EMBO open
Modulation of intracellular ROS levels by TIGAR
controls autophagy
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Karim Bensaad,Eric C Cheung and Karen H Vousden*
The Beatson Institute for Cancer Research,Garscube Estate,Bearsden,Glasgow,UK
The p53-inducible TIGAR protein functions as a fructose-2,6-bisphosphatase,promoting the pentose phosphate pathway and helping to lower intracellular reactive oxy-gen species (ROS).ROS functions in the regulation of many cellular responses,including autophagy—a re-sponse to stress conditions such as nutrient starvation and metabolic stress.In this study,we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress,and functions to inhibit autophagy.The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS,with no clear effects on the
mTOR pathway,and is p53independent.The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels.These results reveal a complex interplay in the regulation of ROS,autophagy and apoptosis in response to TIGAR expression,and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.
The EMBO Journal (2009)28,3015–3026.doi:10.1038/emboj.2009.242;Published online 27August 2009Subject Categories :differentiation &death Keywords :autophagy;p53;ROS;TIGAR
Introduction
Autophagy—a mechanism that results in lysosomal degrada-tion of cytoplasmic constituents—is a critical response to metabolic stress (Meijer and Codogno,2004;Mizushima,2007).Limited autophagy in response to nutrient starvation has been shown to provide a survival function,and specific removal of damaged mitochondria by autophagy can also help prevent the activation of apoptotic pathways (Zhang et al ,2008).However,in some systems,the induction of autophagy has been shown to contribute to,or enhance,the apoptotic response (Crighton et al ,2006).The contribution of autophagy to tumour progression is complex,although evi-dence from animal studies suggests that autophagy can have
an important tumour suppressive function (Qu et al ,2003;Yue et al ,2003;Marino et al ,2007).
Although autophagy in response to nutrient deprivation or metabolic stress is mediated through the regulation of the TSC-mTOR pathway (Reiling and Sabatini,2006),recent studies have also highlighted the important contribution of mitochondrially generated reactive oxygen species (ROS)to this response (Scherz-Shouval et al ,2007;Chen and Gibson,2008;Chen et al ,2008).ROS are produced as a normal by-product of cellular metabolism and function as signalling molecules that are involved in numerous pathways regulating cell proliferation,senescence,apoptosis,necrosis and auto-phagy (Martindale and Holbrook,2002;Balaban et al ,2005).ROS have been shown to induce autophagy through several distinct mechanisms involving the Atg4family of protein proteases,the mitochondrial electron transport chain and catalase (Yu et al ,2006;Chen et al ,2007;Scherz-Shouval et al ,2007).
p53is a tumour suppressor protein that has a critical function in inhibiting cancer development,and mutation in the p53pathway is an extremely common event in most human cancers.p53induces many responses—including cell-cycle arrest,senescence and apoptotic cell death—each of which may contribute to tumour suppression (Murray-Zmijewski et al ,2008).However,in addition to the ability to block cell proliferation,several activities of p53that contribute to cell survival have also been described.These include functions of p53as an anti-oxidant (Sablina et al ,2005)and in the regulation o
f metabolism (Matoba et al ,2006;Bensaad and Vousden,2007).Although the induction of survival signals seems to be inconsistent with the well-understood apoptotic function of p53,it has been suggested that this response may contribute to repair and recovery under conditions of mild stress,whereas more severe damage elicits the apoptotic response (Vousden and Lane,2007).It is not clear how this switch in p53responses is regulated,but this is likely to be an important factor in determining the success of p53-based therapies (Vousden and Prives,2009).Intriguingly,the p53tumour suppressor gene has recently also been shown to function to both induce and inhibit autophagy (Crighton et al ,2006;T asdemir et al ,2008),although the contribution of this response to tumour suppres-sion is not fully resolved.
