0.05

Basal ER stress determines sensitivity to CBD. (a) LX-2 cells were incubated in serum-free media with 0–4 μM CBD for 6 h in the presence or absence of tunicamycin (1 μg/mL) to induce ER stress. Cell death was detected by western blot analysis of cleaved PARP, expressed as fold over vehicle±S.E.M. for n=three independent experiments. (b) Total RNA was extracted from whole cell lysates of either ethanol-fed or control rat HSCs and reverse transcribed to cDNA. Real-time PCR was performed with gene-specific primers for ATF4, ATF6, and CHOP to determine the relative basal mRNA levels, normalized to GAPDH and expressed as fold over control. ^***P<0.001 (Student's t-test). (c) ER stress was induced in HSCs from control rats by incubation with the indicated concentrations of tunicamycin in the presence or absence of 5 μM CBD for 6 h in serum-free media. Cell death and ER stress were detected by western blot analysis of PARP and CHOP expression, respectively. Cleaved PARP is expressed as fold over control±S.E.M. for n=three independent experiments. ^*P<0.05; ^**P<0.01