Knockout of cTAZ promoted ISGs expression in the presence of IFN - α . WT and cTAZ - / - RKO cells were treated with or without of IFN - α ( 50 ng / ml ) for indicated time , the expression of selected genes was quantified by qPCR . WT or cTAZ - / - group was normalized by basal level , respectively . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - test .

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Knockout of cTAZ promoted ISGs expression in the presence of IFN-α. WT and cTAZ-/- RKO cells were treated with or without of IFN-α (50 ng/ml) for indicated time, the expression of selected genes was quantified by qPCR. WT or cTAZ-/- group was normalized by basal level, respectively. Error bars indicate SD, n =3. *P<0.05; **P<0.01; Student's t-test.


STAT1 interacted with cTAZ , and the interaction was enhanced by IFN - α . HEK293A cells were transfected with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour . IP were performed using Flag antibody .

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STAT1 interacted with cTAZ, and the interaction was enhanced by IFN-α. HEK293A cells were transfected with indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour. IP were performed using Flag antibody.


cTAZ attenuated heterodimerization between STAT1 and STAT2 induced by IFN - α . Following transfection , cells were treated with or without IFN - α ( 50 ng / ml ) for 1 hour and subjected to IP assays using Flag antibody .

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cTAZ attenuated heterodimerization between STAT1 and STAT2 induced by IFN-α. Following transfection, cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to IP assays using Flag antibody.


Depletion of cTAZ enhanced the interaction between STAT1 and STAT2 . RKO cell lines ( WT and # 14 ) were treated with IFN - α as in ( B ) . IP assays were performed using STAT1 antibody .

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Depletion of cTAZ enhanced the interaction between STAT1 and STAT2. RKO cell lines (WT and #14) were treated with IFN-α as in (B). IP assays were performed using STAT1 antibody.


cTAZ attenuated interaction between STAT1 and importin α5 ( Im - α5 ) . Following transfection , HEK293A cells were stimulated using IFN - α IP assays were performed using Flag antibody .

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cTAZ attenuated interaction between STAT1 and importin α5 (Im-α5). Following transfection, HEK293A cells were stimulated using IFN-α IP assays were performed using Flag antibody.


Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN - α stimulation . RKO ( WT and cTAZ - / - ) cells treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , and subjected to IF using STAT1 antibody ( green ) . DNA was labeled by DAPI ( blue ) . Scale bar , 50 μm . Quantification was performed using ImageJ software ( right ) . N > C : nuclear ; N = C : nuclear plus cytoplasmic ; N < C : cytoplasmic localization .

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Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. RKO (WT and cTAZ-/-) cells treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF using STAT1 antibody (green). DNA was labeled by DAPI (blue). Scale bar, 50 μm. Quantification was performed using ImageJ software (right). N>C: nuclear; N=C: nuclear plus cytoplasmic; N<C: cytoplasmic localization.


Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN - α stimulation . Cells were treated and subjected to subcellular fractionation .

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Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. Cells were treated and subjected to subcellular fractionation.


cTAZ defective in STAT1 - binding failed to block nuclear translocation of STAT1 . HEK293A stably overexpressing STAT1 were transfected with the indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , and subjected to IF Scale bar , 25 μm .

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cTAZ defective in STAT1-binding failed to block nuclear translocation of STAT1. HEK293A stably overexpressing STAT1 were transfected with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF Scale bar, 25 μm.


cTAZ repressed the antiviral activity of ectopic STAT1 / 2 . HEK293A were transfected with indicated plasmids , infected with gVSV ( MOI = 0 . 01 ) for 12 hours , fixed , and subjected to microscopy ( Left ) . Viral infection and replication were marked by green fluorescence . Scale bar , 100 μm . ( Right ) Quantification of GFP positive cells . About 300 cells ( from 3 different fields ) were analyzed . Error bars indicate SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test .

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cTAZ repressed the antiviral activity of ectopic STAT1/2. HEK293A were transfected with indicated plasmids, infected with gVSV (MOI = 0.01) for 12 hours, fixed, and subjected to microscopy (Left). Viral infection and replication were marked by green fluorescence. Scale bar, 100 μm. (Right) Quantification of GFP positive cells. About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. **P<0.01; ***P<0.001; Student's t-test.


cTAZ and STAT1/2 expression levels of cells


cTAZ effectively repressed antiviral activity of IFN - α . Transfection and infection were performed Top : GFP intensity of cell lysates was measured by a fluorometer . Bottom : viral GFP and ectopic cTAZ protein levels were detected by IB .

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cTAZ effectively repressed antiviral activity of IFN-α. Transfection and infection were performed Top: GFP intensity of cell lysates was measured by a fluorometer. Bottom: viral GFP and ectopic cTAZ protein levels were detected by IB.


Depletion of cTAZ repressed viral replication . RKO cell lines ( WT and two independent cTAZ - / - clones ) were infected with gVSV ( MOI = 0 . 1 ) for 12 hours . Scale bar , 100 μm . The ratio of GFP positive cells were calculated About 300 cells ( from 3 different fields ) were analyzed . Error bars indicate SD . * * * P < 0 . 001 ; Student ' s t - test .

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Depletion of cTAZ repressed viral replication. RKO cell lines (WT and two independent cTAZ -/- clones) were infected with gVSV (MOI = 0.1) for 12 hours. Scale bar, 100 μm. The ratio of GFP positive cells were calculated About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. ***P<0.001; Student's t-test.


Depletion of cTAZ repressed viral replication . Cells were treated GFP intensity of cell lysates was measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test .

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Depletion of cTAZ repressed viral replication. Cells were treated GFP intensity of cell lysates was measured. Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.


Depletion of cTAZ repressed viral replication . Cells were treated the viral genomic RNA was quantified by qPCR ( normalized to human GAPDH ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - test .

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Depletion of cTAZ repressed viral replication. Cells were treated the viral genomic RNA was quantified by qPCR (normalized to human GAPDH). Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.


cTAZ deficiency enhanced ISGs expression . Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test .

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cTAZ deficiency enhanced ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; ***P<0.001; Student's t-test.


Overexpression of cTAZ promotes virus infection . DOX - inducible cTAZ - overexpressing RKO cell lines were pre - treated with DOX for 48 hours ( 1 μg / ml ) . gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test .

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Overexpression of cTAZ promotes virus infection. DOX-inducible cTAZ-overexpressing RKO cell lines were pre-treated with DOX for 48 hours (1 μg/ml). gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.


Overexpression of cTAZ repressed ISGs expression . Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - test .

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Overexpression of cTAZ repressed ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; Student's t-test.


gVSV induced expression of cTAZ . RKO cells were infected with gVSV ( MOI = 0 , 0 . 001 , 0 . 01 , 0 . 01 , 1 ) for 8 hours . Protein expression was determined by IB ( Left ) and quantified ( Right ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA test was used for statistical analysis .

