Figure 3
THC induces autophagy via ER stress–evoked p8 and TRB3
upregulation.
(A and B) Effect of ISP-1 (1 μM) on
THC-induced eIF2α phosphorylation (A; 3 h;
n = 3) and LC3 immunostaining (B, left panels; 18 h;
percentage of cells with LC3 dots relative to the total cell number, mean
± SD; n = 3; scale bar: 20 μm) in U87MG
cells. sip8, p8-selective siRNA; siTRB3, TRB3-selective siRNA. (C)
Effect of THC on p8, ATF4, CHOP, and TRB3 mRNA levels of eIF2α WT and
eIF2α S51A MEFs as determined by real-time quantitative PCR (8 h;
n = 3). Numbers indicate the mean fold increase ±
SD relative to vehicle-treated eIF2α WT MEFs. (D) Top:
Analysis of p8 and TRB3 mRNA levels. Results from a representative RT-PCR
experiment are shown. The numbers indicate gene expression levels as determined by
real-time quantitative PCR (mean fold change ± SD relative to
siC-transfected cells; n = 5). Bottom: Effect of THC on LC3
immunostaining (green) of U87MG cells transfected with siC, sip8, or siTRB3 (18 h;
n = 4). The percentage of cells with LC3 dots relative to
cells cotransfected with a red fluorescent control siRNA is shown in each panel
(mean ± SD). Scale bar: 20 μm. (E) Effect of
THC on LC3 lipidation in U87MG cells transfected with siC, sip8, or siTRB3 (18 h;
n = 6). (F) Effect of THC on LC3 lipidation (top;
18 h; n = 5) and LC3 immunostaining (bottom; 18 h; percentage of
cells with LC3 dots relative to the total cell number, mean ± SD;
n = 4; scale bar: 40 μm) in
p8+/+ or
p8–/– MEFs. *P
< 0.05 and **P < 0.01 compared with
THC-treated U87MG (B), eIF2α WT (C), or
p8+/+ (F) cells and compared with
siC-transfected, THC-treated U87MG cells (D).
