left panels ; Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected i primary OPC ( D ) in absence or in presence of 30 nM YM - 244769 or 100 nM BED D , right panels ; Quantification of the oscillation index i primary OPC ( D ) in absence or in presence of 30 nM YM - 244769 or 100 nM BED 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 10 nM BED - i primary OPC ( d ) . 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 usin D and d , 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 18 - 20 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 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 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 OPC (D) in absence or in presence of 30 nM YM-244769 or 100 nM BED D, right panels; Quantification of the oscillation index i primary OPC (D) in absence or in presence of 30 nM YM-244769 or 100 nM BED 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 10 nM BED -i primary OPC (d). 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 usin D and d, 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 18-20 cells in each group, n=3 biological replicate


E . left panel ; Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in MO3 . 13 cells in presence of siCtl or sincx3 silencing . E , right panel ; Quantification of the oscillation index in MO3 . 13 cells in absence or in presence of sincx3 . e , Quantification of the initial [ Ca2 + ] i increase elicited by D - Asp and measured as ∆ % of peak versus basal values in absence or in presence of sincx3 Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by usin E and e , one way - ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus sictl , ˄ P < 0 . 05 versus D - Asp + sictl . Data are reported as mean of 25 - 30 cells in each group , n = 3 biological

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

E. left panel; Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in MO3.13 cells in presence of siCtl or sincx3 silencing. E, right panel; Quantification of the oscillation index in MO3.13 cells in absence or in presence of sincx3. e, Quantification of the initial [Ca2+]i increase elicited by D-Asp and measured as ∆% of peak versus basal values in absence or in presence of sincx3 Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by usin E and e, one way-ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus sictl, ˄P< 0.05 versus D-Asp + sictl. Data are reported as mean of 25-30 cells in each group, n=3 biological replicate


F . left panel . Superimposed single - cell traces representative of the effect of 100 μM D - Asp on [ Ca2 + ] i detected in primary OPC obtained from wild - type ncx3 + / + , heterozygous ncx3 + / - , and knock - out ncx3 - / - mice . F . right panel . Quantification of the oscillation index elicited by D - Asp in primary mouse OPC obtained from ncx3 + / + , ncx3 + / - , and ncx3 - / - mice . f , Quantification of the initial [ Ca2 + ] i increase measured as ∆ % of peak versus basal values Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using F and f , one way - ANOVA P < 0 . 001 and P = 0 . 003 , respectively , followed by Bonferroni post hoc test , * P < 0 . 05 versus basal value , ˄ P < 0 . 05 versus ncx3 + / + and ncx3 + / - . Data are reported as mean of 10 - 19 cells in each group , n = 3 biological

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

F. left panel. Superimposed single-cell traces representative of the effect of 100 μM D-Asp on [Ca2+]i detected in primary OPC obtained from wild-type ncx3+/+, heterozygous ncx3 +/-, and knock-out ncx3-/- mice. F. right panel. Quantification of the oscillation index elicited by D-Asp in primary mouse OPC obtained from ncx3+/+, ncx3 +/-, and ncx3-/- mice. f, Quantification of the initial [Ca2+]i increase measured as ∆% of peak versus basal values Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using F and f, one way-ANOVA P<0.001 and P=0.003, respectively, followed by Bonferroni post hoc test, *P< 0.05 versus basal value, ˄P< 0.05 versus ncx3+/+ and ncx3+/-. Data are reported as mean of 10-19 cells in each group, n=3 biological replicates


A . Representative inward current traces elicited by 10μM AMPA in human oligodendrocyte MO3 . 13 progenitor cells in the absence or in the presence of 10μM DNQX . The concentration - dependent curve of AMPA ( 1 - 100 μM ) on inward currents is showed at the bottom of panel A Data information : The values represent the mean ± S . E . M from 3 independent experimental sessions . Level of significance was determined by using : in A , one - way ANOVA P < 0 . 001 followed by Bonferroni post hoc test , * P < 0 . 05 versus 1μM AMPA , n = 3 biological replicate

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

A. Representative inward current traces elicited by 10μM AMPA in human oligodendrocyte MO3.13 progenitor cells in the absence or in the presence of 10μM DNQX. The concentration-dependent curve of AMPA (1-100 μM) on inward currents is showed at the bottom of panel A Data information: The values represent the mean ± S.E.M from 3 independent experimental sessions. Level of significance was determined by using: in A, one-way ANOVA P<0.001 followed by Bonferroni post hoc test, *P< 0.05 versus 1μM AMPA, n=3 biological replicate


