C . Representative traces of the inward current ( upper panel ) and the corresponding quantification ( bottom panel ) observed in human oligodendrocyte MO3 . 13 progenitors after : ( I ) the first application of 10μM AMPA alone ; ( II ) the second application of 10μM AMPA alone ; ( III ) the third application of 10μM AMPA in the presence of 10μM DNQX ; ( IV ) the fourth application of 10μM AMPA alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin i one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus versus AMPA ( I ) . Data are reported as mean of at least 25 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces of the inward current (upper panel) and the corresponding quantification (bottom panel) observed in human oligodendrocyte MO3.13 progenitors after: (I) the first application of 10μM AMPA alone; (II) the second application of 10μM AMPA alone; (III) the third application of 10μM AMPA in the presence of 10μM DNQX; (IV) the fourth application of 10μM AMPA alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin i one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus versus AMPA(I). Data are reported as mean of at least 25 cells in each group, n=3 biological replicate


D . Representative traces of the inward current ( upper panel ) and the corresponding quantification ( bottom panel ) observed in human oligodendrocyte MO3 . 13 progenitors after : ( I ) the first application of 100μM D - Asp alone ; ( II ) the second application of 100μM D - Asp alone ; ( III ) the third application of 100μM D - Asp in presence of 10μM DNQX ; ( IV ) the fourth application of 100μM D - Asp alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus D - Asp ( I ) . Data are reported as mean of at least 25 cells for each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative traces of the inward current (upper panel) and the corresponding quantification (bottom panel) observed in human oligodendrocyte MO3.13 progenitors after: (I) the first application of 100μM D-Asp alone; (II) the second application of 100μM D-Asp alone; (III) the third application of 100μM D-Asp in presence of 10μM DNQX; (IV) the fourth application of 100μM D-Asp alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus D-Asp (I). Data are reported as mean of at least 25 cells for each group, n=3 biological replicates


E . Representative inward current traces elicited by 10μM AMPA in rat primary OPC in the absence or in the presence of 10μM DNQX

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

E. Representative inward current traces elicited by 10μM AMPA in rat primary OPC in the absence or in the presence of 10μM DNQX


F , Representative inward current traces elicited by 100μM D - Asp in rat primary OPC in the absence or in presence of 10μM DNQX or 10μM DNQX + 20μM PDC

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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR

F, Representative inward current traces elicited by 100μM D-Asp in rat primary OPC in the absence or in presence of 10μM DNQX or 10μM DNQX + 20μM PDC


G . Representative traces of the inward current ( upper panel ) and the corresponding quantification ( lower panel ) observed in rat primary OPC after : ( I ) the first application of 10μM AMPA alone ; ( II ) the second application of 10μM AMPA alone ; ( III ) the third application of 10μM AMPA in the presence of 10μM DNQX ; ( IV ) the fourth application of 10μM AMPA alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i G , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus versus AMPA ( I ) . Data are reported as mean of at least 25 cells in each group , n = 3 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Representative traces of the inward current (upper panel) and the corresponding quantification (lower panel) observed in rat primary OPC after: (I) the first application of 10μM AMPA alone; (II) the second application of 10μM AMPA alone; (III) the third application of 10μM AMPA in the presence of 10μM DNQX; (IV) the fourth application of 10μM AMPA alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i G, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus versus AMPA(I). Data are reported as mean of at least 25 cells in each group, n=3 biological replicate


H , Representative traces of the inward current ( upper panel ) and the corresponding quantification ( lower panel ) observed in rat primary OPC after : ( I ) the first application of 100μM D - Asp alone ; ( II ) the second application of 100μM D - Asp alone ; ( III ) the third application of 100μM D - Asp in presence of 10μM DNQX ; ( IV ) the fourth application of 100μM D - Asp in presence of 10μM DNQX + 10μM MK801 ; ( V ) the fifth application of 100μM D - Asp in presence of 10μM DNQX + 10μM MK - 801 + 20μM PDC ; ( VI ) the sixth application of 100μM D - Asp alone . Data are expressed as percentage of control Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : i H , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus D - Asp ( I ) . Data are reported as mean of at least 25 cells for each group , n = 3 biological

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

H, Representative traces of the inward current (upper panel) and the corresponding quantification (lower panel) observed in rat primary OPC after: (I) the first application of 100μM D-Asp alone; (II) the second application of 100μM D-Asp alone; (III) the third application of 100μM D-Asp in presence of 10μM DNQX; (IV) the fourth application of 100μM D-Asp in presence of 10μM DNQX + 10μM MK801; (V) the fifth application of 100μM D-Asp in presence of 10μM DNQX + 10μM MK-801+ 20μM PDC; (VI) the sixth application of 100μM D-Asp alone. Data are expressed as percentage of control Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: i H, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus D-Asp (I). Data are reported as mean of at least 25 cells for each group, n=3 biological replicates


B - C . Beam crossing latency during daily training ( left panel , averaged across 3 trials per day ) and average latency to cross the beam over 3 consecutive days ( right panel ) , recorded in control ( open circles ) , cuprizone - ( filled black circles ) and in cuprizone + D - Asp - treated mice ( filled red triangles ) during 3 weeks ( B ) , and 5 weeks ( C ) of cuprizone feeding The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by using left panels ( daily training ) , for each day ( d ) one - way ANOVA P = 0 . 0013 ( B , 1d ) , P = 0 . 00 followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOVA P = 0 . 0001 ( B ) , P < 0 . 0001 ( C ) , followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

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

B-C. Beam crossing latency during daily training (left panel, averaged across 3 trials per day) and average latency to cross the beam over 3 consecutive days (right panel), recorded in control (open circles), cuprizone- (filled black circles) and in cuprizone + D-Asp-treated mice (filled red triangles) during 3 weeks (B), and 5 weeks (C) of cuprizone feeding The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOVA P=0.0013 (B, 1d), P=0.00 followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOVA P=0.0001 (B), P<0.0001 (C ), followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


D . Beam crossing latency during daily training ( left panel , averaged across 3 trials per day ) and average latency to cross the beam over 3 consecutive days ( middle and right panels ) recorded after 2 . 5 weeks of remyelination in control ( open circles ) , vehicle - ( filled black circles ) and in D - Asp - treated mice . D - Asp ( I ) ( filled grey triangles ) refers to the group of mice which received D - Asp only for 2 . 5 weeks after cuprizone withdrawal ; D - Asp ( II ) ( filled red triangles ) refers to the group of mice which received D - Asp during the last week of cuprizone feeding and for 2 . 5 additional weeks after cuprizone withdrawal The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by using left panels ( daily training ) , for each day ( d ) one - way ANOV P = 0 . 0027 ( D , 3d ) , P = 0 . 0025 ( D , 2d followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOV P < 0 . 0001 ( D , for both middle and right panels followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