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gVSV induced expression of cTAZ. RKO cells were infected with gVSV (MOI = 0, 0.001, 0.01, 0.01, 1) for 8 hours. Protein expression was determined by IB (Left) and quantified (Right). Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


IFN - α induced expression of cTAZ . RKO cells were treated with different doses of IFN - α for 8 hours . Protein expression was determined Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA test was used for statistical analysis .

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IFN-α induced expression of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. Protein expression was determined Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


IFN - α induced mRNA level of cTAZ . RKO cells were treated with different doses of IFN - α for 8 hours . RNA levels were measured by qPCR . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA test was used for statistical analysis .

IFN-α induced mRNA level of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. RNA levels were measured by qPCR. Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.


STAT1 / 2 synergistically activated a cTAZ promoter . HEK293A cells were transfected with cTAZ reporter with or without STAT proteins . Promoter activity was determined using luciferase assay , and protein expression was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - test .

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STAT1/2 synergistically activated a cTAZ promoter. HEK293A cells were transfected with cTAZ reporter with or without STAT proteins. Promoter activity was determined using luciferase assay, and protein expression was determined by IB. Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.


STAT1 / 2 binding sites on cTAZ promoter . HEK293A were transfected with cTAZ promoters ( wild type or mutants ) . Luciferase activity and responses to STAT1 / 2 expression were measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; Student ' s t - test .

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STAT1/2 binding sites on cTAZ promoter. HEK293A were transfected with cTAZ promoters (wild type or mutants). Luciferase activity and responses to STAT1/2 expression were measured. Error bars indicate SD, n = 3. **P<0.01; Student's t-test.


STAT1 occupied cTAZ promoter in an IFN - α - sensitive manner . RKO cells were treated with or without IFN - α ( 50 ng / ml ) for 1 hour and subjected to ChIP assays . cTAZ PCR primers were designed to target STAT1 / 2 binding site . ISG15 promoter was included as a positive control .

STAT1 occupied cTAZ promoter in an IFN-α-sensitive manner. RKO cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to ChIP assays. cTAZ PCR primers were designed to target STAT1/2 binding site. ISG15 promoter was included as a positive control.


A Left , time lapse confocal images of PHcrac ( red ) and LimE ( green ) in waves at the basal surface of a giant cell . Right , intensity plot across the blue dotted arrow in image " 30 sec " .

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A Left, time lapse confocal images of PHcrac (red) and LimE (green) in waves at the basal surface of a giant cell. Right, intensity plot across the blue dotted arrow in image "30 sec".


Various biosensors in excitable waves . snapshots of biosensors ( top ) and intensity plots ( bottom ) across the blue dotted arrows , with black plots showing the corresponding biosensors in images above , red ( B ) showing PHcrac in the same cell , Scatter plots of biosensor peak locations relative to the peak of PHcrac ( C ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 cells for RalGDS , RBD , PKBA , , respectively . Scale bars in images above represent 20 μm . Grey arrows in all plots represent the direction of wave propagation .

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Various biosensors in excitable waves. snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, red (B) showing PHcrac in the same cell, Scatter plots of biosensor peak locations relative to the peak of PHcrac (C) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for RalGDS, RBD, PKBA, , respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.


Various biosensors in excitable waves . D , snapshots of biosensors ( top ) and intensity plots ( bottom ) across the blue dotted arrows , with black plots showing the corresponding biosensors in images above , green ( D ) showing LimE . E , Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE ( E ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that the peak precedes that of LimE ) along the direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 cells for , RacGEF1 , PAK1 - GBD , and Coronin , respectively . Scale bars in images above represent 20 μm . Grey arrows in all plots represent the direction of wave propagation .

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Various biosensors in excitable waves. D, snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, green (D) showing LimE. E, Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE (E) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of LimE) along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for , RacGEF1, PAK1-GBD, and Coronin, respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.


F Left , confocal images of PHcrac ( red ) and PAK1 - GBD ( green ) in the same cells treated with LatA . Scale bars represent 10 μm . Right , temporal profiles of normalized mean cytosolic intensities of PHcrac ( red ) and PAK1 - GBD ( green ) ( mean ± s . e . m . , n = 18 cells ) in response to cAMP .

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F Left, confocal images of PHcrac (red) and PAK1-GBD (green) in the same cells treated with LatA. Scale bars represent 10 μm. Right, temporal profiles of normalized mean cytosolic intensities of PHcrac (red) and PAK1-GBD (green) (mean±s.e.m., n=18 cells) in response to cAMP.


A Confocal image of LimE in waves ( scale bar , 20 μm ) .

A Confocal image of LimE in waves (scale bar, 20 μm).


B Top , kymograph of LimE in a fixed region across which a piece of wave propagates , with white arrows pointing to the same puncta . Bottom , box plot of puncta ' s lifetime ( n = 100 puncta ) .

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B Top, kymograph of LimE in a fixed region across which a piece of wave propagates, with white arrows pointing to the same puncta. Bottom, box plot of puncta's lifetime (n=100 puncta).


H A two - dimensional snapshot of a propagating wave ( direction of propagation indicated with the dashed arrow ) . A line scan corresponding to the STEN - CEN profile is shown on the right .

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H A two-dimensional snapshot of a propagating wave (direction of propagation indicated with the dashed arrow). A line scan corresponding to the STEN-CEN profile is shown on the right.


A Time lapse confocal images of PHcrac ( top ) and LimE ( bottom ) in the same giant cell ( scale bar , 20 μm ) . White arrows point to wave breaking events .

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A Time lapse confocal images of PHcrac (top) and LimE (bottom) in the same giant cell (scale bar, 20 μm). White arrows point to wave breaking events.


E Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits Inp54p to membrane induced by rapamycin at time 0 . The white arrow ( - 45 s ) points to a nascent wave before Inp54p recruitment , while the red ( 60 s ) arrow after .

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E Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits Inp54p to membrane induced by rapamycin at time 0. The white arrow (-45 s) points to a nascent wave before Inp54p recruitment, while the red (60 s) arrow after.


F Color coded overlays of LimE before and after Inp54p recruitment . White arrows point to nascent waves at time 0 . G Kymographs of LimE in regions outlined by dotted line boxes in f .

F Color coded overlays of LimE before and after Inp54p recruitment. White arrows point to nascent waves at time 0. G Kymographs of LimE in regions outlined by dotted line boxes in f.


J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment , where rapamycin is added at time 0 to induce Inp54p recruitment . White arrows point to wave patches right before rapamycin addition .

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J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment, where rapamycin is added at time 0 to induce Inp54p recruitment. White arrows point to wave patches right before rapamycin addition.


K Confocal image of PI5K ( left ) and LimE ( right ) at the bottom of the same cell . All scale bars in images represent 20 μm .

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K Confocal image of PI5K (left) and LimE (right) at the bottom of the same cell. All scale bars in images represent 20 μm.


A Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits PKBA to membrane induced by rapamycin at time 0 .

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A Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.


B T - stack of LimE in the region outlined by the colored line box in A .

B T-stack of LimE in the region outlined by the colored line box in A.


