Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM - KO mice . Graph shows mean ± SEM Mander ´ s coefficient indicative of the degree of colocalization of CB1 and Lamp1 ( * P = 0 . 0213 , n = 4 mice per group , Student ' s t - test ) . Scale bar , 5 μm .

B-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum from WT and ASM-KO mice. Graph shows mean ± SEM Mander´s coefficient indicative of the degree of colocalization of CB1 and Lamp1 (*P= 0.0213, n = 4 mice per group, Student's t-test). Scale bar, 5 μm.


Graphs show mean ± SEM SM levels , expressed as percentage of WT values , in extracts containing the same amount of protein of cultured neurons from WT and ASM - KO mice ( E )

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O B-ORGANISM O O O

Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT and ASM-KO mice (E)


Graphs show mean ± SEM SM levels , expressed as percentage of WT values , in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM ( F ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O

Graphs show mean ± SEM SM levels, expressed as percentage of WT values, in extracts containing the same amount of protein of cultured neurons from WT mice incubated or not with 40μΜ SM (F).


Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM ( * * P = 0 . 0021 , n = 5 independent cultures , Student ' s t - test ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM CB1 mRNA levels in cultured neurons from WT mice incubated or not with 40μM SM (**P = 0.0021, n = 5 independent cultures, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM ( n = 2 independent cultures , Student ' s t - test )

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice incubated or not with 40μΜ SM (n = 2 independent cultures, Student's t-test)


Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM . Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units ( left ) or the Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( right ) ( * * * P < 0 . 0001 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

Immunofluorescence images against CB1 and Lamp1 in cultured neurons from WT mice incubated or not with 40 μΜ SM. Graphs show mean ± SEM intensity associated to CB1 in the neuronal processes expressed in arbitrary units (left) or the Mander´s coefficient for the colocalization of CB1 with Lamp1 (right) (***P <0.0001, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle ( veh ) , with 40μM SM ( SM ) , with 0 , 1μM Bafilomycin ( BafA1 ) or with 40μM SM and 0 , 1μM Bafilomyecin ( SM + BafA1 ) . Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units ( n = 2 independent cultures , one - way ANOVA ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cultured neurons from WT mice treated with vehicle (veh), with 40μM SM (SM), with 0,1μM Bafilomycin (BafA1) or with 40μM SM and 0,1μM Bafilomyecin (SM+BafA1). Graph shows mean ± SEM CB1 levels normalized to GAPDH in arbitrary units (n = 2 independent cultures, one - way ANOVA).


Mean ± SEM SM levels in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * P = 0 . 0157 , n = 3 independent cultures , Student ' s t - test ) .

O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (*P = 0.0157, n = 3 independent cultures, Student's t-test).


Mean ± SEM CB1 mRNA levels in cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( * * P = 0 . 0015 , n = 5 independent cultures , Student ' s t - test ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM CB1 mRNA levels in cultured neurons from ASM-KO mice incubated or not with exogenous SMase (**P= 0.0015, n = 5 independent cultures, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) . Images belong to the same blot but to non - consecutive lanes as indicated by the boxes . Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM - KO mice incubated or not with exogenous SMase ( n = 5 independent cultures , Student ' s t - test ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O

Western blot against CB1 and GAPDH (used as loading control). Images belong to the same blot but to non-consecutive lanes as indicated by the boxes. Graph showing mean ± SEM CB1 protein levels normalized to GAPDH in extracts from cultured neurons from ASM-KO mice incubated or not with exogenous SMase (n = 5 independent cultures, Student's t-test).


Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM - KO mice incubated or not with exogenous SMase . DAPI stains cell nuclei . Graph shows mean ± SEM Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( * P = 0 . 0189 , n = 3 independent cultures , > 30 neurons per culture , Student ' s t - test ) . Scale bar , 10 μm .

B-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from ASM-KO mice incubated or not with exogenous SMase. DAPI stains cell nuclei. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (*P = 0.0189, n = 3 independent cultures, > 30 neurons per culture, Student's t-test). Scale bar, 10 μm.


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle or with the indicated concentrations of AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P<0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , the inhibitor of NSM GW , AEA or the combination of GW and AEA ( * * * P < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O O O B-GENEPROD B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA or the combination of GW and AEA (***P <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Western blot against NSM and GAPDH ( used as loading control ) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA ( * P = 0 . 0230 , n = 6 independent cultures , Student ' s t - test ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50μM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , or with JNJ , PF or URB in the presence or absence of AEA ( * * PJNJ = 0 . 0013 , * PPF = 0 . 0143 , * * * PURB = 0 . 0002 , * * PJNJ + AEA = 0 . 0069 , * * PPF + AEA = 0 . 0077 , * * * PURB + AEA < 0 . 0001 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF or URB in the presence or absence of AEA (**PJNJ = 0.0013, *PPF = 0.0143, ***PURB = 0.0002, **PJNJ+AEA = 0.0069, **PPF+AEA = 0.0077, ***PURB+AEA <0.0001, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured neurons from ASM - KO mice treated with vehicle , with PF or with SR141716 + PF ( * PPF = 0 . 0109 , * PSR + PF = 0 . 0343 , n = 3 independent cultures , one - way ANOVA , Bonferroni post hoc ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF or with SR141716 + PF (*PPF = 0.0109, *PSR+PF = 0.0343, n = 3 independent cultures, one - way ANOVA, Bonferroni post hoc).


Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM - KO mice treated with vehicle , AEA , JNJ , PF or URB . Scale bar , 50 μm .

B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

Immunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF or URB. Scale bar, 50 μm.


Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM - KO mice treated with H2O2 ( as positive control ) , vehicle , AEA , JNJ , PF or URB ( n = 3 independent cultures ) .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

Mean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF or URB (n = 3 independent cultures).


