Autophagic flux determination in vivo and ex vivo

Esteban‐Martínez, Lorena; Boya, Patricia

doi:10.1016/j.ymeth.2015.01.008

Cited by 73 publications

(61 citation statements)

References 30 publications

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“…Accordingly, knockdown of Becl1 with two distinct shRNAs (Boya et al., 2005) reduced the levels of LC3-II in Pml −/− MEFs (Figure S1F). Conversely, in vivo blockade of the last step of autophagy (which depends on lysosomal proteases) by means of leupeptin (Esteban-Martínez and Boya, 2015) failed to reduce the difference in LC3-II formation observed between WT and Pml −/− mouse livers (Figure S1G). Similarly, bafilomycin A1 or NH 4 Cl, which both abolish the acidification of lysosomes, caused an increased abundance of LC3-II in both WT and Pml −/− MEFs, yet it did not abolish the difference in LC3-II formation between MEFs with the two genotypes, suggesting that the absence of Pml truly induces an increase in autophagic flux (Figures S1H and S1I).…”

Section: Resultsmentioning

confidence: 99%

PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development

et al. 2016

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SummaryThe precise molecular mechanisms that coordinate apoptosis and autophagy in cancer remain to be determined. Here, we provide evidence that the tumor suppressor promyelocytic leukemia protein (PML) controls autophagosome formation at mitochondria-associated membranes (MAMs) and, thus, autophagy induction. Our in vitro and in vivo results demonstrate how PML functions as a repressor of autophagy. PML loss promotes tumor development, providing a growth advantage to tumor cells that use autophagy as a cell survival strategy during stress conditions. These findings demonstrate that autophagy inhibition could be paired with a chemotherapeutic agent to develop anticancer strategies for tumors that present PML downregulation.

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Section: Resultsmentioning

confidence: 99%

PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development

et al. 2016

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“…We next sought to determine autophagic flux 16 by inhibiting the final steps of autophagy using intraperitoneal leupeptin injections. 37,38 In conditions in which mice received leupeptin during the last 2 h of the experiment, starvation did cause LC3B lipidation, both in leukocytes (Fig. 5A, D, G) and hepatocytes ( Fig.…”

Section: Assessment Of Autophagic Markers After Starvationmentioning

confidence: 96%

Metabolic effects of fasting on human and mouse blood in vivo

et al. 2017

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Starvation is a strong physiological stimulus of macroautophagy/autophagy. In this study, we addressed the question as to whether it would be possible to measure autophagy in blood cells after nutrient deprivation. Fasting of mice for 48 h (which causes »20% weight loss) or starvation of human volunteers for up to 4 d (which causes <2% weight loss) provokes major changes in the plasma metabolome, yet induces only relatively minor alterations in the intracellular metabolome of circulating leukocytes. White blood cells from mice and human volunteers responded to fasting with a marked reduction in protein lysine acetylation, affecting both nuclear and cytoplasmic compartments. In circulating leukocytes from mice that underwent 48-h fasting, an increase in LC3B lipidation (as assessed by immunoblotting and immunofluorescence) only became detectable if the protease inhibitor leupeptin was injected 2 h before drawing blood. Consistently, measurement of an enhanced autophagic flux was only possible if white blood cells from starved human volunteers were cultured in the presence or absence of leupeptin. Whereas all murine leukocyte subpopulations significantly increased the number of LC3B C puncta per cell in response to nutrient deprivation, only neutrophils from starved volunteers showed signs of activated autophagy (as determined by a combination of multi-color immunofluorescence, cytofluorometry and image analysis). Altogether, these results suggest that white blood cells are suitable for monitoring autophagic flux. In addition, we propose that the evaluation of protein acetylation in circulating leukocytes can be adopted as a biochemical marker of organismal energetic status.

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“…However, autophagy is a dynamic process that includes initiation, formation, maturation, and degradation of autophagosomes-a dynamic flow defined as autophagic flux [23]. Increased expression of LC3-II or visualization of autophagic vacuoles may result from either an enhancement of autophagosomal formation or inhibition of autophagosomal degradation [24].…”

Section: Discussionmentioning

confidence: 99%

Intermittent-Hypoxia-Induced Autophagy Activation Through the ER-Stress-Related PERK/eIF2α/ATF4 Pathway is a Protective Response to Pancreatic β-Cell Apoptosis

Song

Tan

Miao

et al. 2018

Cell Physiol Biochem

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Background/Aims: Intermittent hypoxia (IH) causes apoptosis in pancreatic β-cells, but the potential mechanisms remain unclear. Endoplasmic reticulum (ER) stress, autophagy, and apoptosis are interlocked in an extensive crosstalk. Thus, this study aimed to investigate the contributions of ER stress and autophagy to IH-induced pancreatic β-cell apoptosis. Methods: We established animal and cell models of IH, and then inhibited autophagy and ER stress by pharmacology and small interfering RNA (siRNA) in INS-1 cells and rats. The levels of biomarkers for autophagy, ER stress, and apoptosis were evaluated by immunoblotting and immunofluorescence. The number of autophagic vacuoles was observed by transmission electron microscopy. Results: IH induced autophagy activation both in vivo and in vitro, as evidenced by increased autophagic vacuole formation and LC3 turnover, and decreased SQSTM1 level. The levels of ER-stress-related proteins, including GRP78, CHOP, caspase 12, phosphorylated (p)-protein kinase RNA-like ER kinase (PERK), p-eIF2α, and activating transcription factor 4 (ATF4) were increased under IH conditions. Inhibition of ER stress with tauroursodeoxycholic acid or 4-phenylbutyrate partially blocked IH-induced autophagy in INS-1 cells. Furthermore, inhibition of PERK with GSK2606414 or siRNA blocked the ERstress-related PERK/eIF2α/ATF4 signaling pathway and inhibited autophagy induced by IH, which indicates that IH-induced autophagy activation is dependent on this signaling pathway. Promoting autophagy with rapamycin alleviated IH-induced apoptosis, whereas inhibition of autophagy with chloroquine or autophagy-related gene (Atg5 and Atg7) siRNA aggravated pancreatic β-cell apoptosis caused by IH. Conclusion: IH induces autophagy activation through the ER-stress-related PERK/eIF2α/ATF4 signaling pathway, which is a protective response to pancreatic β-cell apoptosis caused by IH.

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Autophagic flux determination in vivo and ex vivo

Cited by 73 publications

References 30 publications

PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development

PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development

Metabolic effects of fasting on human and mouse blood in vivo

Intermittent-Hypoxia-Induced Autophagy Activation Through the ER-Stress-Related PERK/eIF2α/ATF4 Pathway is a Protective Response to Pancreatic β-Cell Apoptosis

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