B . Representative inward current traces elicited by 100μM D - Asp application in MO3 . 13 cells 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 O O O O B-CONTROLLED_VAR

B. Representative inward current traces elicited by 100μM D-Asp application in MO3.13 cells in the absence or in the presence of 10μM DNQX


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 O O O O O O O O O O O O O O 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 O O O O O O O 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 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 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 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 O O O O O O O O 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 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 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 )

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

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

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

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

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 )

O 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 O O O O O O

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)


( D ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated ; All scale bars are 20 μm . n = 3 biological replicates .

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(D) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated; All scale bars are 20 μm. n=3 biological replicates.


( G ) Liver tissues were obtained from the indicated mice and then examined using transmission electron microscopy ( Arrowheads indicate mitochondria ) ; Scale bars are 1 μm and 5 μm . n = 3 biological replicates .

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(G) Liver tissues were obtained from the indicated mice and then examined using transmission electron microscopy (Arrowheads indicate mitochondria); Scale bars are 1 μm and 5 μm. n = 3 biological replicates.


( B ) Gpx4 protein was measured in liver of WT and PPARα - / - mice fed blank solvent or GW7647 ; n = 4 biological replicates .

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) Gpx4 protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647; n = 4 biological replicates.


( E ) Chromatin immunoprecipitation assays were performed on soluble formaldehyde - crosslinked chromatin isolated from untreated and GW7647 - treated WT or PPARα - / - livers with polyclonal anti - PPARα antibodies ( anti - PPARα ) or control IgG . The final DNA extraction was polymerase chain reaction - amplified with a primer pair that covered the sequence in intron 3 of Gpx4 .

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(E) Chromatin immunoprecipitation assays were performed on soluble formaldehyde-crosslinked chromatin isolated from untreated and GW7647-treated WT or PPARα-/- livers with polyclonal anti-PPARα antibodies (anti-PPARα) or control IgG. The final DNA extraction was polymerase chain reaction-amplified with a primer pair that covered the sequence in intron 3 of Gpx4.


( B ) Liver sections were obtained from the indicated mice and stained with probe1 . All scale bars are 20 μm . n = 3 biological replicates .

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(B) Liver sections were obtained from the indicated mice and stained with probe1. All scale bars are 20 μm. n = 3 biological replicates.


( D ) TRF protein was measured in liver of WT and PPARα - / - mice fed blank solvent or GW7647 . n = 4 biological replicates .

(D) TRF protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647. n = 4 biological replicates.


( E ) Liver tissue was obtained from the indicated mice and then examined using transmission electron microscopy ( Arrowheads indicate mitochondria ) . Scale bars are 1 μm and 5 μm . n = 4 biological replicates .

(E) Liver tissue was obtained from the indicated mice and then examined using transmission electron microscopy (Arrowheads indicate mitochondria). Scale bars are 1 μm and 5 μm. n = 4 biological replicates.


( J ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . All scale bars are 20 μm . n = 3 biological replicates .

(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. All scale bars are 20 μm. n = 3 biological replicates.


( E ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . n = 3 biological replicates . All scale bars are 20 μm .

(E) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.


( J ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . n = 3 biological replicates . All scale bars are 20 μm .

(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.


Adult mouse back skin sections immunostained for LYVE1 ( red ) , αSMA ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . LV , lymphatic vessels ; apm , arrector pili muscle .

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

Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle.


Back skin sections of P55 mouse skin immunostained for LYVE1 ( red ) , Tenascin - C ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . TNC , Tenascin - C .

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

Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C.


Adult mouse back skin sections from different postnatal ( P ) days immunostained for LYVE1 ( red ) , Lhx2 ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . epi , epidermis ; der , dermis .

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

Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.


Histogram of the quantification of the LV distance to HFSC positive for Lhx2 . ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Histogram of the quantification of the LV distance to HFSC positive for Lhx2. ). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Back skin sections of P49 mouse skin immunostained for LYVE1 ( red ) , CD34 ( green ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis . White arrowheads denote the proximity of lymphatic capillaries to HFSC .

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

Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC.


