Schematic representation of the expression plasmids for wild type LC3 and mutant LC3ΔG fused to fluorescent proteins at the N-terminus.

<p>(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.</p

The formation of the Atg7-LC3 E1-substrate and Atg3-LC3 E2-substrate intermediates of fluorescent protein-tagged LC3.

<p><b>(A)</b> The formation of the E1-substrate intermediate of Atg7 with fluorescent protein-tagged LC3s. The PK-hLC3<b> (wt) </b>was expressed together with FLAG-tagged human <b>Atg7</b>^C572S in the Huh7.5.1 cells. After preparation of the cell lysate, total proteins were separated on SDS-PAGE. FLAG-hAtg7^C572S and PK-hLC3 were recognized by immunoblotting with appropriate antibodies. As a negative control, mutant PK-hLC3ΔG (Δ<b>G</b>) was expressed. As a loading control,<b> GAPDH</b> was employed. <b>Atg7-PK-hLC3</b> indicated the Atg7-LC3 (E1-substrate) intermediate with <b>Atg7</b>^C572S. <b>(B)</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 Atg3^C264S in the Huh7.5.1 cells. FLAG-hAtg7, Myc-Atg3^C264S and PK-hLC3 were recognized by immunoblotting with appropriate antibodies. As a negative control, mutant PK-hLC3ΔG was expressed. As a loading control, <b>GAPDH</b> was employed. <b>Atg3-PK-hLC3</b> indicated their Atg7-LC3 intermediates with <b>Atg3</b>^C264S.</p

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.</p

Codon usage in original mNeonGreen.

<p>Codon usage in original mNeonGreen.</p

Comparison of the amino acid sequence of mNeonGreen with that of EGFP using Clustal-W alignment.

<p>Comparison of the amino acid sequence of mNeonGreen with that of EGFP using Clustal-W alignment.</p

<i>Homo sapiens</i> codon usage.

<p><i>Homo sapiens</i> codon usage.</p

Optimization of mNeonGreen for <i>Homo sapiens</i> increases its fluorescent intensity in mammalian cells

<div><p>Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in <i>Homo sapiens</i>. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.</p></div

DNA sequence and plasmid maps of mNeonGreen.

<p><b>(A) Comparison of the DNA sequence of humanized mNeonGreen with that of the original one.</b> A pairwise alignment of two DNA sequences of humanized (GenBank Accession No. LC279210) and original mNeonGreen (GenBank Accession No. KC295282) was performed using a CLUSTAL W program (<a href="http://clustalw.ddbj.nig.ac.jp/" target="_blank">http://clustalw.ddbj.nig.ac.jp/</a>). <b>(B) Plasmid maps for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG.</b> Humanized mNeonGreen cDNA with a triple Gly-Gly-Gly-Ser linker was inserted into the <i>Nhe</i>I-<i>Bgl</i>II site of pAcGFP-C1 after removing AcGFP cDNA to create pmNeonGreenHO-G. Humanized mNeonGreen-3xFLAG cDNA was inserted into the <i>Nhe</i>I-<i>Bgl</i>II site of pAcGFP-C1 after removing AcGFP cDNA to create pmNeonGreenHO-3xFLAG. MCS, multicloning sites; CMV, cytomegalovirus.</p

Original and humanized mNeonGreen fluorescence in HEK293.

<p>Plasmids designed for expression of original or humanized mNeonGreen were transfected into HEK293 cells. As a control of transfection efficiency, mCherry2-C1 was employed. Fluorescent intensity of mNeonGreen and mCherry2 was obtained 48 h after transfection using a 2300 EnSpire multimode reader. The data were analyzed with a Welch’s <i>t</i>-test; p < 0.01 in green fluorescent intensity of <b>mNeonGreen</b>; p > 0.7 in red fluorescent intensity of <b>mCherry2</b>. Graphs show the relative fluorescent intensity of both fluorescent proteins (%).</p

Particle Size of Latex Beads Dictates IL-1β Production Mechanism

<div><p>Macrophages (Mϕ) are well documented to produce IL-1β through various signaling pathways in response to small particles such as silica, asbestos and urea crystals, in the presence of lipopolysaccharide (LPS). However, it has not been clear to what extent particle size affects the response. To investigate this point, we stimulated bone marrow-derived macrophages (BMDM) with size-defined latex beads (LxB). Although both nano-sized (20 nm) and micro-sized (1,000 nm) LxB induced IL-1β production, only the nano-sized particles formed large intracellular vacuoles. In contrast, 100 nm LxB did not induce either of the responses. The same cellular responses were also observed in primary microglia cells. Although K⁺ efflux and NLRP3 activation in BMDM were crucial in response to both 20 and 1,000 nm LxB, only IL-1β production by 20 nm LxB was sensitive to cathepsin B and P2X₇, a receptor for ATP. The response by 1,000 nm LxB relied on a robust production of reactive oxygen species (ROS), since IL-1β production was remarkably reduced by ROS inhibitors such as diphenylene iodonium (DPI) and N-acetylcysteine (NAC). In contrast, IL-1β production by 20 nm LxB was augmented by NAC and in BMDM deficient in thioredoxin-binding protein-2 (TBP-2), a negative regulator of the ROS scavenger thioredoxin. These results suggest that the cells responded differently in their secretion of IL-1β depending on particle size, and that there is a range within which neither pathway works.</p> </div