Weekly mean ± SEM body weight of WT and ASM - KO mice treated or not with PF ( * * * P < 0 . 0001 , n = 7 mice per group , statistics reflect the significant difference between the slope of the data in the PF - treated ASM - KO with respect to the other three groups , Linear Regression test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

Weekly mean ± SEM body weight of WT and ASM-KO mice treated or not with PF (***P <0.0001, n = 7 mice per group, statistics reflect the significant difference between the slope of the data in the PF-treated ASM-KO with respect to the other three groups, Linear Regression test).


Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM - KO mice after two months of vehicle or PF treatment ( * PT3 = 0 . 0488 , * PT4 = 0 . 361 , n = 4 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM time spent on the rod in the 4 trials of the rotarod test by WT and ASM-KO mice after two months of vehicle or PF treatment (*PT3 = 0.0488, *PT4 = 0.361, n = 4 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0024 , * * PWT PF Vs KO Veh = 0 . 0016 , * PKO PF Vs KO Veh = 0 . 0391 , n = 9 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM time spent in the new arm of the Y maze test by WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0024, **PWT PF Vs KO Veh = 0.0016, *PKO PF Vs KO Veh = 0.0391, n = 9 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± SEM time of immobility in the Tail suspension test of WT and ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0198 , * * PWT PF Vs KO Veh = 0 . 0067 , n = 6 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM time of immobility in the Tail suspension test of WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0198, **PWT PF Vs KO Veh = 0.0067, n = 6 mice per group, two - way ANOVA, Bonferroni post hoc).


Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM - KO mice after two months of vehicle or PF treatment ( * PWTVeh Vs KO Veh = 0 . 0342 , n = 7 mice per group , two - way ANOVA , Bonferroni post hoc ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM time spent in the open arms of the Elevated Plus Maze by WT and ASM-KO mice after two months of vehicle or PF treatment (*PWTVeh Vs KO Veh = 0.0342, n = 7 mice per group, two - way ANOVA, Bonferroni post hoc).


Percentage of survival with respect to weeks of age in WT and ASM - KO mice treated with vehicle or PF ( * * P = 0 . 0031 , n = 5 mice in WT vehicle , WT PF treated and KO vehicle , 6 mice in KO PF treated , Mantel - Cox )

O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O

Percentage of survival with respect to weeks of age in WT and ASM-KO mice treated with vehicle or PF (**P = 0.0031, n = 5 mice in WT vehicle, WT PF treated and KO vehicle, 6 mice in KO PF treated, Mantel-Cox)


Mean ± SEM AEA levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWT Veh vs KO Veh = 0 . 0063 ; * * * * PWT Veh vs KO PF < 0 . 0001 ; * * * * PWT PF vs KO PF < 0 . 0001 ; * * * * PKO Veh vs KO PF < 0 . 0001 , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-SMALL_MOLECULE O O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM AEA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWT Veh vs KO Veh = 0.0063; ****PWT Veh vs KO PF < 0.0001; ****PWT PF vs KO PF < 0.0001; ****PKO Veh vs KO PF < 0.0001, n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * PKO Veh Vs KO PF = 0 . 0465 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (***PWTVeh Vs KO Veh <0.0001, *PKO Veh Vs KO PF = 0.0465), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Western blot against NSM and GAPDH ( used as loading control ) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( n = 3 mice per group , one - way ANOVA , Bonferroni post hoc ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O O

Western blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (n = 3 mice per group, one - way ANOVA, Bonferroni post hoc).


Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM - KO mice after two months of vehicle or PF treatment ( * * PWTVeh Vs KO Veh = 0 . 0026 , * PKO Veh Vs KO PF = 0 . 0345 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

Mean ± SEM CB1 mRNA levels in cerebellar extracts from WT and ASM-KO mice after two months of vehicle or PF treatment (**PWTVeh Vs KO Veh = 0.0026, *PKO Veh Vs KO PF = 0.0345), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc).


Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM intensity associated to CB1 , expressed as percentage of values in vehicle - treated mice ( n = 4 mice per group , > 20 Purkinje cells per

B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O B-CELL_TYPE I-CELL_TYPE O

Immunofluorescence against CB1 in Purkinje cells of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to CB1, expressed as percentage of values in vehicle-treated mice (n = 4 mice per group, > 20 Purkinje cells per mouse


Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graphs show Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( left ) and mean ± SEM lysosomal size ( right ) ( * * * PWT Veh vs . KO Veh = 0 . 0002 ; * * PKO Veh vs . KO PF = 0 . 0052 , n = 4 mice per group , > 20 lysosomes per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 5 μm .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O 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-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR O B-ORGANISM O O O O O O O O O O O O O O O O O

Immunofluorescence against CB1 and Lamp1 in the Purkinje cell layer of the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graphs show Mander´s coefficient for the colocalization of CB1 with Lamp1 (left) and mean ± SEM lysosomal size (right) (***PWT Veh vs. KO Veh = 0.0002; **PKO Veh vs. KO PF = 0.0052, n = 4 mice per group, >20 lysosomes per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 5 μm.


Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM number of calbindin positive cells per area ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 00642 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 100 μm . Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM intensity associated to GFAP in arbitrary units ( * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0083 ) , n = 4 mice per group , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm . Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM - KO mice after two months of vehicle or PF treatment . Graph shows mean ± SEM number ( left ) and area ( right ) of Iba1 - positive cells ( Left * * * PWTVeh Vs KO Veh < 0 . 0001 , * * PKO PF Vs KO Veh = 0 . 0034 ; Right * * * PWTVeh Vs KO Veh < 0 . 0001 , PKO PF Vs KO Veh = 0 . 00123 ) , n = 4 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

Immunofluorescence against the Purkinje cell marker Calbindin in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number of calbindin positive cells per area (***PWTVeh Vs KO Veh <0.0001, **PKO PF Vs KO Veh = 0.00642), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 100 μm. Immunofluorescence against the astrocytic marker GFAP in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM intensity associated to GFAP in arbitrary units (***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0083), n = 4 mice per group, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm. Immunofluorescence against the microglia marker Iba1 in the cerebellum of WT and ASM-KO mice after two months of vehicle or PF treatment. Graph shows mean ± SEM number (left) and area (right) of Iba1-positive cells (Left ***PWTVeh Vs KO Veh < 0.0001, **PKO PF Vs KO Veh = 0.0034; Right ***PWTVeh Vs KO Veh < 0.0001, PKO PF Vs KO Veh = 0.00123), n = 4 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


H & E staining of cerebellum , hippocampus , cortex and liver from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . Scale bar , 100 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O

H&E staining of cerebellum, hippocampus, cortex and liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Scale bar, 100 μm.


Mean ± SEM SM levels , expressed as percentage of vehicle values , in cerebellar , hippocampal , cortical and liver extracts from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . ( * PKOVeh Vs KO 5 = 0 . 0304 , n = 3 mice per group , one - way ANOVA , Bonferroni post hoc )

O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O

Mean ± SEM SM levels, expressed as percentage of vehicle values, in cerebellar, hippocampal, cortical and liver extracts from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. (*PKOVeh Vs KO 5 = 0.0304, n = 3 mice per group, one - way ANOVA, Bonferroni post hoc)


Immunofluorescences against the microglia marker Iba1 in the cerebellum , hippocampus , cortex or against the macrophage marker F4 / 80 in the liver from WT and ASM - KO mice 48 hours after one single administration of vehicle or the indicated doses of PF . Graphs show mean ± SEM number of microglia per area unit in cerebellum , hippocampus and cortex or mean ± SEM area of macrophages in the liver ( Hippocampus * * PKOVeh Vs KO 5 = 0 . 0015 , Cortex * PKOVeh Vs KO 5 = 0 . 0364 , Liver * PKOVeh Vs KO 5 = 0 . 0152 , n = 6 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-TISSUE O B-TISSUE O B-TISSUE O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O B-CELL_TYPE O O B-TISSUE O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

Immunofluorescences against the microglia marker Iba1 in the cerebellum, hippocampus, cortex or against the macrophage marker F4/80 in the liver from WT and ASM-KO mice 48 hours after one single administration of vehicle or the indicated doses of PF. Graphs show mean ± SEM number of microglia per area unit in cerebellum, hippocampus and cortex or mean ± SEM area of macrophages in the liver (Hippocampus **PKOVeh Vs KO 5 = 0.0015, Cortex *PKOVeh Vs KO 5 = 0.0364, Liver *PKOVeh Vs KO 5 = 0.0152, n = 6 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts from WT and Npc1nmf164 mice ( n = 6 ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD B-ORGANISM O O O O O O

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts from WT and Npc1nmf164 mice (n = 6).


Immunofluorescence images against CB1 and Calbindin in the cerebellum of WT and Npc1nmf164 mice . DAPI stains cell nuclei . Graphs show mean ± SEM intensity associated to CB1 in the Purkinje cells ( indicated by arrows ) expressed in arbitrary units ( * * P = 0 . 0085 , n = 5 mice per group , Student ' s t - test ) . Scale bar , 10 μm .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O B-GENEPROD B-ORGANISM O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

Immunofluorescence images against CB1 and Calbindin in the cerebellum of WT and Npc1nmf164 mice. DAPI stains cell nuclei. Graphs show mean ± SEM intensity associated to CB1 in the Purkinje cells (indicated by arrows) expressed in arbitrary units (**P= 0.0085, n = 5 mice per group, Student's t-test). Scale bar, 10 μm.


Immunofluorescence images against CB1 in Purkinje cells of the cerebelllum of age - matched control and NPC affected children . Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the control values ( 16 and 57 cells in control and NPC , respectively ) . Scale bar , 10 μm .

B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O B-DISEASE O B-ORGANISM O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O

Immunofluorescence images against CB1 in Purkinje cells of the cerebelllum of age-matched control and NPC affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the control values (16 and 57 cells in control and NPC, respectively). Scale bar, 10 μm.


Graph shows mean ± SEM SM levels , expressed as percentage of vehicle values , in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM ( n = 2 different cultures )

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O B-DISEASE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

Graph shows mean ± SEM SM levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n= 2 different cultures)


Graph shows mean ± SEM cholesterol levels , expressed as percentage of vehicle values , in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM ( n = 2 different cultures )

Graph shows mean ± SEM cholesterol levels, expressed as percentage of vehicle values, in cultured fibroblasts from a NPC patient treated with vehicle or with PF at 50μM or 100μM (n=2 different cultures)


Immunofluorescence images of CB1 and Lamp1 in cultured fibroblasts from control subject and NPC patient treated with vehicle or with PF at 100μM . Graph shows mean ± SEM Mander ´ s coefficient for the colocalization of CB1 with Lamp1 ( n = 2 different cultures ) . Scale bar , 5 μm .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-DISEASE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

Immunofluorescence images of CB1 and Lamp1 in cultured fibroblasts from control subject and NPC patient treated with vehicle or with PF at 100μM. Graph shows mean ± SEM Mander´s coefficient for the colocalization of CB1 with Lamp1 (n= 2 different cultures). Scale bar, 5 μm.


