Formation of the puncta of PK-hLC3 during autophagy.

<p>The PK-hLC3 was expressed in Huh7.5.1 cells. The cells were incubated in the Krebs-Ringer buffer for 4 h as starvation conditions in the presence (<b>E64d & pepstatin A</b>) (Da–Dd) or absence (<b>DMSO</b>) (Ca–Cd) of 10 µg/ml E64d and 10 µg/ml pepstatin A (<b>Starvation, 4 h</b>). As nutrient-rich conditions, cells were incubated in the cultured medium (<b>Nutrient-rich, 4 h</b>) (Aa–Ad, Ba–Bd). For induction autophagy by the inhibition of mTOR-signaling pathway, cells were incubated in the cultured medium for 6 h in the presence of 100 nM <b>rapamycin</b> (Fa–Fd) or 100 nM <b>torin1</b> (Ga–Gd). To inhibit the fusion of autophagosome with lysosome, 20 mM ammonium chloride (<b>NH₄Cl</b>) (Ha–Hd) and 20 µg/ml chloroquine (<b>CQ</b>) (Ia–Id) were treated to the cells incubated in the Krebs-Ringer buffer for 4 h. The <b>PK-hLC3ΔG</b> (Ea–Ed and Ja–Jd) was expressed in the cells instead of the PK-hLC3 under the same conditions (Da–Dd and Ia–Id, respectively). The far-red (<b>mKate2)</b> and green (<b>pHluorin)</b> fluorescence in the cells were monitored using a Olympus FluoView FV1000 confocal laser scanning microscope. “<b>Merge</b>” indicates the merging of the green (<b>pHluorin)</b> and far-red images (<b>mKate2</b>), and “<b>DIC+Merge</b>” indicates the overlaying the merged images on the DIC (differential interference contrast) images in the same field. Pearson's correlation coefficient (<b>PCC</b>) analysis with Costes' method was used as a measure of colocalization of mKate2 signals with pHluorin signals. The mean PCC value ± S.E. of at least 20 cells is shown on the bottom.