Figure 2

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