A key mechanism of function of p53is as a transcription factor,and the DRAM proteins have been identified as important mediators of the induction of autophagy by p53(Crighton et al ,2006).However,other p53-target genes that contribute to the regulation of metabolic pathways and oxidative stress may also have a function in the regulation of autophagy.Several p53-inducible genes encode proteins that can function as anti-oxidants,and the constitutive p53-dependent expression of these anti-oxidant proteins under
Received:28May 2009;accepted:22July 2009;published online:27August 2009
*Corresponding author.The Beatson Institute for Cancer Research,Garscube Estate,Switchback Road,Bearsden,Glasgow G611BD,UK.T el.:þ441413302424;Fax:þ441419430372;E-mail:k.vousden@beatson.gla.ac.uk
The EMBO Journal (2009)28,3015–3026|&
2009European Molecular Biology Organization |Some Rights Reserved 0261-4189/09
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normal growth conditions in vivo helps to protect cells from the accumulation of ROS-associated DNA damage (Sablina et al ,2005).This can help to prevent the accumulation of mutations that might not only lead to genomic instability and cancer development,but has also been associated with a role for p53in preventing premature ageing (Matheu et al ,2007).One of the p53-target genes that contributes to the regulation of intracellular ROS levels encodes TIGAR (TP53-induced glycolysis and apoptosis regulator),which indirectly affects ROS through the modulation of the glycolytic pathway (Bensaad et al ,
2006).The TIGAR protein shows similarity to the bisphosphatase domain of PFK-2/FBPase-2(6-phos-phofructo-2-kinase/fructose-2,6-bisphosphatase),an enzyme that has an essential function in the regulation of glycolysis.Recently,TIGAR has been shown to function to hydrolyse fructose-2,6-bisphosphate and fructose-1,6-bisphosphate (Li and Jogl,2009),two activities that lead to the same effects on glycolysis.Expression of TIGAR results in a decreased levels of Fru-2,6-P 2and a decreased glycolytic rate,which in some cells was shown to be pro-apoptotic.However,dampening of flux through the glycolytic pathway by TIGAR also leads to the redirection of glycolytic metabolic intermediates to the oxidative branch of the pentose phosphate pathway.One consequence of this function of TIGAR is an increased NADPH production,which contributes to the scavenging of ROS by reduced glutathione.Induction of this pathway by TIGAR results in decreased intracellular ROS levels and a lower sensitivity of cells to oxidative stress-associated apop-tosis,including that induced by p53(Bensaad et al ,2006).However,ROS levels will also impact autophagy,so we have investigated the effects of TIGAR expression on the autopha-gic and apoptosis response in non-stressed cells and after conditions of nutrient starvation or metabolic stress.
Results
TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress
We have shown previously that TIGAR expression can mod-ulate intracellular ROS levels in response to oxidative stress inducing signals such as DNA damage or p53activation (Bensaad et al ,2006).We extended these studies to examine the effect of TIGAR on intracellular ROS levels after nutrient starvation or metabolic stress in U2OS cell lines that constitu-tively over-expressed ectopic TIGAR,or after siRNA-mediated inhibition of endogenous TIGAR expression (Figure 1).Consistent with our earlier observations,we found that in these tissue culture systems even background levels of ROS
were lower in cells constitutively expressing TIGAR compared with control cells (Figure 1A).Interestingly,nutrient starvation or metabolic stress strongly elevated ROS levels and over-expression of TIGAR effectively inhibited this enhancement of ROS (Figure 1A).Conversely,knockdown of TIGAR expression resulted in an increase in ROS levels,and the increase in ROS induced by nutrient starvation and metabolic stress was further elevated after inhibition of the endogenous TIGAR protein by siRNA knockdown (Figure 1B).
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Figure 1TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.