C Box plots of mean wave speed ( left ) , mean wave patch area ( middle ) , and average number of wave patches per frame ( right ) before and after PKBA recruitment ( red bars indicate median , n = 24 cells ) . Error bars are from the student ' s t - test .

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C Box plots of mean wave speed (left), mean wave patch area (middle), and average number of wave patches per frame (right) before and after PKBA recruitment (red bars indicate median, n=24 cells). Error bars are from the student's t-test.


D Time lapse confocal images of PHcrac at the basal surface of a giant cell , which recruits PKBA to membrane induced by rapamycin at time 0 .

D Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.


E Confocal images of PHcrac ( left ) and PHcrac merged with LimE ( middle , PHcrac as red , LimE as green ) in a giant cell after steady PKBA recruitment . Right , intensity plots of PHcrac ( red ) and LimE ( green ) across the blue box .

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E Confocal images of PHcrac (left) and PHcrac merged with LimE (middle, PHcrac as red, LimE as green) in a giant cell after steady PKBA recruitment. Right, intensity plots of PHcrac (red) and LimE (green) across the blue box.


F Kymograph of PHcrac ( red ) merged with LimE ( green ) along the white dashed line from E .

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F Kymograph of PHcrac (red) merged with LimE (green) along the white dashed line from E.


G Time lapse confocal images of PHcrac at the bottom of a giant cell treated with LatA , where rapamycin is added at time 0 to induce PKBA recruitment . All scale bars above represent 20 μm .

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G Time lapse confocal images of PHcrac at the bottom of a giant cell treated with LatA, where rapamycin is added at time 0 to induce PKBA recruitment. All scale bars above represent 20 μm.


A Left , time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment , induced by rapamycin addition at time 0 . Right , temporal profile of normalized mean LimE intensity at cell cortex ( mean ± s . e . m . , n = 20 cells ) .

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A Left, time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment, induced by rapamycin addition at time 0. Right, temporal profile of normalized mean LimE intensity at cell cortex (mean±s.e.m., n=20 cells).


B Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits RacGEF1ΔN to membrane induced by rapamycin at time 0 .

B Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane induced by rapamycin at time 0.


C T - stack of LimE in the region outlined by the colored line box in B .

C T-stack of LimE in the region outlined by the colored line box in B.


D Right , kymographs of LimE along a line across giant cells after RacGEF1ΔN recruitment . Top left , confocal image with a dotted line as an example used to make kymographs . White arrows in image and kymographs point to examples of actin puncta . Bottom left , box plot of lifetime of puncta ( red bar indicates median , n = 100 puncta from 10 cells ) . All scale bars above represent 20 μm .

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D Right, kymographs of LimE along a line across giant cells after RacGEF1ΔN recruitment. Top left, confocal image with a dotted line as an example used to make kymographs. White arrows in image and kymographs point to examples of actin puncta. Bottom left, box plot of lifetime of puncta (red bar indicates median, n=100 puncta from 10 cells). All scale bars above represent 20 μm.


A Time lapse confocal images of PHcrac at the basal surface of a giant cell , which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin .

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A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin.


B Confocal images of PHcrac ( left ) and PHcrac merged with LimE ( right , PHcrac as red , LimE as green ) in a giant cell after RacGEF1ΔN recruitment .

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B Confocal images of PHcrac (left) and PHcrac merged with LimE (right, PHcrac as red, LimE as green) in a giant cell after RacGEF1ΔN recruitment.


C Kymograph of LimE ( top , green ) and PHcrac ( bottom , red ) across the dotted line in B .

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C Kymograph of LimE (top, green) and PHcrac (bottom, red) across the dotted line in B.


D Confocal image of RalGDS ( left ) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph ( right ) .

D Confocal image of RalGDS (left) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph (right).


E Time lapse confocal images of PHcrac in single cells , which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0 . White arrow points to PHcrac patches at cell cortex .

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E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex.


F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment . Red dashed lines in F indicate rapamycin addition at time 0 .

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F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment. Red dashed lines in F indicate rapamycin addition at time 0.


G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment ( mean ± s . e . m . , n = 20 cells ) . Red dashed lines in F and G indicate rapamycin addition at time 0 .

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G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0.


H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment , with ( solid ) or without ( hollow ) RacGEF1ΔN recruitment ( ~ 6000 cells from 3 independent experiments ) .

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H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments).


I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0 , with ( red ) or without ( blue ) RacGEF1ΔN recruitment ( mean ± s . e . m . , n = 25 cells each ) .

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I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each).


J Confocal images of GflB ( top ) and RapGAP1 ( bottom ) in waves at the basal surface of a giant cell .

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J Confocal images of GflB (top) and RapGAP1 (bottom) in waves at the basal surface of a giant cell.


K Intensity plots of GflB ( blue ) and RapGAP1 ( orange ) across the green dotted arrow in J .

K Intensity plots of GflB (blue) and RapGAP1 (orange) across the green dotted arrow in J.


L Scatter plots of first peak distances , of GflB relative to LimE , RapGAP1 relative to LimE , and RapGAP1 relative to GflB in the same giant cells ( red lines indicate median , n = 19 cells each ) .

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L Scatter plots of first peak distances, of GflB relative to LimE, RapGAP1 relative to LimE, and RapGAP1 relative to GflB in the same giant cells (red lines indicate median, n=19 cells each).


M Confocal images of GflB ( top ) and RapGAP1 ( bottom ) at the basal surface of the same giant cell after RacGEF1ΔN recruitment .

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M Confocal images of GflB (top) and RapGAP1 (bottom) at the basal surface of the same giant cell after RacGEF1ΔN recruitment.


N kymographs of GflB ( top ) and RapGAP1 ( bottom ) across the green dotted lines in m . All scale bars represent 20 μm .

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N kymographs of GflB (top) and RapGAP1 (bottom) across the green dotted lines in m. All scale bars represent 20 μm.


A , B Left , confocal images of LimE ( top ) and kymographs of cortical LimE ( bottom ) in single cells with PKBA ( A ) or RacGEF1ΔN ( B ) steadily recruited ( scale bars , 10 μm ) . Right , color coded overlays of LimE in single cells .

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A, B Left, confocal images of LimE (top) and kymographs of cortical LimE (bottom) in single cells with PKBA (A) or RacGEF1ΔN (B) steadily recruited (scale bars, 10 μm). Right, color coded overlays of LimE in single cells.


C Box plots of roundness ( left ) and cell area ( middle ) comparing wt , cells with RacGEF1ΔN recruited , and spreading phases of oscillators after Inp54p recruitment ( n = 100 cells each ) . Right , box plots of coefficient of variance of cell area with the above conditions in a 10 - minute time window ( n = 30 cells each ) . Red bars indicate median .

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C Box plots of roundness (left) and cell area (middle) comparing wt, cells with RacGEF1ΔN recruited, and spreading phases of oscillators after Inp54p recruitment (n=100 cells each). Right, box plots of coefficient of variance of cell area with the above conditions in a 10-minute time window (n=30 cells each). Red bars indicate median.