Histogram of the quantification of the LV distance to HFSC positive for CD34 . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . C , Catagen ; Te , early Telogen ; Tm , mid Telogen ; Tl , late Telogen ; A , Anagen .

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

Histogram of the quantification of the LV distance to HFSC positive for CD34. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. C, Catagen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen; A, Anagen.


Back skin sections of K15CrePR + / T ; Wls ∆ / ∆ and Control K15CrePR + / + ; Wlsflox / flox mice treated with mifepristone from P7 during 12 weeks ( P90 ) , immunostained for LYVE1 ( red ) , and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis .

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

Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.


Histogram of the quantification of the LV angle relative to the epidermis . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Histogram of the quantification of the LV angle relative to the epidermis. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Distribution of LV and HF at the mouse embryo stage E15 . 5 ( A ) , E16 . 5 ( B ) and E17 . 5 ( C ) . Maximum projection images of whole mount immunofluorescence analyses using LYVE1 ( red ) as lymphatic endothelial marker and counterstained with DAPI ( blue ) . Red arrowheads denote HF placodes . n = 3 - 4 embryos . Scale bar , 50 μm .

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

Distribution of LV and HF at the mouse embryo stage E15.5 (A), E16.5 (B) and E17.5 (C). Maximum projection images of whole mount immunofluorescence analyses using LYVE1 (red) as lymphatic endothelial marker and counterstained with DAPI (blue). Red arrowheads denote HF placodes. n= 3 - 4 embryos. Scale bar, 50 μm.


LV associated with individual HF further organize associating HF triads , which connect with other HF triads across the skin . P70 , 70 d old mice . n = 3 - 4 mice . White arrowheads denote capillaries stemming from HF triads . Scale bar , 100 μm .

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

LV associated with individual HF further organize associating HF triads, which connect with other HF triads across the skin. P70, 70 d old mice. n= 3 - 4 mice. White arrowheads denote capillaries stemming from HF triads. Scale bar, 100 μm.


3D reconstructions of whole skin mounts showing a triad of HF connected by LV . n = 3 - 4 mice . Scale bar , 50 μm . White arrowhead denotes an extending lymphatic capillary from a HF triad .

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

3D reconstructions of whole skin mounts showing a triad of HF connected by LV. n= 3 - 4 mice. Scale bar, 50 μm. White arrowhead denotes an extending lymphatic capillary from a HF triad.


Images of intravital microscopy analyses showing aligned HF rows in the back skin interconnected by LV in the Prox1CreERT2 ; Rosa - LSL - eYPF mice . Dotted boxes denote HF triads ; White arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows . n = 3 - 4 mice . Scale bar , 50 μm .

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

Images of intravital microscopy analyses showing aligned HF rows in the back skin interconnected by LV in the Prox1CreERT2; Rosa-LSL-eYPF mice. Dotted boxes denote HF triads; White arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows. n= 3 - 4 mice. Scale bar, 50 μm.


Adult back skin sections from different postnatal ( P ) days immunostained for LYVE1 ( red ) and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 10 μm . White arrowheads denote LV membrane protrusion and fenestrated areas . LV , lymphatic vessels ; HF , hair follicle .

Adult back skin sections from different postnatal (P) days immunostained for LYVE1 (red) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 10 μm. White arrowheads denote LV membrane protrusion and fenestrated areas. LV, lymphatic vessels; HF, hair follicle.


Histogram of the caliber ( μm ) of LV at different postnatal days . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . A , Anagen ; C , Catagen ; T , Telogen ; Te , early Telogen ; Tm , mid Telogen ; Tl , late Telogen .

Histogram of the caliber (μm) of LV at different postnatal days. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. A, Anagen; C, Catagen; T, Telogen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen.


H & E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 200 μm . epi , epidermis ; der , dermis .

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

H&E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. epi, epidermis; der, dermis.


LYVE1 immunofluorescence ( red ) counterstained with DAPI ( blue ) ( D ) in back skin sections from mice treated intradermally with Control or Clodronate liposomes . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis .

O 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

LYVE1 immunofluorescence (red) counterstained with DAPI (blue) (D) in back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.


Histogram of the caliber ( μm ) of LV ( E ) in back skin sections from mice treated intradermally with Control or Clodronate liposomes .

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

Histogram of the caliber (μm) of LV (E) in back skin sections from mice treated intradermally with Control or Clodronate liposomes.