Graphs show mean ± SEM SM and cholesterol levels expressed as percentage of the values obtained in the vehicle treated mice in the cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg / kg PF ( n = 3 mice )

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O

Graphs show mean ± SEM SM and cholesterol levels expressed as percentage of the values obtained in the vehicle treated mice in the cerebellum of Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF (n=3 mice)


Immunofluorescence images against the microglia marker F4 / 80 in the cerebellum of WT and Npc1nmf164 after 48 hours of a single dose of 5mg / kg PF . Graphs show mean ± SEM number ( upper ) and area ( lower ) of F4 / 80 positive cells ( upper graph * * * * P < 0 . 0001 ; lower graph * * * PWT Veh vs NPC Veh = 0 . 0008 ; * PWT Veh vs . NPC PF = 0 . 0286 ; * * * * PWT PF vs . NPC Veh < 0 . 0001 ; * * PWT PF vs . NPC PF = 0 . 0022 , n = 3 mice per group , > 20 cells analyzed per mouse , one - way ANOVA , Bonferroni post hoc ) . Scale bar , 10 μm .

B-EXP_ASSAY O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O B-DISEASE O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

Immunofluorescence images against the microglia marker F4/80 in the cerebellum of WT and Npc1nmf164 after 48 hours of a single dose of 5mg/kg PF. Graphs show mean ± SEM number (upper) and area (lower) of F4/80 positive cells (upper graph ****P< 0.0001; lower graph ***PWT Veh vs NPC Veh = 0.0008; *PWT Veh vs. NPC PF = 0.0286; ****PWT PF vs. NPC Veh < 0.0001; **PWT PF vs. NPC PF = 0.0022, n = 3 mice per group, > 20 cells analyzed per mouse, one - way ANOVA, Bonferroni post hoc). Scale bar, 10 μm.


A Representative images of E18 . 5 coronal cortical sections from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmids encoding EGFP , EGFP + Cre ( Cre ) or EGFP + Cre + KCC2FL ( Cre + KCC2FL ) . DAPI staining ( blue ) marks cell nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . B Quantification of the number of EGFP + neurons / ROI from embryos electroporated with constructs in ( A ) . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 . Data are presented as mean ± S . E . M . , n ( EGFP ) = 8 embryos ; n ( Cre ) = 8 embryos ; n ( Cre + KCC2FL ) = 13 embryos .

O O O O O O O O B-TISSUE O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O

A Representative images of E18.5 coronal cortical sections from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmids encoding EGFP, EGFP+Cre (Cre) or EGFP+Cre+KCC2FL (Cre+KCC2FL). DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. B Quantification of the number of EGFP+ neurons/ROI from embryos electroporated with constructs in (A). Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01. Data are presented as mean ± S.E.M., n (EGFP) = 8 embryos; n (Cre) = 8 embryos; n (Cre+KCC2FL) = 13 embryos.


C Representative image of cleaved Caspase 3 ( Cas - 3 , upper panel ) and TUNEL ( lower panel ) staining in coronal sections from Kcc2lox / lox cortex at E16 . 5 electroporated with EGFP or EGFP + Cre are shown . Arrowheads point to neurons expressing Cas - 3 ( upper panel ) and TUNEL ( lower panel ) . DAPI staining ( blue ) marks cell nuclei . CP , cortical plate ; VZ , ventricular zone ; SVZ , subventricular zone ; Sp , subplate ; IZ , intermediate zone . Large scale bar : 50 µm , small scale bar : 20 µm . D Quantification of the percentage of EGFP + neurons expressing apoptotic markers at E16 . 5 from embryos electroporated with EGFP ± Cre . Statistical significance was determined using Mann - Whitney U test ( Cas - 3 ) ; and two - tailed t test ( TUNEL ) , * * * P < 0 . 001 . Data are presented as mean ± S . E . M . , n ( - Cre ) = 6 embryos ; n ( + Cre ) = 6 embryos . E The number of EGFP + Cre neurons as a percentage of neurons expressing EGFP alone . Statistical significance was determined using a two - tailed Student ' s t test . Data are presented as mean ± S . E . M . , n = 6 embryos .

O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-TISSUE O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O 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 image of cleaved Caspase 3 (Cas-3, upper panel) and TUNEL (lower panel) staining in coronal sections from Kcc2lox/lox cortex at E16.5 electroporated with EGFP or EGFP+Cre are shown. Arrowheads point to neurons expressing Cas-3 (upper panel) and TUNEL (lower panel). DAPI staining (blue) marks cell nuclei. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; Sp, subplate; IZ, intermediate zone. Large scale bar: 50 µm, small scale bar: 20 µm. D Quantification of the percentage of EGFP+ neurons expressing apoptotic markers at E16.5 from embryos electroporated with EGFP ± Cre. Statistical significance was determined using Mann-Whitney U test (Cas-3); and two-tailed t test (TUNEL), ***P < 0.001. Data are presented as mean ± S.E.M., n (-Cre) = 6 embryos; n (+Cre) = 6 embryos. E The number of EGFP+Cre neurons as a percentage of neurons expressing EGFP alone. Statistical significance was determined using a two-tailed Student's t test. Data are presented as mean ± S.E.M., n = 6 embryos.


A Top : Representative images of E18 . 5 coronal cortical brain sections from Kcc2lox / lox embryos electroporated in utero at E14 . 5 together with plasmid constructs encoding Cre , a fluorescent marker ( EGFP or mRFP , both pseudo - colored in green ) and one of the following constructs : KCC2ΔNTD , KCC2CTD , KCC2R952H , or cofilinS3A . DAPI staining ( blue ) marks cell nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . Bottom : schematic representation of the KCC2 constructs . B Quantification of the number of transfected neurons / ROI from embryos electroporated with constructs in ( A ) . The mean number of transfected neurons from embryos electroporated with Cre + KCC2FL taken from Fig 1B shown as dotted line . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 to Cre + KCC2FL . Data are presented as mean ± S . E . M . , n ( KCC2ΔNTD ) = 7 embryos ; n ( Cre + KCC2CTD ) = 9 embryos ; n ( Cre + KCC2R952H ) = 9 embryos ; n ( Cre + cofilinS3A ) = 8 embryos .