D Phalloidin staining of cells after perturbations . Maximal intensity projections are shown .

D Phalloidin staining of cells after perturbations. Maximal intensity projections are shown.


( a ) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT - α in HCT116 cells .

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(a) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT-α in HCT116 cells.


( b ) Autophagy induced by PFT - α , p53 knockdown , or p53 knockout in HCT116 cells . The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments ( mean ± s . e . m . ; * P 0 . 05 ) . Culture in nutrient - free ( NF ) conditions was used as a positive control .

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(b) Autophagy induced by PFT-α, p53 knockdown, or p53 knockout in HCT116 cells. The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments (mean ± s.e.m.; *P 0.05). Culture in nutrient-free (NF) conditions was used as a positive control.


( c ) Effect of p53 on the maturation of LC3 . Immunoblots are shown for wild - type ( WT ) or p53 − / − HCT116 ( n = 3 ) .

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(c) Effect of p53 on the maturation of LC3. Immunoblots are shown for wild-type (WT) or p53−/− HCT116 (n = 3).


( d ) GFP - LC3 puncta induced by PFT - α , p53 knockdown or knockout . WT and p53 − / − HCT116 cells were transfected with control or p53 - specific siRNAs , re - transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT - α . The percentage of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) is reported ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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(d) GFP-LC3 puncta induced by PFT-α, p53 knockdown or knockout. WT and p53−/− HCT116 cells were transfected with control or p53-specific siRNAs, re-transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT-α. The percentage of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (mean ± s.d., n = 3; *P 0.05).


( e ) GFP - LC3 puncta in p53 − / − MEF , compared with WT MEF transfected with GFP - LC3 ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

(e) GFP-LC3 puncta in p53−/− MEF, compared with WT MEF transfected with GFP-LC3 (mean ± s.d., n = 3; *P 0.05).


( f ) GFP - LC3 dots in tissues from p53 + / + or p53 − / − mice expressing a GFP - LC3 transgene ( mean ± s . d . ; * P 0 . 05 ) , without or after 24 h of starvation . The number of GFP - LC3 dots per area was determined for a minimum of 4 fields ( at × 400 magnification for 4 slides per animal , 3 animals per condition ) .

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(f) GFP-LC3 dots in tissues from p53+/+ or p53−/− mice expressing a GFP-LC3 transgene (mean ± s.d.; *P 0.05), without or after 24 h of starvation. The number of GFP-LC3 dots per area was determined for a minimum of 4 fields (at ×400 magnification for 4 slides per animal, 3 animals per condition).


( g ) TEM pictures of p53 − / − and p53 + / − mouse livers . p53 − / − hepatocytes carry approximately 3 autophagosomes , p53 + / − controls approximately 1 per cell .

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(g) TEM pictures of p53−/− and p53+/− mouse livers. p53−/− hepatocytes carry approximately 3 autophagosomes, p53+/− controls approximately 1 per cell.


( h ) Modulation of DsRed : : LGG - 1 levels by the p53 orthologue CEP - 1 in C . elegans . Representative pictures are shown for WT animals and for nematodes carrying a cep - 1 deletion allele ( gk138 ) . The count of puncta per cell was 6 . 9 ± 3 . 3 for WT , 52 . 2 ± 9 . 4 for cep - 1 ( gk138 ) embryos ( mean ± s . e . m . for 20 individuals , 5 cells per embryo ) . The quantification of DsRed : : LGG - 1 puncta is shown for the indicated genetic backgrounds and conditions ( dots represent pixel intensity for individual embryos ) .

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(h) Modulation of DsRed::LGG-1 levels by the p53 orthologue CEP-1 in C. elegans. Representative pictures are shown for WT animals and for nematodes carrying a cep-1 deletion allele (gk138). The count of puncta per cell was 6.9 ± 3.3 for WT, 52.2 ± 9.4 for cep-1 (gk138) embryos (mean ± s.e.m. for 20 individuals, 5 cells per embryo). The quantification of DsRed::LGG-1 puncta is shown for the indicated genetic backgrounds and conditions (dots represent pixel intensity for individual embryos).


( a ) PFT - α or p53 siRNA - induced GFP - LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5 , Atg10 , Atg12 , Beclin 1 or hVps34 . Culture in nutrient - free ( NF ) conditions was used as a control of autophagy induction .

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(a) PFT-α or p53 siRNA-induced GFP-LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5, Atg10, Atg12, Beclin 1 or hVps34. Culture in nutrient-free (NF) conditions was used as a control of autophagy induction.


( b ) PFT - α or p53 siRNA - mediated GFP - LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1 .

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(b) PFT-α or p53 siRNA-mediated GFP-LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1.


( c ) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells . MCF7 cells stably transfected with a tetracycline - repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression , transfected with GFP - LC3 and subjected to p53 knockdown before quantification of GFP - LC3 puncta . Data in a - c are mean ± s . d . of 3 independent experiments .

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(c) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells. MCF7 cells stably transfected with a tetracycline-repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression, transfected with GFP-LC3 and subjected to p53 knockdown before quantification of GFP-LC3 puncta. Data in a-c are mean ± s.d. of 3 independent experiments.


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - α and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

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(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-α and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - alpha and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-alpha and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( g ) Immunoblot detection of LC3 - I / II in cells treated with PFT - α and / or leupeptin in WT HCT116 cells ( n = 3 ) .

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(g) Immunoblot detection of LC3-I/II in cells treated with PFT-α and/or leupeptin in WT HCT116 cells (n = 3).


( a ) Immunoblot detection of the phosphorylation status of AMPKα , ACCα , TSC2 and p70S6K in HCT116 cells . Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα , ACCα and TSC2 was detected in p53 − / − HCT116 , compared with WT HCT116 cells after culture in complete medium ( n = 5 ) .

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(a) Immunoblot detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in HCT116 cells. Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα, ACCα and TSC2 was detected in p53−/− HCT116, compared with WT HCT116 cells after culture in complete medium (n = 5).


( b ) Quantification of GFP - LC3vac cells in WT and p53 − / − HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK ( mean ± s . d . of 3 independent experiments ; * P 0 . 05 ) . The inset in b demonstrates the efficiency of siRNA - mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis ( n = 3 ) . Note that the antibody recognizes both AMPKα1 and AMPKα2 .

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(b) Quantification of GFP-LC3vac cells in WT and p53−/− HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK (mean ± s.d. of 3 independent experiments; *P 0.05). The inset in b demonstrates the efficiency of siRNA-mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis (n = 3). Note that the antibody recognizes both AMPKα1 and AMPKα2.


( c ) Immunoblot detection of ACCα phosphorylation in WT and p53 − / − HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 ( n = 5 ) ; Co , control .

(c) Immunoblot detection of ACCα phosphorylation in WT and p53−/− HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 (n = 5); Co, control.


( d ) Quantification of GFP - LC3vac cells ( means ± s . d . , n = 3 separate experiments ) in WT HCT116 and p53 − / − HCT116 treated with PFT - α and / or rapamycin .

