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Fig. 5A MCLR activates ER stress and autophagy in TM4 cells. (A) Representative immunoblots showed GRP78 proteins after different treatment. GAPDH was used as the control. Relative protein levels of GRP78 were shown from 4 independent experiments. (B) The mRNA expression of grp78 were evaluated by qRT-PCR (n = 4). (C) The mRNA expression of autophagy related genes atg5, atg12 and lc3b were evaluated by qRT-PCR (n = 4). (D) The expression levels of the autophagy markers ATG5, ATG12, LC3BⅠ and LC3BⅡ were analyzed by Western blot (n = 3). (E) Cell imaging was performed to analyze the colocalization of LysoTracker-stained acidified vesicles and LC3b-positive autophagosomes in control and MCLR-treated cells. Scale bar: 50 µm. *P < 0.05 significantly different from control group. ^P < 0.05 significantly different from 7.5 μg/mL MCLR. ▲P < 0.05 significantly different from 30 μg/mL MCLR.