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

D. Beam crossing latency during daily training (left panel, averaged across 3 trials per day) and average latency to cross the beam over 3 consecutive days (middle and right panels) recorded after 2.5 weeks of remyelination in control (open circles), vehicle- (filled black circles) and in D-Asp- treated mice. D-Asp (I) (filled grey triangles) refers to the group of mice which received D-Asp only for 2.5 weeks after cuprizone withdrawal; D-Asp (II) (filled red triangles) refers to the group of mice which received D-Asp during the last week of cuprizone feeding and for 2.5 additional weeks after cuprizone withdrawal The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOV P=0.0027 (D, 3d), P=0.0025 (D, 2d followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV P<0.0001 (D, for both middle and right panels followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


E - F . Latency to fall during daily training in accelerating rotarod test ( left panel , averaged across 3 trials per day ) and average over 3 consecutive days ( right panel ) recorded in control ( open circles ) , cuprizone - ( filled black circles ) and D - Asp - treated ( filled red triangles ) mice at 3 weeks ( E ) , and 5 weeks ( F ) of cuprizone feeding The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by usin left panels ( daily training ) , for each day ( d ) one - way ANOV P < 0 . 0001 ( E , 3d ) , P = 0 . 0082 ( F , 3d followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOV ) , P = 0 . 0002 ( E ) , P < 0 . 0001 ( F ) followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

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

E-F. Latency to fall during daily training in accelerating rotarod test (left panel, averaged across 3 trials per day) and average over 3 consecutive days (right panel) recorded in control (open circles), cuprizone- (filled black circles) and D-Asp- treated (filled red triangles) mice at 3 weeks (E), and 5 weeks (F) of cuprizone feeding The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by usin left panels (daily training), for each day (d) one-way ANOV P<0.0001 (E, 3d), P=0.0082 (F, 3d followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV ), P=0.0002 (E), P<0.0001 (F) followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


G . Latency to fall during daily training in accelerating rotarod test ( left panel , averaged across 3 trials per day ) and average over 3 consecutive days ( right panel ) recorded after 2 . 5 weeks of remyelination ( REM ) in control , vehicle - and in D - Asp - treated mice . D - Asp ( II ) refers to the group of mice which received D - Asp during the last week of cuprizone feeding and 2 . 5 additional weeks after cuprizone withdrawal The values represent the means ± S . E . M ( n = 8 mice for each group ) Level of significance was determined by using left panels ( daily training ) , for each day ( d ) one - way ANOV P < 0 . 0001 ( G , 1d ) , P = 0 . 0044 ( G , 2d ) , P = 0 . 0038 ( G , 3d ) , followed by Bonferroni post hoc test , # P < 0 . 05 versus cpz at day 1 , § P < 0 . 05 versus cpz at day 2 , * P < 0 . 05 versus cpz at day 3 right panels ( average ) : one - way ANOV P < 0 . 0001 ( G ) , followed by Bonferroni post hoc test * P < 0 . 05 versus control ; ^ P < 0 . 05 versus cpz or vehicle ( veh )

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

G. Latency to fall during daily training in accelerating rotarod test (left panel, averaged across 3 trials per day) and average over 3 consecutive days (right panel) recorded after 2.5 weeks of remyelination (REM) in control, vehicle- and in D-Asp- treated mice. D-Asp (II) refers to the group of mice which received D-Asp during the last week of cuprizone feeding and 2.5 additional weeks after cuprizone withdrawal The values represent the means ± S.E.M (n=8 mice for each group) Level of significance was determined by using left panels (daily training), for each day (d) one-way ANOV P<0.0001 (G, 1d), P=0.0044 (G, 2d), P=0.0038 (G, 3d), followed by Bonferroni post hoc test, #P<0.05 versus cpz at day 1, §P<0.05 versus cpz at day 2, *P<0.05 versus cpz at day 3 right panels (average): one-way ANOV P<0.0001 (G), followed by Bonferroni post hoc test *P<0.05 versus control; ^P<0.05 versus cpz or vehicle (veh)


A . Representative confocal double immunofluorescence images displaying MBP ( red ) and NF200 ( green ) distribution and their coexpression ( white ) in the middle corpus callosum of septostriatal sections of control mice ( a - c ) , and of mice fed with cuprizone for 5 weeks in the absence ( d - f ) or in the presence of D - Asp ( g - i ) . Scale bars in a - i : 20 μm . ( j ) , Densitometric analysis of MBP immunofluorescence signal and ( k ) quantification of MBP - NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice , and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D - Asp Data information The values represent the means ± S . E . M . Level of significance was determined by using in : A , j , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 4 mice for each group ) ; A , k , one - way ANOVA P = 0 . 0162 followed by Newman - Keuls post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 4 mice for each group

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

A. Representative confocal double immunofluorescence images displaying MBP (red) and NF200 (green) distribution and their coexpression (white) in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 20 μm. (j), Densitometric analysis of MBP immunofluorescence signal and (k) quantification of MBP-NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice, and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp Data information The values represent the means ± S.E.M. Level of significance was determined by using in: A, j, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); A, k, one-way ANOVA P=0.0162 followed by Newman-Keuls post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group


B . Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated - mice for 5 weeks in the absence or in the presence of D - Asp , respectively . Data were normalized on the basis of α - tubulin and expressed as percentage of controls Data information The values represent the means ± S . E . M . Level of significance was determined by using i B , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 3 mice for each

O O O O O O O O B-MEASURED_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 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 O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

B. Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively. Data were normalized on the basis of α-tubulin and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i B, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group


C . Representative confocal double immunofluorescence images displaying GFAP ( green ) and Iba1 ( red ) distribution in the middle corpus callosum of septostriatal sections of control mice ( a - c ) , and of mice fed with cuprizone for 5 weeks in the absence ( d - f ) or in the presence of D - Asp ( g - i ) . Scale bars in a - i : 50 μm . ( j - k ) Quantification of GFAP + and Iba1 + cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D - Asp . Data were normalized to the total cell number ( Hoechst signal ) and expressed as percentage of controls Data information The values represent the means ± S . E . M . Level of significance was determined by using i C , j - k , one - way ANOVA P < 0 . 0001 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 4 mice for each

O O O O O O O O B-MEASURED_VAR O O O O B-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 O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 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 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

C. Representative confocal double immunofluorescence images displaying GFAP (green) and Iba1 (red) distribution in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 50 μm. (j-k) Quantification of GFAP+ and Iba1+ cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp. Data were normalized to the total cell number (Hoechst signal) and expressed as percentage of controls Data information The values represent the means ± S.E.M. Level of significance was determined by using i C, j-k, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group


D . Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated - mice for 5 weeks in the absence or in the presence of D - Asp , respectively Data information The values represent the means ± S . E . M . Level of significance was determined by using i D middle panel , one - way ANOVA P = 0 . 0146 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 3 mice for each group ) ; D right panel , one - way ANOVA P = 0 . 0026 followed by Bonferroni post hoc test , * P < 0 . 05 versus control , ^ P < 0 . 05 versus cpz ( n = 3 mice for each group )

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D. Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively Data information The values represent the means ± S.E.M. Level of significance was determined by using i D middle panel, one-way ANOVA P=0.0146 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group); D right panel, one-way ANOVA P= 0.0026 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group)


A . Confocal double immunofluorescence images depicting Olig2 + ( a - e ) and Olig2 + - CC1 + ( f - j ) double labeled cells in the middle corpus callosum of control , cuprizone - treated mice at 5 weeks , in absence ( cpz ) or in presence of D - Asp ( cpz + D - Asp ) , and 2 . 5 weeks after cuprizone withdrawal , in absence ( vehicle ) or in presence of D - Asp ( D - Asp ) . ( k , left panel ) , Quantitative analysis of Olig2 + cells ( red ) and Olig2 + - CC1 + ( blue ) double labeled cells in the middle corpus callosum of control , cuprizone - treated mice , and cuprizone + D - Asp - treated mice at 5 weeks . ( k , right panel ) , Quantitative analysis of Olig2 + cells ( red ) and Olig2 + - CC1 + ( blue ) double labeled cells in the middle corpus callosum of control , vehicle , and D - Asp - treated mice 2 . 5 weeks after cuprizone withdrawal . Arrows point to Olig2 + - CC1 + cells . Arrowheads point to Olig2 + - CC1 + cells . Scale bar in a - j : 50 μm Data information : The values represent the means ± S . E . M . Level of significance was determined by using : in A , k left panel , one - way ANOVA P < 0 . 0001 followed by Newman - Keuls post hoc test , * P < 0 . 05 versus ctrl ; ^ P < 0 . 05 versus cpz ( n = 3 mice for each group ) . A , k right panel , one - way ANOVA P < 0 . 0001 followed by Newman - Keuls post hoc test , * P < 0 . 05 versus ctrl ; ^ P < 0 . 05 versus vehicle ( n = 3 mice for each group

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A. Confocal double immunofluorescence images depicting Olig2+ (a-e) and Olig2+-CC1+ (f-j) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice at 5 weeks, in absence (cpz) or in presence of D-Asp (cpz + D-Asp), and 2.5 weeks after cuprizone withdrawal, in absence (vehicle) or in presence of D-Asp (D-Asp). (k, left panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice, and cuprizone+D-Asp-treated mice at 5 weeks. (k, right panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, vehicle, and D-Asp-treated mice 2.5 weeks after cuprizone withdrawal. Arrows point to Olig2+-CC1+ cells. Arrowheads point to Olig2+-CC1+ cells. Scale bar in a-j: 50 μm Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, k left panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus cpz (n=3 mice for each group). A, k right panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus vehicle (n=3 mice for each group


B . Representative electron microscopy images of middle corpus callosum sections from control mice ( a ) , cuprizone treated - mice for 5 weeks in the absence ( b ) or in the presence of D - Asp ( c ) , and from mice 2 . 5 weeks after cuprizone withdrawal in the absence ( d , vehicle ) or in presence of D - Asp ( e ) . Scale bars in a - e : 1 μ

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B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μ


C . Percentage of myelinated axons in the middle corpus callosum of cuprizone ( blue ) and cuprizone + D - Asp - treated mice ( red ) for 5 weeks Data information : The values represent the means ± S . E . M . Level of significance was determined by using C , two - tailed Student ' s t test , * P < 0 . 05 versus cpz ( n = 4 mice for each

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C. Percentage of myelinated axons in the middle corpus callosum of cuprizone (blue) and cuprizone + D-Asp-treated mice (red) for 5 weeks Data information: The values represent the means ± S.E.M. Level of significance was determined by using C, two-tailed Student's t test, *P<0.05 versus cpz (n=4 mice for each group


D . Scatter plot analysis of myelin thickness , expressed as individual G - ratios against axon diameters , after 2 . 5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle ( blue circle ) or D - Asp - treated mice ( red triangle ) . Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters

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D. Scatter plot analysis of myelin thickness, expressed as individual G-ratios against axon diameters, after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue circle) or D-Asp-treated mice (red triangle). Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters


E . Histogram analysis displaying the G - ratio values of myelinated fibers across the range of axon diameters . Data represent the mean S . E . M . for at least 100 axons from each group Data information : The values represent the means ± S . E . M . Level of significance was determined by usin E , for each axon diameter group , Student ' s t test , * P < 0 . 05 versus vehicle ( n = 4 mice for each

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E. Histogram analysis displaying the G-ratio values of myelinated fibers across the range of axon diameters. Data represent the mean S.E.M. for at least 100 axons from each group Data information: The values represent the means ± S.E.M. Level of significance was determined by usin E, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group


F . Percentage of myelinated axons after 2 . 5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle ( blue ) or D - Asp - treated mice ( red ) . Abbreviations : DEM , demyelination ; REM , remyelination Data information : The values represent the means ± S . E . M . Level of significance was determined by usin F , for each axon diameter group , Student ' s t test , * P < 0 . 05 versus vehicle ( n = 4 mice for each group )

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F. Percentage of myelinated axons after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue) or D-Asp- treated mice (red). Abbreviations: DEM, demyelination; REM, remyelination Data information: The values represent the means ± S.E.M. Level of significance was determined by usin F, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group)


C , D Two types of TNTs were detected in H1299 ( C ) and A549 ( D ) cells , Type 1 , containing F - actin with little or no detectable microtubules ( white box ) and Type2 , containing both microtubules and F - actin ( yellow box ) . Anti - β - tubulin antibody ( green ) and phalloidin ( red ) were used for fluorescent staining . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) . Boxed areas are enlarged and shown in panels on the right .

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C, D Two types of TNTs were detected in H1299 (C) and A549 (D) cells, Type 1, containing F-actin with little or no detectable microtubules (white box) and Type2, containing both microtubules and F-actin (yellow box). Anti-β-tubulin antibody (green) and phalloidin (red) were used for fluorescent staining. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue). Boxed areas are enlarged and shown in panels on the right.