Back skin sections of Controls and K14ΔNβ - cateninER + / T mice immunostained for LYVE1 ( red ) , and counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . epi , epidermis ; der , dermis .

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

Back skin sections of Controls and K14ΔNβ-cateninER+/T mice immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.


Histogram of the LV caliber in skin sections from Controls and K14ΔNβ - cateninER + / T mice . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Histogram of the LV caliber in skin sections from Controls and K14ΔNβ-cateninER+/T mice. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Gene set enrichment plots ( GSEA ) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen ( P69 , Tl ) compared with mid Telogen ( P55 , Tm ) . NES : normalized enrichment score . The false discovery rate ( FDR ; q - value ) is indicated .

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

Gene set enrichment plots (GSEA) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen (P69, Tl) compared with mid Telogen (P55, Tm). NES: normalized enrichment score. The false discovery rate (FDR; q-value) is indicated.


Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen , relative to biological processes , analyzed using the Enrichr platform ( Chen et al . , 2013 ; Kuleshov et al . , 2016 ) . Data shown represent the log2 ( FPKM + 1 ) .

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

Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Data shown represent the log2 (FPKM+1).


Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 ( D ) , DCN ( E ) , PKD1 ( F ) and PLXND1 ( G ) , assessed by the number of mRNA spots present in LYVE1 + cells in the vicinity of HF in P55 and P70 mouse back skin . n = average of 30 LYVE1 + cells / sample , n = 3 mice .

Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 (D), DCN (E), PKD1 (F) and PLXND1 (G), assessed by the number of mRNA spots present in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= average of 30 LYVE1+ cells / sample, n= 3 mice.


Histograms represent the mean fluorescence intensity for Jup ( H ) and Emilin1 ( I ) in LYVE1 + cells in the vicinity of HF in P55 and P70 mouse back skin . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

Histograms represent the mean fluorescence intensity for Jup (H) and Emilin1 (I) in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


H & E staining ( A ) and histogram of the HF length ( B ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA and vehicle ( CSA ) ; and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI treated with tamoxifen , CSA and intradermal Diphtheria Toxin ( CSA + DT ) starting from early Telogen ( P49 ) and analyzed at the end of the treatments ( P58 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Ki67 immunostaining ( C ) and histogram of the number of Ki67 + cells per HF ( D ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA and vehicle ; and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI treated with tamoxifen , CSA and intradermal DT , starting from early Telogen ( P49 ) and analyzed at the end of the treatments ( P58 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


LYVE1 immunostaining ( E ) and histogram of the LV caliber ( F ) analyzed in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA and vehicle ( CSA ) ; and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen , CSA and intradermal DT , starting from early Telogen ( P49 ) and analyzed at the end of the treatments ( P58 ) . Scale bar , 10 μm . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


H & E staining ( G ) and histogram of the HF length ( H ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice ( Control ) , and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen and intradermal DT , starting from Anagen ( P30 ) and analyzed at the end of the treatments ( P37 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 200 μm . Data represent the mean value ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM.


Cleaved caspase 3 immunostaining ( I ) and histogram of the number of cleaved caspase 3 + cells per HF ( J ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice ( Control ) , and Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen and intradermal DT , starting from Anagen ( P30 ) and analyzed at the end of the treatments ( P37 ) . Scale bar , 50 μm . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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

Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Green line : Normalized population expression level averaged over all OxyR , and SoxS - regulated promoters ; red line : average over all LexA - regulated promoters as measured with a promoter - GFP plasmid library in a plate reader . Oxidative stress promoters clearly precede SOS response promoters in response to NIT stress , when measured at the population level Error bars show standard deviation over all detected oxidative stress and SOS promoters , respectively It is not clear if this temporal order correctly reflects the temporal order in single cells . B . Schematic showing response of two different genes ( green and red ) in three different cells ( solid , dashed , dotted line ) . The temporal order observed at the population level correctly reflects the temporal order in each individual cell . C . As B . Here the temporal order at the population level is not the same in every single cell : Although in most cells , the green gene responds before the red one , one cell expresses the green gene before the red one ( dashed lines ) .