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(d) Quantification of GFP-LC3vac cells (means ± s.d., n = 3 separate experiments) in WT HCT116 and p53−/− HCT116 treated with PFT-α and/or rapamycin.


( a ) Cytosolic ATP levels in WT and p53 − / − HCT116 cells in the absence of glucose . Measurements were performed on luciferase - expressing HCT116 , after perfusion of the cells with medium first containing then missing glucose .

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(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose.


( b ) Effect of methylpyruvate ( MetPyr ) on ATP levels in WT p53 − / − HCT116 cells depleted of glucose . ATP levels were measured as in a , in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr .

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(b) Effect of methylpyruvate (MetPyr) on ATP levels in WT p53−/− HCT116 cells depleted of glucose. ATP levels were measured as in a, in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr.


( c ) Effect of autophagy on the ATP levels of WT and p53 − / − HCT116 cells depleted of glucose . Cells were transfected with siRNAs specific for Beclin 1 and AMPKα , or they were treated with rapamycin for 6 h , followed by measurement of ATP levels as in a , before or after glucose withdrawal ( means ± s . d . of triplicates , 3 independent experiments ) .

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(c) Effect of autophagy on the ATP levels of WT and p53−/− HCT116 cells depleted of glucose. Cells were transfected with siRNAs specific for Beclin 1 and AMPKα, or they were treated with rapamycin for 6 h, followed by measurement of ATP levels as in a, before or after glucose withdrawal (means ± s.d. of triplicates, 3 independent experiments).


( d ) Metabolic stress - induced cell death is attenuated in the absence of p53 . HCT116 cells were transfected with control , Atg5 - , Atg10 - , or AMPKα - specific siRNAs and were subjected 48 h later to metabolic stress ( cultured for 48 h in nutrient - free , hypoxic conditions ) and stained with DiOC6 ( 3 ) and PI . The black and white portions of the columns refer to the DiOC6 ( 3 ) low PI − ( dying ) and DiOC6 ( 3 ) low PI + ( dead ) population , respectively .

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(d) Metabolic stress-induced cell death is attenuated in the absence of p53. HCT116 cells were transfected with control, Atg5-, Atg10-, or AMPKα-specific siRNAs and were subjected 48 h later to metabolic stress (cultured for 48 h in nutrient-free, hypoxic conditions) and stained with DiOC6(3) and PI. The black and white portions of the columns refer to the DiOC6(3)low PI− (dying) and DiOC6(3)low PI+(dead) population, respectively.


( e ) Metabolic stress - induced decrease of clonogenic survival was attenuated in the absence of p53 . HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival .

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(e) Metabolic stress-induced decrease of clonogenic survival was attenuated in the absence of p53. HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival.


( f ) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion . MCF7 cells that carry a tetracycline - repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it , transfected with a control siRNA or a p53 - specific siRNA , subjected to metabolic stress , and finally stained with DiOC6 ( 3 ) / PI .

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(f) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion. MCF7 cells that carry a tetracycline-repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it, transfected with a control siRNA or a p53-specific siRNA, subjected to metabolic stress, and finally stained with DiOC6(3)/PI.


( g ) Effect of p53 and autophagy on the survival of metabolically stressed MEFs . WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays . Results in d - g are mean ± s . d . of 3 separate experiments ( * P 0 . 05 ) ; Co , control .

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(g) Effect of p53 and autophagy on the survival of metabolically stressed MEFs. WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays. Results in d-g are mean ± s.d. of 3 separate experiments (*P 0.05); Co, control.


( a , b ) PFT - α - mediated stimulation of autophagy in cytoplasts . GFP - LC3 - transfected HeLa cells were enucleated by density - gradient centrifugation after cytochalasin B treatment and cultured on polylysine - coated coverslips . Mixtures of cells and cytoplasts ( arrows , identified by the lack of Hoechst 33342 staining ) were then exposed to three different autophagy inducers : PFT - α , nutrient starvation and rapamycin . Results are means ± s . d . n = 3 experiments ) .

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(a, b) PFT-α-mediated stimulation of autophagy in cytoplasts. GFP-LC3-transfected HeLa cells were enucleated by density-gradient centrifugation after cytochalasin B treatment and cultured on polylysine-coated coverslips. Mixtures of cells and cytoplasts (arrows, identified by the lack of Hoechst 33342 staining) were then exposed to three different autophagy inducers: PFT-α, nutrient starvation and rapamycin. Results are means ± s.d. n = 3 experiments).


( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 cells . Representative micrographs of cells transfected with plasmids expressing WT , nuclear and cytoplasmic p53 are shown in

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(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells.Representative micrographs of cells transfected with plasmids expressing WT, nuclear and cytoplasmic p53 are shown in d


( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 cells . Quantification of GFP - LC3 puncta of p53 − / − HCT116 cells transiently transfected with p53 mutants is shown in e ( mean ± s . d . , n = 3 experiments , * P 0 . 05 )

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(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells. Quantification of GFP-LC3 puncta of p53−/− HCT116 cells transiently transfected with p53 mutants is shown in e (mean ± s.d., n = 3 experiments, *P 0.05)


( f ) Detection of the phosphorylation status of AMPKα , ACCα , TSC2 and p70S6K in p53 − / − HCT116 cells transiently expressing WT or mutant p53 ( n = 5 ) .

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(f) Detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in p53−/− HCT116 cells transiently expressing WT or mutant p53 (n = 5).


p53 inhibition induces autophagy by ER stress ( a - c ) WT HCT116 cells were transiently transfected with GFP - LC3 , treated with PFT - α for 2 , 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP - LC3 + puncta with the ER marker ERp57 ( a ) or a mitochondrial marker Complex III ( b ) . Note that cells with GFP - LC3 + puncta often contain scarce amounts of ERp57 ( white arrows ) after prolonged incubation with PFT - α . The percentage of colocalization ( means ± s . d . , n = 3 experiments with 50 images per experiment ) was determined by confocal microscopy ( c ) .

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p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c).


( d ) eIF2α phosphorylation in WT or p53 − / − HCT116 cells treated with PFT - α ( n = 5 ) .

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(d) eIF2α phosphorylation in WT or p53−/− HCT116 cells treated with PFT-α (n = 5).


( e , f ) WT HCT116 cells ( e , f ) or p53 − / − HCT116 cells ( f ) were transfected with GFP - LC3 , plus a control siRNA or the indicated combination of IRE1α - and / or p53 - specific siRNA , and treated with PFT - α , thapsigargin , tunicamycin or brefeldin A .

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(e, f) WT HCT116 cells (e, f) or p53−/− HCT116 cells (f) were transfected with GFP-LC3, plus a control siRNA or the indicated combination of IRE1α- and/or p53-specific siRNA, and treated with PFT-α, thapsigargin, tunicamycin or brefeldin A.


( g ) GFP - LC3 - transfected WT or ire1α − / − MEF were treated with PFT - α . Columns in e - g show means ± s . d . of 3 independent experiments ( * P 0 . 05 ) .