C , D TNT formation ( marked by black triangles and white arrowheads ) was imaged in control and shM2 H1299 ( C ) and shM2 A549 ( D ) cells by confocal microscopy . Scale bar : 60 μm . Boxed areas are enlarged and shown at the bottom of the corresponding images . E , F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 ( E ) and shM2 A549 ( F ) cells . 13 images per group ( E ) and 10 per group ( F ) were captured for quantification . Three independent experiments were conducted . G Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

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C, D TNT formation (marked by black triangles and white arrowheads) was imaged in control and shM2 H1299 (C) and shM2 A549 (D) cells by confocal microscopy. Scale bar: 60 μm. Boxed areas are enlarged and shown at the bottom of the corresponding images. E, F Quantification of the percentage of cells forming TNTs in control and shM2 H1299 (E) and shM2 A549 (F) cells. 13 images per group (E) and 10 per group (F) were captured for quantification. Three independent experiments were conducted. G Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


H , I H1299 cells stably expressing the Ctr or GFP - M2 were cultured in the presence of 20 μM 2 - DG for 24 h to induce TNT formation . TNTs were imaged by confocal microscopy ( A ) and the percentage of cells forming TNTs was quantified ( B ) . n = 21 , 20 and 19 images were captured from Ctr , GFP - M2 # 1 and GFP - M2 # 2 , respectively . Three independent experiments were performed . Scale bar : 60 μm . Data information : data were analyzed by one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * * * P < 0 . 001 . Three independent experiments were

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H,I H1299 cells stably expressing the Ctr or GFP-M2 were cultured in the presence of 20 μM 2-DG for 24 h to induce TNT formation. TNTs were imaged by confocal microscopy (A) and the percentage of cells forming TNTs was quantified (B). n=21, 20 and 19 images were captured from Ctr, GFP-M2#1 and GFP-M2#2, respectively. Three independent experiments were performed. Scale bar: 60 μm. Data information: data were analyzed by one-way ANOVA with Bonferroni post-test and shown as mean ±SEM; *** P < 0.001. Three independent experiments were performed


A Time - lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito - GFP were connected by TNTs containing mitochondria . Cells were seeded in collagen - coated dishes and cultured for 24 h before time - lapse life cell images were acquired by confocal microscopy ( also see Movies EV1 and EV2 ) . Fluorescent images ( with bright field ) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells . Scale bar : 20 μm . The moving mitochondria are marked by the white arrows . Boxed areas ( shCtr in yellow dotted boxes ; shM2 in red dotted boxes ) are enlarged and shown in the middle panel . B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . The white arrows marked TNTs , whereas the white triangles marked mitochondria inside TNTs . Scale bar : 25 μm . C Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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A Time-lapse microscopic images showing that H1299 shCtr or shM2 cells stably expressing Mito-GFP were connected by TNTs containing mitochondria. Cells were seeded in collagen-coated dishes and cultured for 24 h before time-lapse life cell images were acquired by confocal microscopy (also see Movies EV1 and EV2). Fluorescent images (with bright field) at different time points show the mitochondria moving along the TNT and entering in one of the two connected cells. Scale bar: 20 μm. The moving mitochondria are marked by the white arrows. Boxed areas (shCtr in yellow dotted boxes; shM2 in red dotted boxes) are enlarged and shown in the middle panel. B Confocal microscopic images of control and shM2 H1299 cells following immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). The white arrows marked TNTs, whereas the white triangles marked mitochondria inside TNTs. Scale bar: 25 μm. C Data information: nuclei were stained with Hoechst 33342 (blue).


C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells . The percentage of TNTs containing mitochondria was determined . n = 168 , 187 and 192 for shCtr , shM2 # 1 and shM2 # 2 groups , respectively ; three independent experiments . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

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C Quantification of TNTs containing mitochondria in shCtr and shM2 H1299 cells. The percentage of TNTs containing mitochondria was determined. n=168, 187 and 192 for shCtr, shM2#1 and shM2#2 groups, respectively; three independent experiments. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


E Representative flow cytometry plot of shCtr and shM2 cells following co - culture for 2 days . Experiments were conducted under three conditions : co - cultured in direct contact in normal glucose medium or in galactose medium or co - cultured through a filter .

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E Representative flow cytometry plot of shCtr and shM2 cells following co-culture for 2 days. Experiments were conducted under three conditions: co-cultured in direct contact in normal glucose medium or in galactose medium or co-cultured through a filter.


F The percentage of mCherry - expressing recipient cells containing Mito - GFP was quantified . Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or fewer recipient cells contained Mito - GFP , and this group is not included in the graph to avoid an overcrowded figure . Three independent experiments were conducted . Data information : data were analyzed using FlowJo analysis software . When co - cultured through the filter in transwells , only 0 . 01 % or

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F The percentage of mCherry-expressing recipient cells containing Mito-GFP was quantified. Twenty thousand events were acquired for each condition and data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or fewer recipient cells contained Mito-GFP, and this group is not included in the graph to avoid an overcrowded figure. Three independent experiments were conducted. Data information: data were analyzed using FlowJo analysis software. When co-cultured through the filter in transwells, only 0.01% or f


A Down - regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton . Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti - Tom20 antibody ( green ) and staining using phalloidin ( red ) . Scale bar : 25 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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A Down-regulating MICAL2PV promotes mitochondrial infiltration into the cortical cytoskeleton. Control and shM2 H1299 cells were subjected to immunofluorescence staining with anti-Tom20 antibody (green) and staining using phalloidin (red). Scale bar: 25 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


C Down - regulating MICAL2PV enhances the mitochondrial localization of Miro1 , Miro2 and KIF5B . Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting . The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β - tubulin protein .

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C Down-regulating MICAL2PV enhances the mitochondrial localization of Miro1, Miro2 and KIF5B. Mitochondria were purified from control and shM2 H1299 cells and subjected to Western blotting. The purity of the mitochondrial fraction was confirmed by detection of mitochondrial ATP5B and the absence of the cytoplasmic β-tubulin protein.


E MICAL2PV interacts with the C - terminal domain of Miro2 containing the transmembrane region . H1299 cells were co - transfected with plasmids encoding Flag - MICAL2PV and Myc - tagged Miro2 mutants as indicated . Following transfection , cells were lysed for immunoprecipitation using the anti - Flag antibody and Western blotting . Please note the Myc - Miro2 ( 410 - 618 ) protein band in lane 4 migrated as a faint band , slightly faster than the IgG light chain .

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E MICAL2PV interacts with the C-terminal domain of Miro2 containing the transmembrane region. H1299 cells were co-transfected with plasmids encoding Flag-MICAL2PV and Myc-tagged Miro2 mutants as indicated. Following transfection, cells were lysed for immunoprecipitation using the anti-Flag antibody and Western blotting. Please note the Myc-Miro2 (410-618) protein band in lane 4 migrated as a faint band, slightly faster than the IgG light chain.