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

Green line: Normalized population expression level averaged over all OxyR, and SoxS-regulated promoters; red line: average over all LexA-regulated promoters as measured with a promoter-GFP plasmid library in a plate reader. Oxidative stress promoters clearly precede SOS response promoters in response to NIT stress, when measured at the population level Error bars show standard deviation over all detected oxidative stress and SOS promoters, respectively It is not clear if this temporal order correctly reflects the temporal order in single cells. B. Schematic showing response of two different genes (green and red) in three different cells (solid, dashed, dotted line). The temporal order observed at the population level correctly reflects the temporal order in each individual cell. C. As B. Here the temporal order at the population level is not the same in every single cell: Although in most cells, the green gene responds before the red one, one cell expresses the green gene before the red one (dashed lines).


Single - cell gene expression time traces from the dnaK promoter in response to TET stress , normalized to the median full response . Response times were determined as the time point at which a threshold expression level was reached This threshold was low enough so that most cells exceed it and high enough to avoid false positives due to the low signal - to - noise ratio at time point zero . Brown line : median of all cells . Time traces are from one microcolony . B . Histogram of the response times for the dnaK promoter with mean µt = 1 . 87 h and standard deviation σt = 0 . 37 h , and fit of an Erlang distribution with shape parameter n = 37 Response times are from two microcolonies .

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

Single-cell gene expression time traces from the dnaK promoter in response to TET stress, normalized to the median full response. Response times were determined as the time point at which a threshold expression level was reached This threshold was low enough so that most cells exceed it and high enough to avoid false positives due to the low signal-to-noise ratio at time point zero. Brown line: median of all cells. Time traces are from one microcolony. B. Histogram of the response times for the dnaK promoter with mean µt = 1.87 h and standard deviation σt = 0.37 h, and fit of an Erlang distribution with shape parameter n = 37 Response times are from two microcolonies.


C . Standard deviation σt versus mean response time µt for 23 different promoters in three antibiotic stress conditions ( TMP , TET , and NIT ) . The standard deviation of the response time σt grows with the mean response time µt and does not fall below a ' precision limit ' ( dashed line ) that increases linearly with a slope ~ 0 . 165 . The dotted line indicates the upper bound to timing variability where σt = µt , see text . The promoter dnaK under TET has low timing variability , whereas the promoters recA , fpr , osmC , and wrbA under TMP stress have high timing variability . The response time mean and standard deviation are from subsampling of descendants of single cells that were present at the time of stress addition Subsampling for each promoter was done from at least 2 microcolonies and the descendants of at least 17 individual cells present at the time of stress addition .

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 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 B-CONTROLLED_VAR 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

C. Standard deviation σt versus mean response time µt for 23 different promoters in three antibiotic stress conditions (TMP, TET, and NIT). The standard deviation of the response time σt grows with the mean response time µt and does not fall below a 'precision limit' (dashed line) that increases linearly with a slope ~0.165. The dotted line indicates the upper bound to timing variability where σt = µt, see text. The promoter dnaK under TET has low timing variability, whereas the promoters recA, fpr, osmC, and wrbA under TMP stress have high timing variability. The response time mean and standard deviation are from subsampling of descendants of single cells that were present at the time of stress addition Subsampling for each promoter was done from at least 2 microcolonies and the descendants of at least 17 individual cells present at the time of stress addition.


E . Standard deviation σt versus mean response time µt for the LlacO - 1 promoter induced with different IPTG concentrations .

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E. Standard deviation σt versus mean response time µt for the LlacO-1 promoter induced with different IPTG concentrations.


D . Response times of the LlacO - 1 promoter ( Lutz & Bujard , 1997 ) , induced with 1 mM IPTG at time zero , measured with YFP and CFP constructs in the same cell ( r = 0 . 81 ± 0 . 04 , p = 6 · 10 - 12 ) .

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D. Response times of the LlacO-1 promoter (Lutz & Bujard, 1997), induced with 1 mM IPTG at time zero, measured with YFP and CFP constructs in the same cell (r = 0.81 ± 0.04, p = 6 · 10-12).