O O O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

A Top: Representative images of E18.5 coronal cortical brain sections from Kcc2lox/lox embryos electroporated in utero at E14.5 together with plasmid constructs encoding Cre, a fluorescent marker (EGFP or mRFP, both pseudo-colored in green) and one of the following constructs: KCC2ΔNTD, KCC2CTD, KCC2R952H, or cofilinS3A. DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. Bottom: schematic representation of the KCC2 constructs. B Quantification of the number of transfected neurons/ROI from embryos electroporated with constructs in (A). The mean number of transfected neurons from embryos electroporated with Cre+KCC2FL taken from Fig 1B shown as dotted line. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Cre+KCC2FL. Data are presented as mean ± S.E.M., n (KCC2ΔNTD) = 7 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos; n (Cre+cofilinS3A) = 8 embryos.


A Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmid constructs encoding EGFP or EGFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted line . EGFP signal is shown as green pseudocolor . DAPI staining ( blue ) marks cell nuclei . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . B Number of EGFP + Cre neurons migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * * P < 0 . 001 to EGFP . Data are presented as mean ± S . E . M . , n ( EGFP + Cre ) = 8 embryos ; n ( EGFP ) = 8 embryos .

O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O

A Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding EGFP or EGFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. EGFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. B Number of EGFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to EGFP. Data are presented as mean ± S.E.M., n (EGFP +Cre) = 8 embryos; n (EGFP) = 8 embryos.


C Representative image of E18 . 5 coronal brain sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmid constructs encoding mRFP or mRFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . mRFP signal is shown as green pseudocolor . DAPI staining ( blue ) marks cell nuclei . D Number of mRFP + Cre neurons migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * * P < 0 . 001 to mRFP . Data are presented as mean ± S . E . M . , n ( mRFP + Cre ) = 5 embryos ; n ( mRFP ) = 5 embryos .

O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O

C Representative image of E18.5 coronal brain sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding mRFP or mRFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. mRFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. D Number of mRFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to mRFP. Data are presented as mean ± S.E.M., n (mRFP+Cre) = 5 embryos; n (mRFP) = 5 embryos.


E Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos co - electroporated in utero at E14 . 5 with plasmids encoding a fluorescent marker ( green ) together and Cre + KCC2FL , Cre + KCC2CTD , or Cre + KCC2R952H . DAPI staining ( blue ) marks cell nuclei . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . F Number of transfected neurons migrating above ( II - IV , left ) and below ( V - VI / IZ - SVZ , right ) the upper border of layer V in embryos electroporated with constructs in ( E ) normalized to respective data from embryos electroporated with Cre . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 to Cre . Data are presented as mean ± S . E . M . , n ( Cre + KCC2FL ) = 13 embryos ; n ( Cre + KCC2CTD ) = 9 embryos ; n ( Cre + KCC2R952H ) = 9 embryos .

O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

E Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos co-electroporated in utero at E14.5 with plasmids encoding a fluorescent marker (green) together and Cre+KCC2FL, Cre+KCC2CTD, or Cre+KCC2R952H. DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. F Number of transfected neurons migrating above (II-IV, left) and below (V-VI/IZ-SVZ, right) the upper border of layer V in embryos electroporated with constructs in (E) normalized to respective data from embryos electroporated with Cre. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 to Cre. Data are presented as mean ± S.E.M., n (Cre+KCC2FL) = 13 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos.


A , B , C The layer thickness and neuronal number within each layer was assessed using layer - specific markers in Kcc2 - / - and Kcc2 + / + embryos at E18 . 5 . Cux1 was used to label layers II - IV ( A ) , Ctip2 to label layer V ( B ) , and Tbr1 to label layer VI ( C ) . Dashed white lines in the representative images in ( A - C ) indicate upper and lower layer boundaries . Sp , subplate . Statistical significance was determined using Kruskal - Wallis test with Dunn ' s post hoc ( neuronal numbers ) and one - way ANOVA with Holm - Sidak ' s post hoc ( layer thickness ) . Data are presented as mean ± S . E . M . , n = 11 embryos for both genotypes . Scale bar = 50 µm .

O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O B-TISSUE I-TISSUE 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-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O 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, B, C The layer thickness and neuronal number within each layer was assessed using layer-specific markers in Kcc2-/-and Kcc2+/+ embryos at E18.5. Cux1 was used to label layers II-IV (A), Ctip2 to label layer V (B), and Tbr1 to label layer VI (C). Dashed white lines in the representative images in (A-C) indicate upper and lower layer boundaries. Sp, subplate. Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc (neuronal numbers) and one-way ANOVA with Holm-Sidak's post hoc (layer thickness). Data are presented as mean ± S.E.M., n = 11 embryos for both genotypes. Scale bar = 50 µm.


D Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) in Kcc2 - / - and Kcc2 + / + embryos electroporated in utero at E14 . 5 with a plasmid encoding EGFP ( green ) . DAPI staining ( blue ) marks cell nuclei . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . E Neurons from the brain sections in ( D ) were quantified and the total number of EGFP + neurons in the Kcc2 - / - sections is presented as a percentage of pooled Kcc2 + / + and Kcc2 + / - values . Statistical significance was determined using one - tailed Student ' s t test , * P < 0 . 05 . Data are presented as mean ± S . E . M . , n ( Kcc2 + / + + Kcc2 + / - ) = 14 embryos ; n ( Kcc2 - / - ) = 9 embryos . F Number of EGFP + neurons in Kcc2 - / - embryos migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective pooled data from Kcc2 + / + and Kcc2 + / - embryos . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 to Kcc2 + / + + Kcc2 + / - . Data are presented as mean ± S . E . M . , n ( Kcc2 + / + + Kcc2 + / - ) = 14 embryos ; n ( Kcc2 - / - ) = 9 embryos .