F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration . H1299 cells were co - transfected with Myc - tagged Miro2 and GFP - tagged MCAL2PV ( GFP - M2 ) or control vector . Following transfection , cells were fixed and stained with anti - Tom20 ( red ) and anti - Myc antibodies ( magenta ) . Confocal microscopic images were taken under the same objective and the same microscope settings . In cells co - expressing the GFP vector and Myc - Miro2 ( upper panels ) , most of Miro2 signals ( magenta ) are co - colocalized with mitochondria ( red Tom20 signals ) . However , a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co - expressing GFP - M2 and Myc - Miro2 ( marked by the yellow arrowheads ) . Mitochondria in cells expressing GFP - M2 show the perinuclear distribution pattern , whereas mitochondria in cells transfected by Myc - Miro2 without GFP - M2 ( marked by the yellow stars ) exhibit an infiltration pattern into the cortical cytoplasm . Scale bar : 20 μm .

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F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration. H1299 cells were co-transfected with Myc-tagged Miro2 and GFP-tagged MCAL2PV (GFP-M2) or control vector. Following transfection, cells were fixed and stained with anti-Tom20 (red) and anti-Myc antibodies (magenta). Confocal microscopic images were taken under the same objective and the same microscope settings. In cells co-expressing the GFP vector and Myc-Miro2 (upper panels), most of Miro2 signals (magenta) are co-colocalized with mitochondria (red Tom20 signals). However, a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co-expressing GFP-M2 and Myc-Miro2 (marked by the yellow arrowheads). Mitochondria in cells expressing GFP-M2 show the perinuclear distribution pattern, whereas mitochondria in cells transfected by Myc-Miro2 without GFP-M2 (marked by the yellow stars) exhibit an infiltration pattern into the cortical cytoplasm. Scale bar: 20 μm.


A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . Following transfection , cells were stained with anti - Tom20 antibody ( green ) and phalloidin ( red ) . Confocal microscopic images are shown . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


F Down - regulating Miro2 inhibits mitochondrial transport in shM2 cells . Mito - GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2 . These cells were then co - cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito - GFP was quantified . Twenty thousand events were analyzed for each condition Data information : Data were analyzed using two - way ANOVA with Bonferroni post - test and presented as mean ± SEM ; * P < 0 . 05 , * * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant . Three independent experiments were conducted .

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F Down-regulating Miro2 inhibits mitochondrial transport in shM2 cells. Mito-GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. These cells were then co-cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito-GFP was quantified. Twenty thousand events were analyzed for each condition Data information: Data were analyzed using two-way ANOVA with Bonferroni post-test and presented as mean ± SEM; * P < 0.05, *** P < 0.01, *** P < 0.001, ns, not significant. Three independent experiments were conducted.


A Stable H1299 cells ( shCtr or shM2 ) were treated by 5 - FU ( 50 μM , 24 h ) and then cultured alone or co - cultured with non - drug treated vehicle - treated cells stably expressing mito - mCherry in the absence or presence of Cyto B ( 500 nM , 24 h ) . The percentage of dead cells in each group was quantified . Three independent experiments were conducted . Data information : data were analyzed using two - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

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A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


D Confocal microscopic images of TNTs in each group , white arrowheads and yellow triangle in enlarged views ( zoom - in panels , zoom ) marked the TNTs formed between non - drug treated ( mCherry positive ) and drug - treated cells . Scale bar : 25 μm .

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D Confocal microscopic images of TNTs in each group, white arrowheads and yellow triangle in enlarged views (zoom-in panels, zoom) marked the TNTs formed between non-drug treated (mCherry positive) and drug-treated cells. Scale bar: 25 μm.


D MICAL2PV mutants containing the MO domain are active in depolymerizing F - actin . Following transfection of plasmids expressing different HA - MICAL2PV mutants ( # 1 , # 2 , or # 4 ) into shM2 cells , confocal microscopy was performed following staining with phalloidin ( red ) and anti - HA antibody ( green ) . F - actin signals were reduced in cells expressing different mutants ( HA - positive green cells marked by dashed lines ) . Scale bar : 20 μm . Data information : nuclei were stained with Hoechst 33342 ( blue ) .

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D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green). F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).


G , H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation . Stable shM2 H1299 ( G ) or A549 ( H ) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site ( MOmut : amino acid residues 93 - 98 GxGxxG mutated to WxWxxW ) or the control vector ( Ctr ) . Following transfection , Western blotting and quantification of TNT formation were carried out as in panel E . 10 images were captured for quantification in each group . Three independent experiments were performed . Data information : Data were analyzed using a one - way ANOVA with Bonferroni post - test and shown as mean ± SEM ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant .

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G, H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation. Stable shM2 H1299 (G) or A549 (H) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site (MOmut: amino acid residues 93-98 GxGxxG mutated to WxWxxW) or the control vector (Ctr). Following transfection, Western blotting and quantification of TNT formation were carried out as in panel E. 10 images were captured for quantification in each group. Three independent experiments were performed. Data information: Data were analyzed using a one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.


( A ) HEK293 cells were transfected with DAPK ΔCaM or with a control vector ( pcDNA3 - luciferase , LUC ) , together with shRNAs targeting beclin 1 or HcRed , and with GFP - LC3 plasmid . After 72 h , cells were counted and lysates were prepared . ( A ) The percentage of cells with punctate GFP - LC3 fluorescence per total GFP - LC3 - positive cells was quantified . Data presented are the mean ± s . d . from a triplicate of 100 transfected cells . The asterisks denote a significance level of P = 0 . 001 .

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(A) HEK293 cells were transfected with DAPK ΔCaM or with a control vector (pcDNA3-luciferase, LUC), together with shRNAs targeting beclin 1 or HcRed, and with GFP-LC3 plasmid. After 72 h, cells were counted and lysates were prepared. (A) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3-positive cells was quantified. Data presented are the mean±s.d. from a triplicate of 100 transfected cells. The asterisks denote a significance level of P=0.001.


( B ) Western blot analysis was performed using the indicated antibodies .

(B) Western blot analysis was performed using the indicated antibodies.


( C ) Representative GFP - LC3 staining of cells transfected with the control shRNA ( HcRed ) together with pcDNA3 ‐ luciferase or DAPKΔCaM . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GFP , green fluorescent protein ; HEK , human embryonic kidney ; shRNA , short hairpin RNA .

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(C) Representative GFP-LC3 staining of cells transfected with the control shRNA (HcRed) together with pcDNA3‐luciferase or DAPKΔCaM. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; shRNA, short hairpin RNA.


( A ) Flag ‐ tagged DAPK ( 100 ng ) was incubated with GST ( 900 ng ) or GST - beclin ‐ 1 ( 750 ng ) in the presence of Ca2 + , calmodulin and [ γ ‐ 33P ] ATP for 30 min or 60 min . Phosphorylated proteins were visualized by X ‐ ray film exposure , and GST / GST - beclin ‐ 1 levels were visualized by Ponceau S staining . The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples .

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(A) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST-beclin‐1 (750 ng) in the presence of Ca2+, calmodulin and [γ‐33P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST-beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples.