A . Normalized expression from the promoters ybjC and recA in response to NIT stress , measured at the population level using GFP reporters 4 μg / mL NIT was added at time zero . The oxidative stress promoter ybjC clearly precedes the SOS response promoter recA , when measured at the population level Error bars show standard deviation of three replicate experiments measured on different days . B . Normalized ybjC and recA expression over time in response to the addition of 4 μg / mL NIT at time zero in single cells from one microcolony . Dashed line : threshold used to determine response times Lower panels , upper row : Dual - color images of the oxidative stress reporter ybjC ( green ) and the SOS reporter recA ( red ) in single cells at 4 different time points after NIT addition in one microcolony . Note that the ybjC signal ( green ) is stronger at t = 2h , whereas the recA signal ( red ) is only apparent at t = 4h and 6h . Lower row : Constitutively expressed mCherry used for segmentation . C . Response times for ybjC and recA from individual cells with dual reporters ( Figure 3 ) , combined from two microcolonies . All response times for recA are higher than for ybjC and response times for both reporters are strongly correlated ( r = 0 . 74 ± 0 . 04 , p = 3 . 8 · 10 - 6 ) . The error of the correlation coefficient is from subsampling of descendants of single cells that were present at the time of NIT

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 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 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 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 O O O O O O O O O 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

A. Normalized expression from the promoters ybjC and recA in response to NIT stress, measured at the population level using GFP reporters 4 μg/mL NIT was added at time zero. The oxidative stress promoter ybjC clearly precedes the SOS response promoter recA, when measured at the population level Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized ybjC and recA expression over time in response to the addition of 4 μg/mL NIT at time zero in single cells from one microcolony. Dashed line: threshold used to determine response times Lower panels, upper row: Dual-color images of the oxidative stress reporter ybjC (green) and the SOS reporter recA (red) in single cells at 4 different time points after NIT addition in one microcolony. Note that the ybjC signal (green) is stronger at t = 2h, whereas the recA signal (red) is only apparent at t = 4h and 6h. Lower row: Constitutively expressed mCherry used for segmentation. C. Response times for ybjC and recA from individual cells with dual reporters (Figure 3), combined from two microcolonies. All response times for recA are higher than for ybjC and response times for both reporters are strongly correlated (r = 0.74 ± 0.04, p = 3.8 · 10-6). The error of the correlation coefficient is from subsampling of descendants of single cells that were present at the time of NIT addition


A . Expression from the gadB and recA promoters , measured at the population level using GFP reporters over time ; 0 . 5 μg / mL TMP was added at time zero . The acid stress response ( gadB ) clearly precedes the SOS response ( recA ) . Error bars show standard deviation of three replicate experiments measured on different days . B . Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0 . 5 μg / mL TMP at time zero shown for one microcolony . Dashed line : threshold used to determine response times ( Methods ) . Lower panels , upper row : Dual - color images of gadB ( blue ) and recA ( red ) in single cells at 4 different time points . Most cells are predominantly either red or blue , i . e . acid stress and SOS response are typically not both strongly expressed in the same cells . Lower row : Constitutively expressed mCherry used for segmentation . C . Scatter plot of response times for gadB and recA in single cells ; there is no clear temporal order and no significant correlation ( r = 0 . 08 ± 0 . 08 , p = 0 . 68 ) ; data is combined from three microcolonies . Error is from subsampling of descendants of single cells present at the time point of TMP

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

A. Expression from the gadB and recA promoters, measured at the population level using GFP reporters over time; 0.5 μg/mL TMP was added at time zero. The acid stress response (gadB) clearly precedes the SOS response (recA). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0.5 μg/mL TMP at time zero shown for one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of gadB (blue) and recA (red) in single cells at 4 different time points. Most cells are predominantly either red or blue, i.e. acid stress and SOS response are typically not both strongly expressed in the same cells. Lower row: Constitutively expressed mCherry used for segmentation. C. Scatter plot of response times for gadB and recA in single cells; there is no clear temporal order and no significant correlation (r = 0.08 ± 0.08, p = 0.68); data is combined from three microcolonies. Error is from subsampling of descendants of single cells present at the time point of TMP addition


D . Expression of gadB and recA in inosine - supplemented medium , averaged over 13 single cells in a microcolony . Insets : Dual color image of gadB and recA and segmentation image at t = 6h .

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D. Expression of gadB and recA in inosine-supplemented medium, averaged over 13 single cells in a microcolony. Insets: Dual color image of gadB and recA and segmentation image at t = 6h.


( B ) Dot - plot showing the fold change ( log2 ) in number of reads between vehicle and AZD8186 - treated conditions vs the p - value of the difference between the two treatment conditions for each shRNA . 9 out of 18 shRNAs targeting EGFR showed a p - value < 0 . 2 and are highlighted in the plot . The plot was generated considering the results from biological triplicate of the experiment .