(A )ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5and TIGAR#7)or control cells (clones Cont#1and Cont#3)left untreated,after 6h of nutrient starvation or 24h of metabolic stress.ROS levels were measured by flow cytometry after DCF treatment.The results are expressed as the mean DCF fluorescence (and standard deviation),from three independent experiments.(B )Basal,nutrient starvation-induced (5h)or metabolic stress-induced (18h)ROS levels in U2OS cells in the presence of either scrambled,TIGAR siRNA1or TIGAR siRNA2,measured by flow cytometry after DCF treatment.The results are expressed as the mean intensity of cell fluorescence (and standard deviation).*represents significant difference from control conditions (P o 0.05).
Figure 2TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(A )(Left panel)Confocal microscopic images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7)or control cells (clone Cont#1)and infected with an adenovirus expressing GFP-LC3for 16h.Cells were then left untreated,exposed to nutrient starvation for 6h or to metabolic stress for 24h.(Right panel)Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta from three independent experiments.The mean values with standard deviation are presented.(B )(Left panel)Confocal microscopic images of the fluorescence in U2OS cells stably expressing GFP-LC3and transfected with scrambled or TIGAR siRNAs.After 48h transfection,cells wer
e then left untreated,exposed to nutrient starvation for 5h or to metabolic stress for 18h.(Right panel)Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta from three independent experiments.The mean values with standard deviation are presented.(C )(Left panel)Western blot showing the expression levels of endogenous LC3-I,LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs,and 48h later exposed to nutrient starvation for 0,2.5and 6h.(Middle panel)Western blot showing the expression levels of endogenous LC3-I,LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5and TIGAR#7)or control cells (clones Cont#1and Cont#3)and left untreated.(Right panel)Western blot showing the expression levels of p62,COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated.Actin expression was examined as a loading control.(D )Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0,1,3,5and 8h;*represents significant difference from control conditions (P o 0.05);#represents a lack of significant difference from control conditions (P 40.05).
Regulation of autophagy by TIGAR K Bensaad et al
TIGAR expression modulates autophagy
Recent results have shown that the autophagic response to nutrient starvation or metabolic stress involves ROS(Scherz-Shouval et al,2007).T o determine whether changes in TIGAR expression and the consequent modulation of ROS levels can affect autophagy,we examined the response of cells to nutrient starvation or metabolic stress,two signals that have been shown to effectively induce an autophagic re-sponse(Munafo and Colombo,2001;Jin and White,2007). Autophagy was monitored by measuring the formation of autophagosomes,as measured in cells by the accumulation of GFP-tagged LC3puncta byfluorescence microscopy (Klionsky et al,2008)(Figure2).As expected,either nutrient starvation or metabolic stress resulted in a strong
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Regulation of autophagy by TIGAR
K Bensaad et al
of autophagy,which was significantly reduced in the TIGAR over-expressing cells(Figure2A).We have found previously that siRNA depletion of TIGAR expression in U2OS cells sensitized them to ROS-dependent apoptotic signals,and therefore we investigated the effect of TIGAR knockdown on the induction of autophagy.Interestingly,removal of TIGAR enhanced autophagy in unstressed cells,as well as in response to nutrient starvation or metabolic stress (Figure2B).There was a very close correlation between the activation of autophagy and the elevation of ROS levels after knockdown of TIGAR(Figures1B and2B).The increase in autophagy after siRNA-mediated inhibition of TIGAR expression was seen in various cell lines,including other transformed cells and untransformed primary epithelial cells (Supplementary Figure1).
T o further validate the effects of TIGAR expression on autophagy,we analysed several other parameters of this process(Klionsky et al,2008).The lipidation of the ubiqui-tin-like protein LC3during the process of autophagy can also be used as a marker.The modification of LC3-I to form LC3-II during autophagy was measured by western blot,in which the increase in modification and conversion to LC3-II (indicative of autophagy)correlated with levels of TIGAR expression after treatment with different siRNAs(Figure2C).Conversely,less LC3-II was formed in cells over-expressing TIGAR(Figure2
C).The degradation of p62,which serves as a link between LC3and ubiquitinated substrates,and the mitochondrial protein COX-IV also serve as markers of auto-phagy(Klionsky et al,2008).A decrease in the levels of both of these proteins was observed after inhibition of endogenous TIGAR(Figure2C).