O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O

D Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) in Kcc2-/- and Kcc2+/+ embryos electroporated in utero at E14.5 with a plasmid encoding EGFP (green). DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. E Neurons from the brain sections in (D) were quantified and the total number of EGFP+ neurons in the Kcc2-/- sections is presented as a percentage of pooled Kcc2+/+ and Kcc2+/- values. Statistical significance was determined using one-tailed Student's t test, *P < 0.05. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos. F Number of EGFP+ neurons in Kcc2-/- embryos migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective pooled data from Kcc2+/+ and Kcc2+/- embryos. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Kcc2+/+ + Kcc2+/-. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.


( A ) Homer1c - GFP signal ( magenta ) and immunostaining for surface AMPARs ( cyan ) and SP ( green ) in neurons treated for 48h with TTX , or untreated ( UT ) . Scale bars : 10 µm ( low magnification ) , 5 µm ( insets ) . Dotted lines indicate the outline of dendrites . Arrowheads indicate SP + synapses . ( B ) Percentage of SP + synapses for untreated ( UT ) and TTX - treated neurons ( dot plots represent different cells ; UT : n = 26 ; TTX : n = 33 , n indicates the number of cells , from 2 cultures ) . % SP + spines : * * P = 0 . 019 ( Mann Whitney test ) . Data information : Data are represented as mean ± SEM .

O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O 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-SUBCELLULAR O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O 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) Homer1c-GFP signal (magenta) and immunostaining for surface AMPARs (cyan) and SP (green) in neurons treated for 48h with TTX, or untreated (UT). Scale bars: 10 µm (low magnification), 5 µm (insets). Dotted lines indicate the outline of dendrites. Arrowheads indicate SP+ synapses. (B) Percentage of SP+ synapses for untreated (UT) and TTX-treated neurons (dot plots represent different cells; UT: n = 26; TTX: n = 33, n indicates the number of cells, from 2 cultures). % SP+ spines: **P = 0.019 (Mann Whitney test). Data information: Data are represented as mean ± SEM.


( C ) AMPAR synaptic fluorescence intensity for SP + versus SP - synapses in UT and TTX - treated neurons . AMPAR synaptic fluorescence intensity was normalized to SP - or small synapses , respectively , from UT condition . ( UT : SP - , n = 1455 , SP + , n = 516 ; TTX : SP - , n = 1529 , SP + , n = 724 , n indicates the number of synapses , from 2 cultures ) . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-CELL_TYPE O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) AMPAR synaptic fluorescence intensity for SP+ versus SP- synapses in UT and TTX-treated neurons. AMPAR synaptic fluorescence intensity was normalized to SP- or small synapses, respectively, from UT condition. (UT: SP-, n = 1455, SP +, n = 516; TTX: SP-, n = 1529, SP+, n = 724, n indicates the number of synapses, from 2 cultures). **P < 0.01, ****P < 0.0001, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Example images of a FRAP experiment : SP - and SP + spines from the same cultured hippocampal neuron transfected with SEP - GluA2 ( gray and color - coded ) + RFP - SP ( red ) 10s before , and 0 , 370 , 750 s after the photobleaching . Scale bars : 1 µm . Dotted lines represent the outline of dendrites . Arrowheads indicate dendritic spines which were targeted for photobleaching . ( B ) SEP - GluA2 fluorescence intensity at SP - vs SP + spines . Each pair of dot plots represents average fluorescence for SP - and SP + spines from a same neuron ( 4 cultures ) . * * P = 0 . 0061 ( two - tailed paired Student ' s t test ) . ( C ) Quantification of FRAP dynamics for SP - vs SP + spines . Recovery curves represent SEP - GluA2 fluorescence average per cell ( n = 14 cells , from 4 cultures ) . The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test . The F test indicates that the traces are best fitted by two divergent models ( P < 0 . 0001 ) . ( D ) Quantification of the recovery fraction 750 s after the photobleaching . Each pair of dot plots represents average recovery for SP - and SP + spines from a same neuron ( 4 cultures ) . * * P = 0 . 0067 ( Wilcoxon matched - pairs signed rank test ) . Data information : Data are represented as mean ± SEM .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O 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-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O 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-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O 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) Example images of a FRAP experiment: SP- and SP+ spines from the same cultured hippocampal neuron transfected with SEP-GluA2 (gray and color-coded) + RFP-SP (red) 10s before, and 0, 370, 750 s after the photobleaching. Scale bars: 1 µm. Dotted lines represent the outline of dendrites. Arrowheads indicate dendritic spines which were targeted for photobleaching. (B) SEP-GluA2 fluorescence intensity at SP- vs SP+ spines. Each pair of dot plots represents average fluorescence for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0061 (two-tailed paired Student's t test). (C) Quantification of FRAP dynamics for SP- vs SP+ spines. Recovery curves represent SEP-GluA2 fluorescence average per cell (n = 14 cells, from 4 cultures). The two traces were fitted using double exponential components equations and the convergence of the traces to a common fit was tested using the extra sum of squares F test. The F test indicates that the traces are best fitted by two divergent models (P < 0.0001). (D) Quantification of the recovery fraction 750 s after the photobleaching. Each pair of dot plots represents average recovery for SP- and SP+ spines from a same neuron (4 cultures). **P = 0.0067 (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Immunoblot analysis of SP in lysates from COS - 7 cells which were untransfected , transfected with RFP - SP + empty vector or co - transfected with SP - shRNA - GFP + RFP - SP ( knock - down ) or shRNA - resistant RFP - SP ( rescue ) . β - actin and GFP signals illustrate equal protein loading and SP - shRNA expression level , respectively . ( B ) Quantification of SP expression from 6 different experiments , normalized to the β - actin signal . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data represent mean ± SEM .