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(g) GFP-LC3-transfected WT or ire1α−/− MEF were treated with PFT-α. Columns in e-g show means ± s.d. of 3 independent experiments (*P 0.05).


Kinetics of p53 degradation ( a ) and induction of GFP - LC3 puncta ( b ) in WT HCT116 cells cultured in complete medium in the presence of rapamycin ( Rapa ) , tunicamycin ( Tn ) , lithium ( Li ) or in nutrient - free medium ( NF ) .

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Kinetics of p53 degradation (a) and induction of GFP-LC3 puncta (b) in WT HCT116 cells cultured in complete medium in the presence of rapamycin (Rapa), tunicamycin (Tn), lithium (Li) or in nutrient-free medium (NF).


( c ) WT HCT116 cells transfected with control or HDM2 - targeted siRNAs were cultured in the presence or absence of MG132 for 3 h , followed by the treatment with autophagy inducers for 6 h ( Tp , thapsigargin ; other abbreviations as in b ) . The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP - LC3 in vacuoles ( GFP - LC3vac ) was determined .

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(c) WT HCT116 cells transfected with control or HDM2-targeted siRNAs were cultured in the presence or absence of MG132 for 3 h, followed by the treatment with autophagy inducers for 6 h (Tp, thapsigargin; other abbreviations as in b). The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP-LC3 in vacuoles (GFP-LC3vac) was determined.


( d ) Effects of HDM2 inhibitors on p53 protein levels and autophagy . WT HCT116 were left untreated or treated with Nutlin - 3 , RITA and / or the indicated autophagy inducers , followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP - LC3 redistribution .

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(d) Effects of HDM2 inhibitors on p53 protein levels and autophagy. WT HCT116 were left untreated or treated with Nutlin-3, RITA and/or the indicated autophagy inducers, followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP-LC3 redistribution.


( e ) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53 − / − HCT116 cells .

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(e) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53−/− HCT116 cells.


( f ) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53 . Nutlin - 3 and RITA were added to p53 − / − HCT116 cells , followed by quantification of GFP - LC3 puncta .

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(f) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53. Nutlin-3 and RITA were added to p53−/− HCT116 cells, followed by quantification of GFP-LC3 puncta.


( g ) Effect of WT and ubiquitination - resistant p53 on autophagy . p53 − / − HCT116 cells were transfected with GFP - LC3 alone or in combination with different concentrations of pcDNA3 . 1 , WT p53 or p53 lacking the ubiquitination site at amino acids 13 - 19 ( p53ΔI ) , then treated with rapamycin , tunicamycin or thapsigargin . Thereafter , p53 expression and GFP - LC3 puncta were quantified .

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(g) Effect of WT and ubiquitination-resistant p53 on autophagy. p53−/− HCT116 cells were transfected with GFP-LC3 alone or in combination with different concentrations of pcDNA3.1, WT p53 or p53 lacking the ubiquitination site at amino acids 13-19 (p53ΔI), then treated with rapamycin, tunicamycin or thapsigargin. Thereafter, p53 expression and GFP-LC3 puncta were quantified.


( h ) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors . In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1 , p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin , tunicamycin or thapsigargin .

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(h) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors. In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1, p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin, tunicamycin or thapsigargin.


A . RT - PCR of CNPase ( left ) and MBP ( right ) mRNAs expression in MO3 . 13 precursors under control conditions and following 10 - 200 μM D - Asp exposure for 3 days . Graphs show quantification of ratio of CNPase , and MBP to L19 Data information : The values represent the means ± S . E . M . Level of significance was determined by using : in A , left panel , one - way ANOVA P < 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control ( n = 3 ) ; A , right panel , one - way ANOVA P < 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control ( n = 3

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A. RT-PCR of CNPase (left) and MBP (right) mRNAs expression in MO3.13 precursors under control conditions and following 10-200 μM D-Asp exposure for 3 days. Graphs show quantification of ratio of CNPase, and MBP to L19 Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, left panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control (n=3); A, right panel, one-way ANOVA P<0.0001 followed by Tukey's post hoc test, *P<0.05 versus control (n=3


B . Western blotting ( left ) and densitometric analysis ( right ) of MBP expression in absence or in presence of 10 - 200 μΜ D - Asp exposure for 5 days Data information : The values represent the means ± S . E . M . Level of significance was determined by using ) ; B , one - way ANOVA P = 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( n =

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B. Western blotting (left) and densitometric analysis (right) of MBP expression in absence or in presence of 10-200 μΜ D-Asp exposure for 5 days Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); B, one-way ANOVA P=0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control (n=3


C . Cell growth analysis of human MO3 . 13 oligodendrocytes in absence or in presence of 200 μM D - Asp for 1 - 5 days . The density of M03 . 13 oligodendrocytes was daily recorded through trypan blue dye exclusion . Mean of daily measurements were recorded . The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0 Data information : The values represent the means ± S . E . M . Level of significance was determined by using C , for each day , two - tailed Student ' s t test , * P < 0 . 05 versus control at 3 days ( n = 5 )

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C. Cell growth analysis of human MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 1-5 days. The density of M03.13 oligodendrocytes was daily recorded through trypan blue dye exclusion. Mean of daily measurements were recorded. The data of each experimental group were normalized to the density of cells plated at day 0 and expressed as percentage of ctrlday0 Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, for each day, two-tailed Student's t test, *P<0.05 versus control at 3 days (n=5)


D . FACS - based cell cycle distribution analysis after PI incorporation of MO3 . 13 oligodendrocytes in absence or in presence of 200 μM D - Asp for 3 days . Representative FACS plots of biological replicates are shown ( n = 5 independent experimental sessions )

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D. FACS-based cell cycle distribution analysis after PI incorporation of MO3.13 oligodendrocytes in absence or in presence of 200 μM D-Asp for 3 days. Representative FACS plots of biological replicates are shown (n=5 independent experimental sessions)


E . FACS - based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D - Asp for 1 day . Representative FACS plots of biological replicates are shown ( n = 3 independent experimental sessions )

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E. FACS-based cell cycle distribution analysis after PI incorporation of rat primary OPC in absence or in presence of 100 μM D-Asp for 1 day. Representative FACS plots of biological replicates are shown (n=3 independent experimental sessions)


F . Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin - 594 ( a - b and c - d , respectively ) in rat primary OPC cultured in absence or in presence of 100 μM D - Asp for 4 days . ( e ) , Single representative MBP + + cell . Scale bars : 50 μm in a - d ; 10 μm in e . ( f - g ) ; Quantitative analysis of Alexa - 594 - phalloidin - positive oligodendrocytes ( f ) or MAG + and MBP + + oligodendrocytes ( g ) scored in control and D - Asp exposed cells . Alexa - 594 - phalloidin - positive oligodendrocytes were scored in 4 categories , according to their morphological complexity . In each category , data were normalized on the total number of oligodendrocytes Data information : The values represent the means ± S . E . M . Level of significance was determined by usin F , for each category two - tailed Student ' s t test , * P < 0 . 05 versus control ( n = 3 )