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(B) Dot-plot showing the fold change (log2) in number of reads between vehicle and AZD8186-treated conditions vs the p-value of the difference between the two treatment conditions for each shRNA. 9 out of 18 shRNAs targeting EGFR showed a p-value <0.2 and are highlighted in the plot. The plot was generated considering the results from biological triplicate of the experiment.


( C MDA - MB - 468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin . EGFR mRNA was then measured by RT - qPCR ( C ) Average ± SD of triplicates and representative of three independent experiments .

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(C MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. EGFR mRNA was then measured by RT-qPCR (C) Average ± SD of triplicates and representative of three independent experiments.


D ) MDA - MB - 468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin . cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 ( D ) . Average ± SD of triplicates and representative of three independent experiments .

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D) MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 (D). Average ± SD of triplicates and representative of three independent experiments.


( E MDA - MB - 468 were treated with serial dilutions of gefitinib ( E ) in the presence of vehicle , AZD8186 ( 0 . 25 μM ) , GDC0941 ( 0 . 25 μM ) or MK2206 ( 0 . 45 μM ) , as indicated . Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor - treated condition to the viability in the absence of gefitinib Average ± SD of triplicates and representative of two independent experiments .

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(E MDA-MB-468 were treated with serial dilutions of gefitinib (E) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of gefitinib Average ± SD of triplicates and representative of two independent experiments.


F ) MDA - MB - 468 were treated with serial dilutions of lapatinib ( F ) in the presence of vehicle , AZD8186 ( 0 . 25 μM ) , GDC0941 ( 0 . 25 μM ) or MK2206 ( 0 . 45 μM ) , as indicated . Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor - treated condition to the viability in the absence of lapatinib . Average ± SD of triplicates and representative of two independent experiments .

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F) MDA-MB-468 were treated with serial dilutions of lapatinib (F) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of lapatinib. Average ± SD of triplicates and representative of two independent experiments.


( G ) Viability of six PTEN - null VS five PTEN - WT TNBC cell lines - not carrying other known mutations in PIK3CA , PIK3CB or PIK3R1 genes - treated with PI3Kβi ( AZD8186 90 nM ) , EGFRi ( gefitinib 3 μM ) alone or in combination for 6 days . Mean of 3 independent experiments ± SD . Statistical significance of two - tailed unpaired student t - test in PTEN - null PI3Kbi versus PI3Kbi + EGFRi * * P = 0 . 0059 , PTEN - null EGFRi versus PI3Kbi + EGFRi * * P = 0 . 0047 , PTEN - null PI3Kbi + EGFRi versus PTEN - WT PI3Kbi + EGFRi * P = 0 . 0459 , PTEN - WT PI3Kbi versus PI3Kbi + EGFRi * P = 0 . 0108 , PTEN - WT EGFRi versus PI3Kbi + EGFRi n . s . P = 0 . 1926 . PTEN - null cell lines used in the experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 and BT - 549 ; PTEN - WT cell lines were : MDA - MB - 157 , MDA - MB - 231 , HCC1187 , HCC1428 and HCC1806 .

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(G) Viability of six PTEN-null VS five PTEN-WT TNBC cell lines - not carrying other known mutations in PIK3CA, PIK3CB or PIK3R1 genes - treated with PI3Kβi (AZD8186 90 nM), EGFRi (gefitinib 3 μM) alone or in combination for 6 days. Mean of 3 independent experiments ± SD. Statistical significance of two-tailed unpaired student t-test in PTEN-null PI3Kbi versus PI3Kbi+EGFRi **P=0.0059, PTEN-null EGFRi versus PI3Kbi+EGFRi **P=0.0047, PTEN-null PI3Kbi+EGFRi versus PTEN-WT PI3Kbi+EGFRi *P=0.0459, PTEN-WT PI3Kbi versus PI3Kbi+EGFRi *P=0.0108, PTEN-WT EGFRi versus PI3Kbi+EGFRi n.s. P=0.1926. PTEN-null cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395 and BT-549; PTEN-WT cell lines were: MDA-MB-157, MDA-MB-231, HCC1187, HCC1428 and HCC1806.


