Arrows: Avi, arrow heads: Avd. (B) The number of autophagic vacuoles (AVs, which includes equally Avi and Avd), Avi and Avd for every 100 m2 cytosol was quantified. Data are offered as 
signifies SE (much more than 15 cell sections). p<0.05, 7721 vs HepG2 (Student t test). (C) 7721 and HepG2 cells were treated with metformin for 24 hours. Total cell lysates were subjected to immunoblot analysis. Representative blots are shown from three independent experiments.To determine whether the feedback activation of Akt and lack of autophagic flux that we observed in 7721 cells after metformin treatment would also occur in other HCC cells, 97L and LM3, two other HCC cells were treated with metformin. Similar to 7721 cells, we found that metformin treatment decreased the levels of phosphorylated S6 but increased the phosphorylated levels of Akt (S473) in 97L and LM3 cells (Fig 6A). Metformin treatment also failed to induce autophagic flux in 97L and LM3 cells (Fig 6B), which was also consistent with the results that we observed in 7721 cells. These results suggest that 97L and LM3 cells may behave similar to 7721 cells but are different from HepG2 cells. Indeed, results from MTT assay revealed that 97L and LM3 cells were also resistant to metformin-induced growth inhibition compared to HepG2 cells. When all these four different HCC cells were treated with metformin in the presence of CQ, CQ treatment further reduced the cell growth compared to metformin alone (Fig 6C). These data suggest that inhibition of autophagy may further sensitize 7721, 97L, LM3 and HepG2 cells to metformin-induced cell growth inhibition even though metformin did not induce autophagic flux in these cells, which was likely due to high basal autophagy levels in these cells. Taken together, our results suggest that the basal autophagy level and feedback activation of Akt in different HCC cells may account for their different sensitivities in response to metformin.Fig 5. Metformin does not induce autophagic flux in 7721 and HepG2 cells during prolonged culture conditions. (A) 7721 and HepG2 cells were treated with metformin (10 mM) in the presence or absence of CQ (20 M) for 24, 48 and 72 hours. CQ was added at the last four hours before the cells were harvested. Total cell lysates were subjected to immunoblot analysis. Representative blots are shown from three independent experiments. (B) 7721 and HepG2 cells were cultured in full DMEM medium for 0 (after cells were trypsinized and attached overnight) or 24 hours (culture for 24 hours after overnight attachment) or cultured in EBSS buffer for 2 hours. Total cell lysates were subjected to immunoblot analysis. Representative blots are shown from three independent experiments.Fig 6. Pharmacological Suppression of autophagy sensitizes 7721, 97L, LM3 and HepG2 cells to metformin-induced inhibition of cell growth. (A) 97L and LM3 cells were treated with metformin (10 mM) for 24 hours. Total cell lysates were 18421270subjected to immunoblot analysis and representative blots from 3 independent experiments are shown. (B) 97L and LM3 cells were treated with metformin (10 mM) in the presence or absence of CQ (20 M) for 24 hours.