( B ) Flag ‐ tagged DAPK ( 60 ng ) was incubated with Flag ‐ tagged beclin 1 ( 250 ng ) , which was purified from HEK293T cells , and a kinase assay was performed for 60 min . Where indicated ( + LiCl ) , beclin ‐ 1 ‐ bound beads were first washed stringently in 0 . 5 M LiCl and 0 . 5 M KCl . Phosphorylated proteins were visualized by X ‐ ray film exposure , and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies . DAPK , death ‐ associated protein kinase ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney .

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(B) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney.


( Aa ) Protein extracts from COS7 cells , HEK293T cells , or no extracts were added to bacterially produced GST or GST - beclin ‐ 1 . The pulled ‐ down proteins , as well as the total cell extracts , were blotted with the indicated antibodies . ( Ab ) Ponceau S staining of GST and GST - beclin ‐ 1 to which the extracts were added .

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(Aa) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST-beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (Ab) Ponceau S staining of GST and GST-beclin‐1 to which the extracts were added.


( B ) HEK293 cells were co ‐ transfected with Flag ‐ tagged beclin 1 or with Flag ‐ tagged beclin 1 lacking the Bcl ‐ 2 ‐ binding domain ( ΔBD ) together with Bcl ‐ XL and HA ‐ tagged DAPK . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted with DAPK , Bcl ‐ XL and beclin 1 antibodies .

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(B) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐XL and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐XL and beclin 1 antibodies.


( C ) HEK293 cells were transfected with Flag - beclin ‐ 1 or Flag - GFP , and the Flag ‐ tagged proteins were immunoprecipitated using Flag antibodies , and eluted with an excess of Flag peptides . The blot was reacted with DAPK antibodies or with Flag antibodies at different time

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(C) HEK293 cells were transfected with Flag-beclin‐1 or Flag-GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures


( A ) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30 ° C with increasing concentrations ( 5 - 10 nmol ) of a peptide corresponding to the BH3 domain of beclin 1 ( aa 108 - 127 ) and with the same peptide in which Thr 119 was substituted by alanine . An in vitro kinase assay was performed , and the reactions were applied to Whatman filters . Total levels of TCA ‐ insoluble counts were measured and plotted against substrate concentration . Data are the mean ± s . d . of three experiments .

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(A) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5-10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108-127) and with the same peptide in which Thr 119 was substituted by alanine. An in vitro kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments.


( C ) Flag ‐ tagged DAPK ( 60 ng ) was incubated with GST - WT beclin 1 or with GST - T119A beclin 1 ( 1000 ng ) in the presence of Ca2 + , calmodulin and ATP for 30 min . GST ‐ beclin 1 levels were visualized by Ponceau S staining , and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody ( Western blot ) .

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(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).


( D ) HEK293 cells were co ‐ transfected with Flag ‐ tagged beclin 1 with or without ΔCaM DAPK . After 24 h , beclin 1 was immunoprecipitated from cells , using Flag antibodies , and the immunoprecipitates were reacted with phosphoThr 119 antibodies . The Ponceau staining shows equal amounts of immunoprecipitated beclin 1 . The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney ; TCA , trichloro ‐ acetic acid .

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(D) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.


( A ) HEK293 cells were transfected with Flag ‐ tagged beclin 1 and Bcl ‐ XL with or without haemagglutinin ‐ tagged DAPK . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies .

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(A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.


( B ) HEK293 cells were co ‐ transfected with Flag ‐ tagged T119A or T119E beclin 1 mutants and Bcl ‐ XL . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies .

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(B) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐XL. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.


( C ) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP - LC3 plasmid . After 24 h , cells were counted and lysates were prepared . ( a ) Representative GFP - LC3 staining . ( b ) The percentage of cells with punctate GFP - LC3 fluorescence per total GFP - LC3 ‐ positive cells was quantified . Data presented are the mean ± s . d . from a triplicate experiment with 100 transfected cells . The asterisks denote significance level : * P = 0 . 01 ; * * P = 0 . 005 . ( c ) Western blot analysis was performed using the indicated antibodies .

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(C) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP-LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (a) Representative GFP-LC3 staining. (b) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: *P=0.01; **P=0.005. (c) Western blot analysis was performed using the indicated antibodies.


( D ) HEK293 cells were transfected with Bcl ‐ XL , Flag ‐ tagged beclin 1 ( WT ) or Flag ‐ tagged T119A beclin 1 mutant with or without haemagglutinin ‐ tagged activated DAPK ( ΔCaM ) . Beclin 1 was immunoprecipitated using Flag antibodies , and the co ‐ immunoprecipitated proteins , as well as the total cell extracts , were blotted using the indicated antibodies . Levels of Bcl ‐ XL were quantified using NIH image software , and the ratio between immunoprecipitated and expressed Bcl ‐ XL was calculated .

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(D) HEK293 cells were transfected with Bcl‐XL, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐XL were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐XL was calculated.


( E ) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl ‐ XL , Flag ‐ tagged beclin 1 and ΔCaM or pcDNA3 ‐ luciferase as a control . The images in ( c ) and ( d ) were taken at higher magnifications of the ΔCaM treatment ( see the scale bars ) . ' AV ' indicates autophagic vacuoles . CaM , calmodulin ; DAPK , death ‐ associated protein kinase ; GFP , green fluorescent protein ; GST , glutathione S ‐ transferase ; HEK , human embryonic kidney ; NIH , National Institutes of Health .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O

(E) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐XL, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (c) and (d) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). 'AV' indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.


( A ) Expression levels of NAT4 analyzed by qRT - PCR using total RNA extracted from a wild - type ( BY4741 ) and Pste5 - NAT4 strain grown in 2 % ( NCR ) , or 0 . 1 % glucose ( CR ) . NAT4 levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM ( Standard Error of the Mean ) of 3 independent experiments . * * p ≤ 0 . 01 ; calculated by unpaired two - tailed Student ' s t - test .

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

(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student's t-test.


( B ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown in the same conditions as in ( A ) . Chromatin was immunoprecipitated using an antibody against N - acH4 and analyzed by qRT - PCR using primers for the different rDNA regions . Enrichment was normalized to Histone H4 levels . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 compared values to wild - type NCR conditions ; arrowhead p ≤ 0 . 05 compared values to wild - type CR

O O O O O O O O O O O O B-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 B-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 O O O O O O O O O O O O O O O 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) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown in the same conditions as in (A). Chromatin was immunoprecipitated using an antibody against N-acH4 and analyzed by qRT-PCR using primers for the different rDNA regions. Enrichment was normalized to Histone H4 levels. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01 compared values to wild-type NCR conditions; arrowhead p ≤ 0.05 compared values to wild-type CR conditions


( C ) Replicative lifespan ( RLS ) for BY4741 wild - type and nat4Δ strains . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Replicative lifespan (RLS) for BY4741 wild-type and nat4Δ strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( D ) Mean RLS for wild - type and nat4 ∆ strains using different genetic backgrounds . Values outside of parenthesis indicate mean lifespan , while values in parenthesis indicate the number of cells examined .