O O O B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of SP in lysates from COS-7 cells which were untransfected, transfected with RFP-SP + empty vector or co-transfected with SP-shRNA-GFP + RFP-SP (knock-down) or shRNA-resistant RFP-SP (rescue). β-actin and GFP signals illustrate equal protein loading and SP-shRNA expression level, respectively. (B) Quantification of SP expression from 6 different experiments, normalized to the β-actin signal. *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( C ) Homer - DsRed ( green ) and immunostained endogenous SP ( red ) in dendrites from neurons transfected with either empty vector , scramble shRNA or SP - shRNA with GFP reporter ( blue ) . Scale bar : 10 µm . ( D ) Average SP intensity for same conditions ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates the number of cells , from 3 cultures ; normalized to empty vector condition ) . * * P < 0 . 01 , * * * * P < 0 . 0001 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . ( E ) Fraction of SP + synapses for same conditions . ( EV : n = 47 ; Scr - shRNA : n = 53 ; SP - shRNA : n = 62 , n indicates the number of cells from 3 cultures ) . * P < 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data represent mean ± SEM .

O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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) Homer-DsRed (green) and immunostained endogenous SP (red) in dendrites from neurons transfected with either empty vector, scramble shRNA or SP-shRNA with GFP reporter (blue). Scale bar: 10 µm. (D) Average SP intensity for same conditions (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells, from 3 cultures; normalized to empty vector condition). **P < 0.01, ****P < 0.0001 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (E) Fraction of SP+ synapses for same conditions. (EV: n = 47; Scr-shRNA: n = 53; SP-shRNA: n = 62, n indicates the number of cells from 3 cultures). *P < 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( F ) Homer1c - DsRed ( red ) and immunostained surface AMPARs ( green ) in dendrites from neurons transfected with either SP - shRNA - GFP or empty vector ( EV ) with GFP reporter ( blue ) and treated with TTX or left untreated ( UT ) . Scale bar : 5 µm . ( G ) AMPAR synaptic fluorescence intensity normalized to untreated empty vector ( EV ) condition ( EV : UT , n = 42 , TTX , n = 33 ; SP - shRNA : UT , n = 30 , TTX , n = 31 , n indicates the number of cells , from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data represent mean ± SEM .

O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Homer1c-DsRed (red) and immunostained surface AMPARs (green) in dendrites from neurons transfected with either SP-shRNA-GFP or empty vector (EV) with GFP reporter (blue) and treated with TTX or left untreated (UT). Scale bar: 5 µm. (G) AMPAR synaptic fluorescence intensity normalized to untreated empty vector (EV) condition (EV: UT, n = 42, TTX, n = 33; SP-shRNA: UT, n = 30, TTX, n = 31, n indicates the number of cells, from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data represent mean ± SEM.


( A ) Micrographs showing Homer1c - GFP ( green ) and immunostaining for surface AMPARs ( blue ) and endogenous SP ( red ) in neurons transfected with miR - 124 or control miR - 67 ( miR - Ctrl ) , and treated with TTX , or left untreated ( UT ) . Scale bar : 5 µ ( B ) Percentage of SP + synapses for same conditions as in ( A ) ( miR - Ctrl : UT , n = 25 , TTX , n = 26 ; miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates the number of cells , from 3 cultures ) . * * * * P < 0 . 0001 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . ( C ) Homer1c - GFP intensity for same condition as in ( A ) , normalized to untreated miR - Ctrl ( miR - Ctrl : UT , n = 25 , TTX , n = 26 ; miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates the number of cells , from 3 cultures ) . ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . ( D ) AMPAR synaptic fluorescence intensity for same condition as in ( A ) , normalized to untreated miR - Ctrl ( miR - Ctrl : UT , n = 25 , TTX , n = 26 , miR - 124 : UT , n = 30 , TTX , n = 30 , n indicates the number of cells , from 3 cultures ) . * * * P < 0 . 001 , * P < 0 . 01 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing Homer1c-GFP (green) and immunostaining for surface AMPARs (blue) and endogenous SP (red) in neurons transfected with miR-124 or control miR-67 (miR-Ctrl), and treated with TTX, or left untreated (UT). Scale bar: 5 µ (B) Percentage of SP+ synapses for same conditions as in (A) (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ****P < 0.0001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (C) Homer1c-GFP intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26; miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). (D) AMPAR synaptic fluorescence intensity for same condition as in (A), normalized to untreated miR-Ctrl (miR-Ctrl: UT, n = 25, TTX, n = 26, miR-124: UT, n = 30, TTX, n = 30, n indicates the number of cells, from 3 cultures). ***P < 0.001, *P < 0.01, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Micrographs showing dendrites from neurons transfected with Homer1c - DsRed ( magenta ) and SEP - GluA2 constructs containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( green ) and treated with TTX for 48 h , or left untreated ( UT ) . Scale bar : 10 µm . ( B ) SEP - GluA2 synaptic fluorescence intensity for each condition , normalized to GluA2 - 3 ' UTR - WT untreated neurons ( 3 ' UTR - WT : UT , n = 29 , TTX , n = 37 ; 3 ' UTR - MUT : UT , n = 18 , TTX , n = 14 ; n indicates the number of cells , from 3 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-SUBCELLULAR O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing dendrites from neurons transfected with Homer1c-DsRed (magenta) and SEP-GluA2 constructs containing wild-type (WT) or mutated (MUT) 3'UTR (green) and treated with TTX for 48 h, or left untreated (UT). Scale bar: 10 µm. (B) SEP-GluA2 synaptic fluorescence intensity for each condition, normalized to GluA2-3'UTR-WT untreated neurons (3'UTR-WT: UT, n = 29, TTX, n = 37; 3'UTR-MUT: UT, n = 18, TTX, n = 14; n indicates the number of cells, from 3 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( C ) Micrographs showing dendrites from neurons transfected with Homer1c - BFP ( magenta ) , GFP - SP - shRNA ( gray ) and a rescue RFP - SP construct containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR ( geen ) and treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( D ) Percentage of SP + synapses for each condition ( 3 ' UTR - WT : UT , n = 26 , TTX , n = 28 ; 3 ' UTR - MUT : UT , n = 26 , TTX , n = 24 ; n indicates the number of cells , from 3 cultures ) . * * P < 0 . 01 , * * * P < 0 . 001 , ns , not significant , P > 0 . 05 ( two - way ANOVA test followed by Tukey ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-SUBCELLULAR O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing dendrites from neurons transfected with Homer1c-BFP (magenta), GFP-SP-shRNA (gray) and a rescue RFP-SP construct containing wild-type (WT) or mutated (MUT) 3'UTR (geen) and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (D) Percentage of SP+ synapses for each condition (3'UTR-WT: UT, n = 26, TTX, n = 28; 3'UTR-MUT: UT, n = 26, TTX, n = 24; n indicates the number of cells, from 3 cultures). **P < 0.01, ***P < 0.001, ns, not significant, P > 0.05 (two-way ANOVA test followed by Tukey's multiple comparison test). Data information: Data are represented as mean ± SEM.