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F. Confocal microscopic images displaying the coexpression of MAG or MBP with Phalloidin-594 (a-b and c-d, respectively) in rat primary OPC cultured in absence or in presence of 100 μM D-Asp for 4 days. (e), Single representative MBP++ cell. Scale bars: 50 μm in a-d; 10 μm in e. (f-g); Quantitative analysis of Alexa-594-phalloidin-positive oligodendrocytes (f) or MAG+ and MBP++ oligodendrocytes (g) scored in control and D-Asp exposed cells. Alexa-594-phalloidin-positive oligodendrocytes were scored in 4 categories, according to their morphological complexity. In each category, data were normalized on the total number of oligodendrocytes Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each category two-tailed Student's t test, *P<0.05 versus control (n=3)


B . Maximum intensity projection of z - stack confocal images displaying MBP ( red ) and NF200 ( green ) immunoreactivities in 7 DIV cerebellar organotypic slices cultured in absence ( b - d ) or in presence of 100 μM D - Asp ( f - h ) . Panels d and h display colocalized points ( white ) . Panels a and e show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence ( a ) or in presence of 100 μM D - Asp ( e ) . Scale bars in a and e : 200 μm ; in b - d and f - h : 50 μm

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B. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in 7 DIV cerebellar organotypic slices cultured in absence (b-d) or in presence of 100 μM D-Asp (f-h). Panels d and h display colocalized points (white). Panels a and e show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a and e: 200 μm; in b-d and f-h: 50 μm


C . Scatter plot histogram analysis of the myelination index in 7 DIV cerebellar slices cultured in absence or in presence of 100 μΜ D - Asp Data information : The values represent the means ± S . E . M . Level of significance was determined by using : in C , two - tailed Student ' s t test , * P < 0 . 05 versus control ( n = 4 animals , 3 - 4 slices per group

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C. Scatter plot histogram analysis of the myelination index in 7 DIV cerebellar slices cultured in absence or in presence of 100 μΜ D-Asp Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in C, two-tailed Student's t test, *P<0.05 versus control (n=4 animals, 3-4 slices per group


D . Western blotting analysis of MBP protein levels from homogenates of organotypic cerebellar slices culture in absence or in presence of 100 μΜ D - Asp . Data were normalized on the basis of β - actin and expressed as percentage of controls Data information : The values represent the means ± S . E . M . Level of significance was determined by using D , two - tailed Student ' s t test , * P < 0 . 05 versus controls ( n =

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D. Western blotting analysis of MBP protein levels from homogenates of organotypic cerebellar slices culture in absence or in presence of 100 μΜ D-Asp. Data were normalized on the basis of β-actin and expressed as percentage of controls Data information: The values represent the means ± S.E.M. Level of significance was determined by using D, two-tailed Student's t test, *P<0.05 versus controls (n=3


F . Maximum intensity projection of z - stack confocal images displaying MBP ( red ) and NF200 ( green ) immunoreactivities in control cerebellar organotypic slices ( b - d ) , and in cerebellar slices at 6 days after LPC exposure ( dpl ) in absence ( f - h ) or in presence 100 μM D - Asp ( j - l ) . Panels d , h , and l display colocalized points ( white ) . Panels a , e , and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence ( a ) or in presence of 100 μM D - Asp ( e ) . Scale bars in a , e , i : 200 μm ; in b - d , f - h , and j - l : 50

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F. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 6 days after LPC exposure (dpl) in absence (f-h) or in presence 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h, and j-l: 50 μm


G . Western blotting and quantitative analysis of MBP protein levels from homogenates of organotypic cerebellar slices at 6 dpl cultured in absence or in presence of 100 μΜ D - Asp Data information : The values represent the means ± S . E . M . Level of significance was determined by usin G , one - way ANOVA P = 0 . 003 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus LPC ( n =

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G. Western blotting and quantitative analysis of MBP protein levels from homogenates of organotypic cerebellar slices at 6 dpl cultured in absence or in presence of 100 μΜ D-Asp Data information: The values represent the means ± S.E.M. Level of significance was determined by usin G, one-way ANOVA P=0.003 followed by Bonferroni post hoc test, *P<0.05 versus control, ˄P< 0.05 versus LPC (n=3


H . Scatter plot histogram analysis of the remyelination index in cerebellar explants at 6dpl after LPC exposure cultured in absence or in presence of 100 μΜ D - Asp . Data were normalized to vehicle control Data information : The values represent the means ± S . E . M . Level of significance was determined by using ) ; H , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus controls , ˄ P < 0 . 05 versus LPC ( n = 4 animals , 3 - 4 slices per group )

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H. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 6dpl after LPC exposure cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control Data information: The values represent the means ± S.E.M. Level of significance was determined by using ); H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group)


I . Maximum intensity projection of z - stack confocal images displaying MBP ( red ) and NF200 ( green ) immunoreactivities in control cerebellar organotypic slices ( b - d ) , and in cerebellar slices at 10 dpl in absence ( f - h ) or in presence of 100 μM D - Asp ( j - l ) . Panels d , h , and l display colocalized points ( white ) . Panels a , e , and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence ( a ) or in presence of 100 μM D - Asp ( e ) . Scale bars in a , e , i : 200 μm ; in b - d , f - h , and j - l : 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Maximum intensity projection of z-stack confocal images displaying MBP (red) and NF200 (green) immunoreactivities in control cerebellar organotypic slices (b-d), and in cerebellar slices at 10 dpl in absence (f-h) or in presence of 100 μM D-Asp (j-l). Panels d, h, and l display colocalized points (white). Panels a, e, and i show representative low magnification images of 7 DIV cerebellar slices cultured cultures in absence (a) or in presence of 100 μM D-Asp (e). Scale bars in a, e ,i: 200 μm; in b-d, f-h,and j-l: 50 μm


J . Scatter plot histogram analysis of the remyelination index in cerebellar explants at 10 dpl cultured in absence or in presence of 100 μΜ D - Asp . Data were normalized to vehicle control Data information : The values represent the means ± S . E . M . Level of significance was determined by usin J , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus controls , ˄ P < 0 . 05 versus LPC ( n = 4 animals , 3 - 4 slices per group )

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

J. Scatter plot histogram analysis of the remyelination index in cerebellar explants at 10 dpl cultured in absence or in presence of 100 μΜ D-Asp. Data were normalized to vehicle control Data information: The values represent the means ± S.E.M. Level of significance was determined by usin J, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus controls, ˄P< 0.05 versus LPC (n=4 animals, 3-4 slices per group)


A . RT - PCR of CNPase mRNA expression in oligodendrocyte MO3 . 13 progenitors under control conditions and following 200 μM D - Asp exposure for 3 days , in absence or in presence of 10 μM MK - 801 ( left panel ) , or 30 nM YM - 244769 or 100 nM BED ( right panel ) . Graphs show quantification of ratio of CNPase to L19 Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : in A , left , one - way ANOVA P = 0 . 009 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 ) ; A , right , one - way ANOVA P = 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