( H ) Synergy score for combinations of serial dilutions of PI3Kβi ( AZD8186 ) plus EGFRi ( gefitinib ) tested on six PTEN - null and five PTEN - WT TNBC cell lines for 6 days in three independent experiments . The score was obtained analysing the viability data through the software Chalice Analyser . Mean of the synergy scores ± SD . Statistical significance of Mann Whitney two - tailed test * P = 0 . 0303 .

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(H) Synergy score for combinations of serial dilutions of PI3Kβi (AZD8186) plus EGFRi (gefitinib) tested on six PTEN-null and five PTEN-WT TNBC cell lines for 6 days in three independent experiments. The score was obtained analysing the viability data through the software Chalice Analyser. Mean of the synergy scores ± SD. Statistical significance of Mann Whitney two-tailed test *P=0.0303.


( I ) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups " PTEN - low " when falling in the lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in all other cases . Comparison of the expression of EGFR between the PTEN low or mut and the PTEN - WT groups . Data presented in a box and whisker plot with the central band indicating the median , the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD . P value calculated by unpaired t - test .

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(I) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of EGFR between the PTEN low or mut and the PTEN-WT groups. Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD. P value calculated by unpaired t-test.


( A ) Tumor volume of MDA - MB - 468 mammary fat - pad xenografts treated with vehicle , AZD8186 ( 50 mg / kg , og twice / day ) , erlotinib ( 50 mg / kg IP once / day ) alone or in combination ( 5 - 6 mice per group , mean ± SEM ) . Statistical significance of two - way ANOVA statistical test * * P = 0 . 0028 and * * * P < 0 . 0001 . ( B ) Tumor volume of HCC - 70 mammary fat - pad xenografts treated with vehicle , AZD8186 ( 150 mg / kg , og once / day ) , erlotinib ( 50 mg / kg IP once / day ) alone or in combination ( 6 - 7 mice per group , mean ± SEM ) . Growth curves were compared using two - way ANOVA statistical test . Statistical significance of two - way ANOVA statistical test * * * P = 0 . 0006 and * * * * P < 0 . 0001 .

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(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001. (B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001.


( C ) Waterfall representation of changes in the volume of individual HCC - 70 tumors during the treatment . Statistical comparison between different treatment groups by Mann - Whitney test * * P = 0 . 0023 and * * * P = 0 . 0006 .

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(C) Waterfall representation of changes in the volume of individual HCC-70 tumors during the treatment. Statistical comparison between different treatment groups by Mann-Whitney test **P=0.0023 and ***P=0.0006.


( D ) Change in the body weight of mice harbouring HCC70 xenograft tumors ( 6 - 7 mice per group , mean ± SEM ) during 21 days of treatment .

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(D) Change in the body weight of mice harbouring HCC70 xenograft tumors (6-7 mice per group, mean±SEM) during 21 days of treatment.


( E ) A cell line derived from a mammary tumor spontaneously developed in a Wap - cre : Ptenfl / fl : Tp53fl / fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6 / J recipient mice . Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure . When tumours reached an average volume of 100mm3 , they were treated with vehicle or a combination of AZD8186 ( 150mg / kg , og once / day ) and erlotinib ( 50mg / kg IP once / day ) . Tumours were then measured during the treatment ( 6 mice per group , mean ± SEM ) . Statistical significance of two - way ANOVA statistical test * * * * P < 0 . 0001 .

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(E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.


( A ) MDA - MB - 468 cells were treated for 24h with vehicle , PI3Kβi ( AZD8186 250 nM ) , EGFRi ( gefitinib 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in combination . The cell lysates were probed with the indicated antibodies .

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(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.


( B ) MDA - MB - 468 cells were treated for 24h with vehicle , AKTi ( MK2206 450 nM ) , gefitinib ( 3 μM ) or cetuximab ( 100 μg / ml ) , alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( C , D ) HCC70 ( C ) and ZR - 75 - 1 ( D ) parental cells or PI3Kβi - Res ( derivative cells with acquired resistance to AZD8186 ) , or AKTi - Res ( acquired resistance models to MK2206 ) were treated with vehicle , PI3Kβi ( AZD8186 250 nM ) or AKTi ( MK2206 1 μM ) for 24 hours . The whole cell lysates were then probed with the indicated antibodies . Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P - S6 and S6 tot in ( D ) have been cut and reassembled for figure purposes .

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(C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes.