Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells

Ylä-Anttila, Päivi; Vihinen, Helena; Jokitalo, Eija; Eskelinen, Eeva‐Liisa

doi:10.1016/s0076-6879(08)03610-0

Cited by 239 publications

(205 citation statements)

References 18 publications

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“…21,26,44 The results (Figures 5-7) are consistent with our findings in diabetic kidneys. TXNIP expression is ubiquitous and is induced by a variety of cellular stresses, including high intracellular glucose.…”

Section: Discussionsupporting

confidence: 91%

“…[38][39][40][41][42] The lipidation of LC3 by the phospholipid phosphatidylethanolamine to forms LC3-II associates with forming autophagosome membranes and the conversion of LC3 to LC3-II is well accepted as a marker of autophagy induction, and p62 protein degraded by autophagy inversely reflects the autophagic flux, therefore the protein levels of LC3-II and p62 were examined to monitor autophagy and autophagic flux in this study. 21,22,26,[43][44][45][46] We found significantly increased expression of LC3 and p62 in diabetic kidneys compared with non-diabetic controls (Figures 1 and 2). The quantitative and meaningful assay for examining autophagy is the measurement of autophagosome formation.…”

Section: Discussionmentioning

confidence: 70%

“…The samples were prepared for transmission electron microscopic analysis as reported previously. 25,26 Briefly, cells were washed with pre-warmed PBS twice, and then fixed in 2% glutaraldehyde in PBS for 60 min at room temperature. Fixed cells were washed with PBS three times and then postfixed with 1% in Figure 3 The mRNA and protein expression of TXNIP in diabetic mice.…”

Section: The Measurement Of Autophagy Using Transmissionmentioning

confidence: 99%

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Thioredoxin-interacting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux

Huang

Lin

Cheng

et al. 2014

Laboratory Investigation

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Thioredoxin-interacting protein (TXNIP) expression is ubiquitous and is induced by a variety of cellular stresses, including high intracellular glucose. TXNIP is associated with activation of oxidative stress and tubulointerstitial fibrosis in diabetic nephropathy. Autophagy is a major pathway that delivers damaged proteins and organelles to lysosomes to maintain cellular homeostasis. This study aimed to investigate the dysregulation of autophagy and the regulation of TXNIP on autophagy in renal proximal tubular cells (PTCs) under diabetic conditions. The formation of autophagosomes was measured using transmission electron microscopy, and LC3-II, and the effectiveness of autophagic clearance was determined by p62 expression in diabetic kidney and in human PTCs exposed to high glucose (HG). The results collectively demonstrated increased expression of TXNIP, LC3/LC3-II and p62 in renal tubular cells of mice with diabetic nephropathy and in cultured human PTCs exposed to HG (30 mM/l) for 48 h compared with control. The formation of autophagic vacuoles was increased in HG-induced cells. Furthermore, silencing of TXNIP by siRNA transfection reduced autophagic vacuoles and the expression of LC3-II and p62 in human PTCs exposed to HG compared with control and partially reversed the accumulation of LC3-II and p62 induced by bafilomycin A 1 (50 nM/l), a pharmacological inhibitor of autophagy which blocks the fusion of autophagosomes with lysosomes and impairs the degradation of LC3-II and p62. Collectively, these results suggest that hyperglycemia leads to dysfunction of autophagy in renal tubular cells and decreases autophagic clearance. HG-induced overexpression of TXNIP may contribute to the dysfunction of tubular autophagy in diabetes.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 70%

Section: The Measurement Of Autophagy Using Transmissionmentioning

confidence: 99%

See 1 more Smart Citation

Thioredoxin-interacting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux

Huang

Lin

Cheng

et al. 2014

Laboratory Investigation

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“…Quantitative counting of autophagic compartments was performed using a transmission electron microscope (JEOL 1200 EX II or JEOL 1400 EX, Electron microscopy unit, Institute of Biotechnology, University of Helsinki) as described previously. 40 For immuno-electron microscopy NRK or HeLa cells were grown on 3.5-cm plates to semiconfluency. MYC-RAB24 plasmid was transfected with Lipofectamine 2000 or TransIT-LTI (Mirus, MIR 2300) reagent according to the manufacturer's instructions one day after seeding the cells.…”

Section: Western Blottingmentioning

confidence: 99%

RAB24 facilitates clearance of autophagic compartments during basal conditions

et al. 2015

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RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.

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“…The quantification of autophagosomes and autophagolysosomes were performed as previously described. 20 …”

Section: Transmission Electron Microscopy Analysismentioning

confidence: 99%

HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells

et al. 2010

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Autophagy, a tightly regulated lysosome-dependent catabolic pathway, is important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. However, the role of autophagy in leukemia still remains largely unknown. Here we show that high-mobility group box 1 (HMGB1), the best characterized damage-associated molecular pattern, was released from leukemia cell lines after chemotherapy-induced cytotoxicity and activated autophagy to protect against injury. Treatment with HMGB1-neutralizing antibodies increased the sensitivity of leukemia cells to chemotherapy; whereas, exogenous HMGB1 rendered these cells more resistant to drug-induced cytotoxicity. Moreover, exogenous HMGB1 increased autophagy as evaluated by increased expression of the autophagic marker microtubule-associated protein light chain 3-II, degradation of sequestosome 1 (p62) and autophagosome formation. Furthermore, knockdown or pharmacological inhibition of either phosphoinositide 3-kinase-III or extracellular signal-regulated kinase kinase mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase inhibited HMGB1-induced autophagy. Taken together, these results suggest that HMGB1 release after chemotherapy is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia.

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Chapter 10 Monitoring Autophagy by Electron Microscopy in Mammalian Cells

Cited by 239 publications

References 18 publications

Thioredoxin-interacting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux

Thioredoxin-interacting protein mediates dysfunction of tubular autophagy in diabetic kidneys through inhibiting autophagic flux

RAB24 facilitates clearance of autophagic compartments during basal conditions

HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells

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