T o determine whether TIGAR expression is regulated after starvation or metabolic stress,we examined protein levels in various cell lines over a time course of treatment(Figure2D; Supplementary Figure2).These results did not show a clear difference in overall levels of TIGAR expression,consistent with our observation that TIGAR depletion enhances auto-phagy even under control conditions(Figure2B).Glucose starvation has been shown to induce a p53-dependent cell-cycle arrest(Jones et al,2005),which might be expected to induce TIGAR expression.However,under the short-time course examined here,in which a clear autophagic response was seen,we did not note strong activation of p53as measured by increase in p53levels(Figure3C),or enhanced expression of another p53-target gene,p21(data not shown). However,the expression of TIGAR in these cells is,to some extent,dependent on basal levels of p53,as siRNA-mediated reduction of p53leads to a drop in TIGAR levels(Figure
3C).
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Figure3TIGAR expression modulates autophagy independently of p53.(A)Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta.U2OS cells stably expressing GFP-LC3were transfected with scrambled or TIGAR siRNAs,and48h after transfection, cells were left untreated(t0)or treated with Bafilomycin A1(100nM)for1or2h(t1and t2).The percentage of cells with GFP-LC3puncta was calculated at the indicated time points.Data are shown as the mean and standard deviation from three independent experiments.
(B)Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta.U2OS cells stably expressing GFP-LC3were cotransfected with scrambled or TIGAR siRNAs,and scrambled or p53siRNA.After48h transfection,cells were left untreated or exposed for5h to nutrient starvation.The percentage of cells with GFP-LC3puncta was calculated,and data are shown as the mean and standar
d deviation from three independent experiments.(C)Western blot showing the expression levels of endogenous p53and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs,and scrambled or p53siRNA,and48h later exposed to nutrient starvation for5h.Actin expression was examined as a loading control.(D)Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta.U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs,and scrambled or DRAM siRNA1/2.After48h transfection,cells were left untreated or exposed for5h to nutrient starvation.The percentage of cells with GFP-LC3puncta was calculated,and data are shown as the mean and standard deviation from three independent experiments;*represents significant difference from starved control conditions(P o0.05); @represents significant difference from untreated control conditions(P o0.05);#represents a lack of significant difference from control conditions(P40.05).
Regulation of autophagy by TIGAR
K Bensaad et al
TIGAR expression modulates autophagy independently of p53
The formation of GFP-LC3vesicles is a convenient way to measure autophagy,but could result from eit
her the in-creased rate of autophagosome formation or an inhibition in their turnover(Klionsky et al,2008).T o determine whether TIGAR expression is promoting the degradation or inhibiting the formation of autophagosomes,we examined the accumu-lation of LC3vesicles in cells treated with bafilomycin A1, which prevents degradation of autophagic vacuoles by in-hibiting fusion between autophagosomes and lysosomes (Y amamoto et al,1998).If the effect of TIGAR knockdown is to drive accumulation of vesicles through inhibiting their maturation,we would expect bafilomycin A1treatment to neutralize the effect of TIGAR.However,even in the presence of bafilomycin A1,there was still a clear increase in auto-phagosome formation after inhibition of TIGAR expression (Figure3A).T aken together,these results suggest that TIGAR can have a function in inhibiting the formation of autophagosomes rather than activating their degradation, and that removal of TIGAR promotes the autophagic response.
Earlier studies have shown that p53can contribute to both the induction and inhibition of autophagy,and that nutrient starvation/metabolic stress can induce p53. The ability of p53to regulate autophagy has been shown to be dependent both on direct cytoplasmic activities of p53,as well as on the activity of p53-inducible genes such as DRAM,which promotes autophagy(Crighton et al,2006; T asdemir et al,2008).As TIGAR is a p53-target gene that seems to have a function in limiting autophagy,
we were interested to determine the interplay between p53and TIGAR,or DRAM and TIGAR,in the regulation of this process.The enhanced autophagic response to TIGAR inhibition was clearly retained in cells depleted of p53,indicating that p53is not required for TIGAR-dependent modulation of autophagy(Figure3B).However, p53was clearly required for the autophagic response to nutrient starvation in cells that retain TIGAR expression (Figure3B).These results suggest a balance in which p53has a function in enhancing autophagy in these cells(possibly through regulation of DRAM expression), with TIGAR serving to dampen this response by decreasing ROS levels.As p53regulates TIGAR expression,TIGAR levels were significantly lower after siRNA-mediated depletion of p53(Figure3C).However,even these reduced TIGAR levels were sufficient to limit autophagy in both untreated cells or in response to starvation,as shown by the enhanced autophagy after inhibition of TIGAR expression in p53siRNA-treated cells(Figure3B). T o more directly assess the function of DRAM in the regula-tion of autophagy,we used siRNA to deplete cells of DRAM expression(Crighton et al,2006)(Figure3D). As expected,inhibition of DRAM expression reduced the autophagic response under all conditions,although starvation still enhanced autophagy in the absence of DRAM,suggesting that other p53-dependent genes may have a function in promoting autophagy under these condi-tions.Knockdown of TIGAR enhanced autophagy regardless of the presence or absence of DRAM,showing that these two proteins function independently to promote and inhibit autophagy,respectively.Modulation of ROS by TIGAR correlates with modulation of autophagy
T o determine whether the autophagy induced in our cell systems was dependent on increased ROS,we modulated ROS levels directly by treatment with N-acetyl cystein(NAC) and L-ascorbic acid,direct scavengers of ROS(Figure4A)or hydrogen peroxide(H2O2)to enhance intracellular ROS levels (Figure4B).Both nutrient starvation and metabolic stress-induced autophagy were lowered by the anti-oxidant treat-ment(Figure4A).We were,however,unable to completely prevent the autophagic response by NAC and ascorbate treatment,suggesting that some ROS-independent autophagy was also being induced in these cells after these treatments. Treatment of cells with increasing concentrations of H2O2 enhanced intracellular ROS(Figure4B)to levels comparable with those seen after knockdown of TIGAR(Figure1B). Interestingly,enhanced ROS in response to H2O2also pro-moted autophagy,even in the absence of further stresses,to levels very similar to those seen after TIGAR depletion (Figure2B).
These results suggest that the changes in ROS levels seen after alterations in TIGAR expression may be responsible for the effects on autophagy.Further support for this model was provided by the observation that although anti-oxidant treat-ment with NAC and ascorbate effectively lowered autophagy in response to nutrient starvation or metabolic stress,this treatment had little further effect on decreasing autophagy in TIGAR over-expressing cells,in which autophagy in response to either nutr
ient starvation or metabolic stress was already lower compared with control(Figure4C).The earlier de-scribed effects of TIGAR in lowering intracellular Fru-2,6-P2 levels,promoting the pentose phosphate pathway and decreasing intracellular ROS levels,are consistent with the ability of TIGAR to carry out the bisphosphatase function of the bifunctional enzyme PFK-2/FBPase-2(Bensaad et al, 2006;Li and Jogl,2009).We have shown previously that the activities of TIGAR can be mimicked by the expression of the isolated bisphosphatase domain(FBPase-2)from PFK-2/ FBPase-2.Accordingly,expression of the isolated bis-phosphatase domain also inhibited starvation or metabolic stress-induced autophagy to levels comparable to that seen after over-expression of TIGAR(Figure4C).Furthermore,the effect of expression of the isolated FBPase-2domain was also lost after anti-oxidant treatment,as seen for TIGAR (Figure4C).Similar results were obtained in cells stably over-expressing TIGAR(data not shown)and in cells treated with a number of other anti-oxidants(Figure4D).These results are,therefore,consistent with a function for TIGAR in preventing autophagy by lowering ROS levels,as the effect of TIGAR is greatly diminished when ROS are removed by another mechanism(NAC and ascorbate).Conversely,the autophagic response induced after inhibition of endogenous TIGAR expression by treatment with siRNA was reduced after anti-oxidant treatment(Figure4E),which correlated with a decrease in ROS levels after anti-oxidant treatment.
