Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls

Tanida, Isei; Yamaji, Taiki; Ueno, Takashi; Ishiura, Shoichi; Kominami, Eiki; Hanada, Kentaro

doi:10.4161/auto.5233

Cited by 80 publications

(81 citation statements)

References 17 publications

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“…Similar results were obtained in HCT116shp38 cells when compared with control cells (Figure 5c). As a negative control for this approach, we used the mutant GFP-LC3DGly120 (Tanida et al, 2008;Supplementary Information 5), showing a diffuse pattern lacking the characteristic aggregates observed in the wild type. To corroborate our observation with GFP-LC3 over-expression, immunocytochemistry was performed to detect endogenous Atg-12, another key component of the Atg family.…”

Section: P38mapk Is Activated In Response To 5-fu/5-dfur and Mediatesmentioning

confidence: 99%

RETRACTED ARTICLE: P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance

et al. 2011

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5-Fluorouracil (5-FU), together with other drugs such as oxaliplatin, is one of the most important pharmacological agents in the treatment of colorectal cancer. Although mitogen-activated protein kinases (MAPKs) have been extensively connected with resistance to platinum compounds, no role has been established in 5-FU resistance. Here we demonstrate that p38MAPK activation is a key determinant in the cellular response to 5-FU. Thus, inhibition of p38MAPKa by SB203580 compound or by short-hairpin RNA interference-specific knockdown correlates with a decrease in the 5-FU-associated apoptosis and chemical resistance in both HaCaT and HCT116 cells. Activation of p38MAPK by 5-FU was dependent on canonical MAP2K, MAPK kinase (MKK)-3 and MKK6. In addition, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) showed a redundancy of function for the final activation of p38MAPK. Resistance associated with p38MAPK inhibition correlates with an autophagic response that was mediated by a decrease in p53-driven apoptosis, without effect onto p53-dependent autophagy. Moreover, the results with colorectal cancer-derived cell lines with different p53 status and patterns of resistance to 5-FU suggest that de novo and acquired resistance was controlled by similar mechanisms. In summary, our data demonstrate a critical role for the p38MAPK signaling pathway in the cellular response to 5-FU by controlling the balance between apoptosis and autophagy.

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Section: P38mapk Is Activated In Response To 5-fu/5-dfur and Mediatesmentioning

confidence: 99%

RETRACTED ARTICLE: P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance

et al. 2011

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“…50 In our study, nearly all of the LC3 G120A puncta examined exhibited rapid exchange. However, similar to the results obtained with atg5 −/− MEFs, in puromycin-treated HeLa cells expressing mCherry-LC3 G120A alone, puncta showing no fluorescence recovery after pcDNA3.1(+)-mRFP/mCerulean/mCitrine.…”

Section: Chemicalsmentioning

confidence: 90%

LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells

Wang¹,

Chen²,

Yang³

2013

Autophagy

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LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells

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“…The plasmid constructs used in our experiments were obtained from Addgene (Cambridge, MA). pcDNA-EGFP, native EGFP; EGFP-LC3 (pEX-EGFP-hLC3B), EGFP fused to the human LC3 protein; and EGFP-ΔLC3 (pEX-EGFP-mhLC3B G120A, a mutant form of LC3 that does not localize to autophagosomes when cells are starved) [24,25]. Autophagy was induced in transfected MCF-7 cells by 3 hour amino acid starvation using Hank's Balanced Salt Solution (HBSS) (Life Technologies Corp., Carlsbad, CA).…”

Section: Cell Culture Transfection and Autophagy Inductionmentioning

confidence: 99%

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Gohar

Cao

Jenkins

et al. 2013

Biomed. Opt. Express

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Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.

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Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls

Cited by 80 publications

References 17 publications

RETRACTED ARTICLE: P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance

RETRACTED ARTICLE: P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance

LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

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