A. RT-PCR of CNPase mRNA expression in oligodendrocyte MO3.13 progenitors under control conditions and following 200 μM D-Asp exposure for 3 days, in absence or in presence of 10 μM MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel). Graphs show quantification of ratio of CNPase to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, left, one-way ANOVA P=0.009 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); A, right, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3


B . RT - PCR of MBP mRNA expression in MO3 . 13 cells under control conditions and following D - Asp exposure for 3 days , in absence or in presence of 10 μΜ MK - 801 ( left panel ) , or 30 nM YM - 244769 or 100 nM BED ( right panel ) Graphs show quantification of ratio of MBP to L19 Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin B , left , one - way ANOVA P = 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 ) ; B , right , one - way ANOVA P = 0 . 0004 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n =

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

B. RT-PCR of MBP mRNA expression in MO3.13 cells under control conditions and following D-Asp exposure for 3 days, in absence or in presence of 10 μΜ MK-801 (left panel), or 30 nM YM-244769 or 100 nM BED (right panel) Graphs show quantification of ratio of MBP to L19 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin B, left, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3); B, right, one-way ANOVA P=0.0004 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3


C . ( a - j ) ; Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D - Asp for 3 days , in absence ( a - c ) or in presence of 10 μM MK - 801 ( d - f ) , or 30 nM YM - 244769 ( g - i ) or 100 nM BED ( j - l ) C . ( m - n ) ; Quantitative analysis of NG2 + and MAG + cells in rat primary OPC cultured in presence of D - Asp for 3 days , in absence or in presence of 10 μM MK - 801 , 30 nM YM - 244769 or 100 nM BED Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using C , m and C , n , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. (a-j); Confocal images displaying the expression of NG2 and MAG proteins in rat primary OPC cultured in presence of D-Asp for 3 days, in absence (a-c) or in presence of 10 μM MK-801 (d-f), or 30 nM YM-244769 (g-i) or 100 nM BED (j-l) C. (m-n); Quantitative analysis of NG2+ and MAG+ cells in rat primary OPC cultured in presence of D-Asp for 3 days, in absence or in presence of 10 μM MK-801, 30 nM YM-244769 or 100 nM BED Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C, m and C, n, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3)


D . RT - PCR of NCX3 mRNA expression under control conditions and following 10 - 200 μM D - Asp exposure or 100 nM PMA for 3 days Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i D , one - way ANOVA P = 0 . 0001 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 )

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

D. RT-PCR of NCX3 mRNA expression under control conditions and following 10-200 μM D-Asp exposure or 100 nM PMA for 3 days Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i D, one-way ANOVA P=0.0001 followed by Tukey's post hoc test, *P< 0.05 versus control, ˄P< 0.05 versus D-Asp (n=3)


E . RT - PCR of NCX3 mRNA expression following 200 μM D - Asp exposure , in absence or in presence of 10 μM MK - 801 Data information : The values represent the means ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using E , one - way ANOVA P = 0 . 0083 followed by Tukey ' s post hoc test , * P < 0 . 05 versus control , ˄ P < 0 . 05 versus D - Asp ( n = 3 ) , n . s , not

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

E. RT-PCR of NCX3 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801 Data information: The values represent the means ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using E, one-way ANOVA P=0.0083 followed by Tukey's post hoc test, *P<0.05 versus control, ˄P< 0.05 versus D-Asp (n=3), n.s, not significant


F . RT - PCR of NCX1 mRNA expression following 200 μM D - Asp exposure , in absence or in presence of 10 μM MK - 801 . Graphs show quantification of ratio of NCX1 and NCX3 to L19

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

F. RT-PCR of NCX1 mRNA expression following 200 μM D-Asp exposure, in absence or in presence of 10 μM MK-801. Graphs show quantification of ratio of NCX1 and NCX3 to L19


left panels . Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in MO3 . 13 cells ( A in absence or in presence of 10 μM MK - 801 or 150 μM APV right panels . Quantification of the oscillation index in MO3 . 13 cells ( A in absence or in presence of 10 μM MK - 801 or 150 μM APV Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 10 μM MK - 801 or 150 μM AP in M03 . 13 cells ( a MK - 801 and APV were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : in A and a , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 23 - 30 cells in each group , n = 3 biological replicate

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

left panels. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells (A in absence or in presence of 10 μM MK-801 or 150 μM APV right panels. Quantification of the oscillation index in MO3.13 cells (A in absence or in presence of 10 μM MK-801 or 150 μM APV Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 10 μM MK-801 or 150 μM AP in M03.13 cells (a MK-801 and APV were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A and a, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus control (basal value), ˄P<0.05 versus D-Asp. Data are reported as mean of 23-30 cells in each group, n=3 biological replicate


left panels . Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected i primary OP ( B ) in absence or in presence of 10 μM MK - 801 or 150 μM APV right panels . Quantification of the oscillation index i primary OPC ( B ) in absence or in presence of 10 μM MK - 801 or 150 μM APV Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 10 μM MK - 801 or 150 μM APV - i primary OPC ( b ) . MK - 801 and APV were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using B and b , one way - ANOVA , P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 20 - 23 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

left panels. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected i primary OP (B) in absence or in presence of 10 μM MK-801 or 150 μM APV right panels. Quantification of the oscillation index i primary OPC (B) in absence or in presence of 10 μM MK-801 or 150 μM APV Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 10 μM MK-801 or 150 μM APV -i primary OPC (b). MK-801 and APV were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using B and b, one way-ANOVA, P<0.001 followed by Bonferroni post hoc test, *P<0.05 versus control (basal value), ˄P< 0.05 versus D-Asp. Data are reported as mean of 20-23 cells in each group, n=3 biological replicate


left panels ; Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in MO3 . 13 cells ( C in absence or in presence of 30 nM YM - 244769 or 100 nM BED right panels ; Quantification of the oscillation index in MO3 . 13 cells ( C in absence or in presence of 30 nM YM - 244769 or 100 nM BED d , Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp measured as ∆ % of peak versus basal values in absence or in presence of 30 nM YM - 244769 or 100 nM BED in M03 . 13 cells ( c YM - 244769 or BED were preincubated 10 minutes before registration Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using C and c , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * p < 0 . 05 versus control ( basal value ) , ˄ P < 0 . 05 versus D - Asp . Data are reported as mean of 19 - 30 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

left panels; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells (C in absence or in presence of 30 nM YM-244769 or 100 nM BED right panels; Quantification of the oscillation index in MO3.13 cells (C in absence or in presence of 30 nM YM-244769 or 100 nM BED d, Quantification of the initial [Ca2+]i increase elicited by D-Asp measured as ∆% of peak versus basal values in absence or in presence of 30 nM YM-244769 or 100 nM BED in M03.13 cells (c YM-244769 or BED were preincubated 10 minutes before registration Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using C and c, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *p< 0.05 versus control (basal value), ˄ P< 0.05 versus D-Asp. Data are reported as mean of 19-30 cells in each group, n=3 biological replicate