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 O O O O O O O O O O O O O

(D) Mean RLS for wild-type and nat4∆ strains using different genetic backgrounds. Values outside of parenthesis indicate mean lifespan, while values in parenthesis indicate the number of cells examined.


( E ) Mean RLS for BY4741 wild - type and nat4Δ strains under NCR and CR conditions .

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(E) Mean RLS for BY4741 wild-type and nat4Δ strains under NCR and CR conditions.


( F ) Mean RLS for BY4741 wild - type and Pste5 - NAT4 strains under NCR and CR conditions .

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

(F) Mean RLS for BY4741 wild-type and Pste5-NAT4 strains under NCR and CR conditions.


( B ) ChIP experiments were performed in SF10 wild - type , nat4 ∆ , NET1 - TAP and NET1 - TAP nat4 ∆ strains using an anti - IgG antibody . The enrichment from the antibody was normalized to input . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * * p ≤ 0 . 01 compared nat4 ∆ to WT values .

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

(B) ChIP experiments were performed in SF10 wild-type, nat4∆, NET1-TAP and NET1-TAP nat4∆ strains using an anti-IgG antibody. The enrichment from the antibody was normalized to input. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared nat4∆ to WT values.


( C ) Quantitative real - time PCR analysis of rDNA copy number for young and old cells in BY4741 wild - type , nat4Δ and fob1Δ strains . Error bars , SEM of 3 independent experiments . Statistical significance was determined by unpaired two - tailed Student ' s t test : * * p ≤ 0 . 01 compared to corresponding WT values .

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 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

(C) Quantitative real-time PCR analysis of rDNA copy number for young and old cells in BY4741 wild-type, nat4Δ and fob1Δ strains. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared to corresponding WT values.


( D ) Polysome profiling analysis performed in BY4741 wild - type and nat4Δ strains . The plots are representative of two independent experiments .

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(D) Polysome profiling analysis performed in BY4741 wild-type and nat4Δ strains. The plots are representative of two independent experiments.


( C ) Gene expression analysis of selected upregulated stress - response genes ( according to results in 3A ) in BY4741 wild - type and nat4 ∆ strains grown under NCR and CR conditions . Expression levels were normalized to TAF10 , whose expression remains unchanged . Error bars , SEM of 3 independent experiments . p - values were obtained comparing results to wild - type under NCR conditions , and calculated by unpaired two - tailed Student ' s t - test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene expression analysis of selected upregulated stress-response genes (according to results in 3A) in BY4741 wild-type and nat4∆ strains grown under NCR and CR conditions. Expression levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained comparing results to wild-type under NCR conditions, and calculated by unpaired two-tailed Student's t-test: * p ≤ 0.05; ** p ≤ 0.01.


( D ) Gene expression analysis showing fold induction levels ( Log2 ) of selected genes comparing BY4741 wild - type and STE5p - NAT4 strains under NCR ( 2 % glucose ) and CR ( 0 . 1 % glucose ) conditions . Expression levels were normalized to TAF10 , whose expression remains unchanged .

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 B-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

(D) Gene expression analysis showing fold induction levels (Log2) of selected genes comparing BY4741 wild-type and STE5p-NAT4 strains under NCR (2% glucose) and CR (0.1% glucose) conditions. Expression levels were normalized to TAF10, whose expression remains unchanged.


( A ) RLS analysis for BY4741 wild - type and nat4 - E186A strains . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) RLS analysis for BY4741 wild-type and nat4-E186A strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: *** p ≤ 0.001; **** p ≤ 0.0001.


( B ) Mean RLS for YSC5106 wild - type , nat4 ∆ and H4S1D strains .

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(B) Mean RLS for YSC5106 wild-type, nat4∆ and H4S1D strains.


( C ) Gene expression analysis of the indicated stress - induced genes in YSC5106 wild - type , nat4 ∆ and H4S1D strains . Expression levels were normalized to RPP0 whose expression remains unchanged . Error bars , SEM of 3 independent experiments . p - values were obtained by comparing nat4 ∆ and H4S1D to wild - type values and calculated by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

(C) Gene expression analysis of the indicated stress-induced genes in YSC5106 wild-type, nat4∆ and H4S1D strains. Expression levels were normalized to RPP0 whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained by comparing nat4∆ and H4S1D to wild-type values and calculated by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01.


( D ) Gene expression analysis of the indicated stress - induced genes in BY4742 wild - type , nat4 ∆ and nat4 ∆ [ hNAA40 ] strains . Expression levels were normalized to ACT1 , whose expression remains unchanged .

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

(D) Gene expression analysis of the indicated stress-induced genes in BY4742 wild-type, nat4∆ and nat4∆[hNAA40] strains. Expression levels were normalized to ACT1, whose expression remains unchanged.


( A ) RLS analysis for YSC5106 wild - type , nat4 ∆ , H4R3K and double mutant strains . Values in parenthesis indicate mean lifespan . Statistical significance was determined by one - way ANOVA test : * * * * p ≤ 0 . 0001 .

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

(A) RLS analysis for YSC5106 wild-type, nat4∆, H4R3K and double mutant strains. Values in parenthesis indicate mean lifespan. Statistical significance was determined by one-way ANOVA test: **** p ≤ 0.0001.


( B ) Gene expression analysis of the indicated stress - induced genes in YSC5106 WT , nat4 ∆ , H4R3K and double mutant strains . Expression levels were normalized to RPP0 , whose expression remains unchanged .

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

(B) Gene expression analysis of the indicated stress-induced genes in YSC5106 WT, nat4∆, H4R3K and double mutant strains. Expression levels were normalized to RPP0, whose expression remains unchanged.


( A ) Gene expression analysis of the indicated stress - induced genes in young and old cells isolated from YSC5106 WT , nat4 ∆ and H4R3K nat4 ∆ strains . Expression levels were normalized to ACT1 , whose expression remains unchanged . Error bars , SEM of 3 independent experiments . P - values were calculated by unpaired two - tailed Student ' s t test : * p ≤ 0 . 05 compared to young wild - type ; arrowhead p ≤ 0 . 05 compared to old wild - type .

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

(A) Gene expression analysis of the indicated stress-induced genes in young and old cells isolated from YSC5106 WT, nat4∆ and H4R3K nat4∆ strains. Expression levels were normalized to ACT1, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. P-values were calculated by unpaired two-tailed Student's t test: * p ≤ 0.05 compared to young wild-type; arrowhead p ≤ 0.05 compared to old wild-type.


( B ) RLS analysis for WT , nat4 ∆ , pnc1 ∆ and double mutant strains in BY4741 genetic background . Values in parenthesis ( here and hereafter ) indicate mean lifespan . Statistical significance ( here and hereafter ) was determined by one - way ANOVA test : * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ns = non - significant .