TIGAR does not clearly affect the mTOR signalling pathway
Although we have concentrated on a function for TIGAR in regulating autophagy through the control of ROS,it is possi-ble that other functions of TIGAR might control other path-ways important in the regulation of autophagy.The most
Regulation of autophagy by TIGAR
K Bensaad et al
obvious of these is signalling through mTOR,the inhibition of which has been shown to be a critical component driving the activation of autophagy in response to nutrient starvation
(Meijer and Codogno,2004).A decrease in mTOR signalling in response to nutrient deprivation can be assessed by a reduction in phosphorylation of the downstream target
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Figure 4Modulation of ROS by TIGAR correlates with the modulation of autophagy.(A )U2OS cells wer
e left untreated,exposed to nutrient starvation for 6h or to metabolic stress for 24h,with or without treatment with AO1(NAC (2mM)and L -ascorbic acid (2mM))for 24h.The percentage of cells with GFP-LC3puncta was calculated,and data are shown as the mean and standard deviation from three independent experiments.(B )(Left panel)ROS levels in U2OS cells left untreated or treated with 0.5or 1mM of H 2O 2for 24h.ROS levels were measured by flow cytometry after DCF treatment.The results are expressed as the mean DCF fluorescence (and standard deviation)from three independent experiments.(Right panel)Quantitation of the percentage of GFP-LC3puncta positive cells for cells treated as described above.Data are shown as the mean and standard deviation from three independent experiments.(C )Quantitation of the percentage of GFP-LC3puncta positive cells.U2OS cells stably expressing GFP-LC3were transfected with vector pCHER1A expressing the mCherry gene as control,or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2.After 48h transfection,cells were left untreated,exposed to nutrient starvation for 6h or to metabolic stress for 24h,with or without treatment with AO1(NAC (2mM)and L -ascorbic acid (2mM))for 24h.The percentage of cells with GFP-LC3puncta was calculated,and data are shown as the mean and standard deviation from three independent experiments.(D )Quantitation of the percentage of GFP-LC3puncta positive cells.Cells were left untreated,exposed to nutrient starvation for 6h with or without treatment with AO1(NAC (2mM)and L -ascorbic acid (2mM)),AO2(glutathione ethyl ester (4mM))or AO
3(ethyl pyruvate (4mM))for 24h.U2OS cells stably expressing GFP-LC3were transfected with vector pCHER1A expressing the mCherry gene as control,or expression plasmid for Flag-tagged-TIGAR.After 48h transfection,cells were treated.The percentage of cells with GFP-LC3puncta was calculated,and data are shown as the mean and standard deviation from three independent experiments.(E )(Left panel)Quantitation of the percentage of GFP-LC3puncta positive cells.Cells were left untreated,exposed to nutrient starvation for 5h or to metabolic stress for 18h,with or without treatment with AO1(NAC (2mM)and L -ascorbic acid (2mM))for 24h.U2OS cells stably over-expressing GFP-LC3in the presence of either scrambled,TIGAR siRNA1or TIGAR siRNA2.(Right panel)Basal or nutrient starvation-induced (5h)ROS levels in U2OS cells in the presence of either scrambled,TIGAR siRNA1or TIGAR siRNA2with or without treatment with AO1(NAC (2mM)and L -ascorbic acid (2mM))for 24h,measured by flow cytometry after DCF treatment.The results are expressed as the mean intensity of cell fluorescence (and standard deviation).*represents significant difference from control conditions (P o 0.05);#represents a lack of significant difference from control conditions (P 40.05).
Regulation of autophagy by TIGAR K Bensaad et al

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