Figure 1.
The pH sensitivity of the green fluorescence of EGFP, mWasabi, and super-ecliptic pHluorin.
The green fluorescent proteins, EGFP (A–D), mWasabi (E–I), and pHluorin (J–P), were expressed in the Huh7.5.1 cells. After fixation and permeabilization of the cells, the cells were buffered at pH 5.5 (D, H, I, M, N, P), 6.0 (C, G, L, O), 6.5 (B, F, K), and 7.2 (A, E, J). The green fluorescence of each of the proteins in the cells were obtained with Biozero BZ-8000 using the filter set for GFP. The Images in A–H and J–N were obtained under the same conditions of the fluorescent microscope. The image in I was exposed three times longer than that in H. The images in O and P were exposed ten times longer than those in the respective L and M. In H, cells were incubated at pH 5.5, and the pH was changed to 7.2. Bar indicates 10 µm.
Figure 2.
The pH sensitivity of the pHluorin-mKate2 tandem fluorescent protein.
The pHluorin-mKate2 tandem fluorescent protein was expressed in the Huh7.5.1 cells. After fixation and permeabilization, cells were buffered at pH 5.5 (D, I), 6.0 (C, H), 6.5 (B, G), and 7.2 (A, F). In E and J, cells were incubated at pH 5.5, and the pH was changed to pH 7.2. The green and far-red fluorescence in the cells were obtained with Biozero BZ-8000 using the filter sets for GFP (A–E) and Texas Red (F–J). Bar indicates 10 µm.
Figure 3.
Schematic representation of the expression plasmids for wild type LC3 and mutant LC3ΔG fused to fluorescent proteins at the N-terminus.
(A) The expression plasmids for wild type PK-hLC3, and mutant PK-hLC3ΔG under the control of chicken ß-actin (CAG) promoter. The name designated to each plasmid is shown in the left panel. (B) The plasmids for lentiviral packaging and transient expression for wild type PK-hLC3 and mutant PK-hLC3ΔG under the control of human polyubiquitin C (hUbC) promoter.
Figure 4.
The formation of the Atg7-LC3 E1-substrate and Atg3-LC3 E2-substrate intermediates of fluorescent protein-tagged LC3.
(A) The formation of the E1-substrate intermediate of Atg7 with fluorescent protein-tagged LC3s. The PK-hLC3 (wt) was expressed together with FLAG-tagged human Atg7C572S in the Huh7.5.1 cells. After preparation of the cell lysate, total proteins were separated on SDS-PAGE. FLAG-hAtg7C572S and PK-hLC3 were recognized by immunoblotting with appropriate antibodies. As a negative control, mutant PK-hLC3ΔG (ΔG) was expressed. As a loading control, GAPDH was employed. Atg7-PK-hLC3 indicated the Atg7-LC3 (E1-substrate) intermediate with Atg7C572S. (B) The formation of the E2-substrate intermediate of Atg3 with fluorescent protein-tagged LC3s. The PK-hLC3 was expressed together with wild type FLAG-tagged Atg7 and mutant Myc-Tagged Atg3C264S in the Huh7.5.1 cells. FLAG-hAtg7, Myc-Atg3C264S and PK-hLC3 were recognized by immunoblotting with appropriate antibodies. As a negative control, mutant PK-hLC3ΔG was expressed. As a loading control, GAPDH was employed. Atg3-PK-hLC3 indicated their Atg7-LC3 intermediates with Atg3C264S.
Figure 5.
Formation of the puncta of PK-hLC3 during autophagy.
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 (E64d & pepstatin A) (Da–Dd) or absence (DMSO) (Ca–Cd) of 10 µg/ml E64d and 10 µg/ml pepstatin A (Starvation, 4 h). As nutrient-rich conditions, cells were incubated in the cultured medium (Nutrient-rich, 4 h) (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 rapamycin (Fa–Fd) or 100 nM torin1 (Ga–Gd). To inhibit the fusion of autophagosome with lysosome, 20 mM ammonium chloride (NH4Cl) (Ha–Hd) and 20 µg/ml chloroquine (CQ) (Ia–Id) were treated to the cells incubated in the Krebs-Ringer buffer for 4 h. The PK-hLC3ΔG (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 (mKate2) and green (pHluorin) fluorescence in the cells were monitored using a Olympus FluoView FV1000 confocal laser scanning microscope. “Merge” indicates the merging of the green (pHluorin) and far-red images (mKate2), and “DIC+Merge” indicates the overlaying the merged images on the DIC (differential interference contrast) images in the same field. Pearson's correlation coefficient (PCC) 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.