O O O O O O O O B-CONTROLLED_VAR 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) RLS analysis for WT, nat4∆, pnc1∆ and double mutant strains in BY4741 genetic background. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. ns = non-significant.


( C ) Mean RLS in BY4741 wild - type , msn2 ∆ msn4 ∆ double and msn2 ∆ msn4 ∆ nat4 ∆ triple mutant strains .

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

(C) Mean RLS in BY4741 wild-type, msn2∆ msn4∆ double and msn2∆ msn4∆ nat4∆ triple mutant strains.


( D ) Mean RLS in BY4741 wild - type , nat4 ∆ , fob1 ∆ sir2 ∆ double and fob1 ∆ sir2 ∆ nat4 ∆ triple mutant strains .

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(D) Mean RLS in BY4741 wild-type, nat4∆, fob1∆ sir2∆ double and fob1∆ sir2∆ nat4∆ triple mutant strains.


( E ) ChIP analysis performed in BY4741 wild - type , nat4Δ and Pste5 - NAT4 strains grown under NCR and CR conditions . Chromatin was immunoprecipitated using antibodies against N - acH4 and Histone H4 , and analyzed by qRT - PCR with the indicated primers . PNC1 - P ( Promoter ) , PNC1 - S ( Transcriptional Start ) , PNC1 - E ( End of gene ) . The enrichment from the antibody was normalized to Histone H4 .

O O O O O O O O O O O O B-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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

(E) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown under NCR and CR conditions. Chromatin was immunoprecipitated using antibodies against N-acH4 and Histone H4, and analyzed by qRT-PCR with the indicated primers. PNC1-P (Promoter), PNC1-S (Transcriptional Start), PNC1-E (End of gene). The enrichment from the antibody was normalized to Histone H4.


MDM were incubated with 1 , 25D3 for 4 h before infection with HIV and / or M . tuberculosis ( TB ) for 3 h , washed then incubated with or without 1 , 25D3 for 7 days . ( A ) Top , extracellular release of HIV p24 antigen into the cell supernatant at days 0 , 4 and 7 was detected by ELISA . Bottom , MDM were harvested and stained for HIV p17 . Histograms are shown for a representative donor .

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

MDM were incubated with 1,25D3 for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h, washed then incubated with or without 1,25D3 for 7 days. (A) Top, extracellular release of HIV p24 antigen into the cell supernatant at days 0, 4 and 7 was detected by ELISA. Bottom, MDM were harvested and stained for HIV p17. Histograms are shown for a representative donor.


( B ) Top , cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase . Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7 . Bottom , MDM were harvested and stained for mycobacterium . Histograms are shown for a representative donor .

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(B) Top, cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase. Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7. Bottom, MDM were harvested and stained for mycobacterium. Histograms are shown for a representative donor.


( C ) Cell lysates from panel B were diluted and run in a MGIT 960 . Bar and line graphs are reported as mean ± s . e . m . of three independent experiments performed in triplicate . § P < 0 . 05 ; * P < 0 . 001 .

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(C) Cell lysates from panel B were diluted and run in a MGIT 960. Bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.


HIV and / or M . tuberculosis ( TB ) infected MDM were treated for 7 days with 100 pmol / L 1 , 25D3 . ( A ) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β - actin performed .

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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O

HIV and/or M. tuberculosis (TB) infected MDM were treated for 7 days with 100 pmol/L 1,25D3. (A) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β-actin performed.


B ) Cells were incubated with 10 µg / mL pepstatin A for 4 h on day 7 prior to lysis . Immunoblots of LC3B isoforms using antibody to LC3B or β - actin .

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

B) Cells were incubated with 10 µg/mL pepstatin A for 4 h on day 7 prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.


C ) Flow cytometry analysis of saponin - resistant LC3B - II in macrophages after 1 , 25D3 treatment for 7 d . Representative histograms of cells displaying saponin - resistant LC3B - II from three donors are shown .

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C) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.


( D ) Flow cytometry analysis of saponin - resistant LC3B - II in macrophages after 1 , 25D3 treatment for 7 d followed by 10 µg / mL pepstatin A for a further 4 h . Representative histograms of cells displaying saponin - resistant LC3B - II from three donors are shown .

O O O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

(D) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d followed by 10 µg/mL pepstatin A for a further 4 h. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.


( E ) Immunoblots of p62 using antibody to p62 or β - actin 7 d after macrophages treated with 1 , 25D3 .

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(E) Immunoblots of p62 using antibody to p62 or β-actin 7 d after macrophages treated with 1,25D3.


( F ) At 7 d post - infection , aliquots of supernatant taken before the addition of WST - 1 were tested for lactate dehydrogenase ( LDH ) spectrophotometrically using the LDHPLUS assay . For the last hour cells were incubated with WST - 1 , and the reduction of the WST - 1 reagent to its formazan product was monitored spectrophotometrically .

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(F) At 7 d post-infection, aliquots of supernatant taken before the addition of WST-1 were tested for lactate dehydrogenase (LDH) spectrophotometrically using the LDHPLUS assay. For the last hour cells were incubated with WST-1, and the reduction of the WST-1 reagent to its formazan product was monitored spectrophotometrically.


1 , 25D3 induces autophagy in human macrophages co - infected with HIV and M . tuberculosis . HIV ( A ) , M . tuberculosis ( TB ) ( B ) , and HIV / TB dual infected MDM ( C ) were treated for 7 days with 100 pmol / L 1 , 25D3 , harvested and stained for HIVp17 , mycobacteria , and saponin - resistant LC3B - II . Representative density plots from three donors are shown . ( D ) HIV / M . tuberculosis infected MDM ( C ) were treated for 7 days with 100 pmol / L 1 , 25D3 , harvested and stained for HIVp17 , mycobacteria , and saponin - resistant LC3B - II . Left , representative density plots from three donors from HIV / M . tuberculosis infected MDM are shown for HIV / M . tuberculosis co - infection . Right , histograms of saponin - resistant LC3B - II in macrophages that were HIV + M . tuberculosis − or HIV + day 7 + at day 7 post - infection .

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 B-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 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-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 O O O O O O O O O O O O O O O B-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 O O

1,25D3 induces autophagy in human macrophages co-infected with HIV and M. tuberculosis.HIV (A), M. tuberculosis (TB) (B), and HIV/TB dual infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Representative density plots from three donors are shown. (D) HIV/M. tuberculosis infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Left, representative density plots from three donors from HIV/M. tuberculosis infected MDM are shown for HIV/M. tuberculosis co-infection. Right, histograms of saponin-resistant LC3B-II in macrophages that were HIV+M. tuberculosis− or HIV+day 7+ at day 7 post-infection.