( E ) Micrographs showing Homer1c - GFP ( magenta ) and immunostaining for surface AMPARs ( cyan ) and endogenous SP ( green ) in neurons transfected with or without 50 nM SP TSB - LNA and treated with TTX , or left untreated ( UT ) . Scale bar : 10 µm . ( F ) Percentage of SP + synapses for each condition ( Control : UT , n = 36 , TTX , n = 52 ; SP TSB - LNA : UT , n = 62 , TTX , n = 29 ; n indicates the number of cells from 3 cultures ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . ( G ) AMPAR synaptic fluorescence intensity for each condition , normalized to control untreated neurons ( Control : UT , n = 30 , TTX , n = 36 ; SP TSB - LNA : UT , n = 37 , TTX , n = 20 ; n indicates the number of cells from 2 cultures ) . * P < 0 . 05 , ns , not significant , P > 0 . 05 ( Kruskal - Wallis test followed by Dunn ' s multiple comparison test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing Homer1c-GFP (magenta) and immunostaining for surface AMPARs (cyan) and endogenous SP (green) in neurons transfected with or without 50 nM SP TSB-LNA and treated with TTX, or left untreated (UT). Scale bar: 10 µm. (F) Percentage of SP+ synapses for each condition (Control: UT, n = 36, TTX, n = 52; SP TSB-LNA: UT, n = 62, TTX, n = 29; n indicates the number of cells from 3 cultures). *P < 0.05, **P < 0.01, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). (G) AMPAR synaptic fluorescence intensity for each condition, normalized to control untreated neurons (Control: UT, n = 30, TTX, n = 36; SP TSB-LNA: UT, n = 37, TTX, n = 20; n indicates the number of cells from 2 cultures). *P < 0.05, ns, not significant, P > 0.05 (Kruskal-Wallis test followed by Dunn's multiple comparison test). Data information: Data are represented as mean ± SEM.


( A ) Micrographs showing puro - PLA staining of newly synthesized SP ( green ) in neurons immunostained for MAP - 2 ( magenta ) and DAPI ( blue ) and treated with TTX for 24 h or left untreated . The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody . Scale bars : 30 µm ( large view ) , 5 µm ( insets ) .

O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(A) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons immunostained for MAP-2 (magenta) and DAPI (blue) and treated with TTX for 24 h or left untreated. The images on bottom panels show no staining in the absence of puromycin treatment or when omitting SP primary antibody. Scale bars: 30 µm (large view), 5 µm (insets).


( C ) Density of SP puro - PLA puncta , normalized to the untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n represents the number of cells , from 3 cultures ) . * P < 0 . 05 ( Mann - Whitney test ) . ( D ) SP puro - PLA cluster fluorescence intensity normalized to the untreated condition ( UT ) ( UT : n = 62 ; TTX : n = 57 , n indicates the number of cells , from 3 cultures ) . * * * * P < 0 . 0001 ( Mann - Whitney test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-GENEPROD B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O 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-GENEPROD B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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) Density of SP puro-PLA puncta, normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n represents the number of cells, from 3 cultures). *P < 0.05 (Mann-Whitney test). (D) SP puro-PLA cluster fluorescence intensity normalized to the untreated condition (UT) (UT: n = 62; TTX: n = 57, n indicates the number of cells, from 3 cultures). ****P < 0.0001 (Mann-Whitney test). Data information: Data are represented as mean ± SEM.


( E ) Micrographs showing puro - PLA staining of newly synthesized SP ( green ) in neurons transfected with Homer1c - GFP ( magenta ) and treated with TTX for 24 h or left untreated . Scale bar : 5 µm . Arrowheads indicate puro - PLA + spines . ( F ) Percentage of SP puro - PLA + synapses ( UT , n = 26 , TTX , n = 33 ; n indicates the number of cells , from 2 cultures ) . * * P < 0 . 01 , ns , not significant ( Mann - Whitney test ) . ( G ) Homer1c - GFP integrated fluorescence intensity at SP puro - PLA - vs SP puro - PLA + synapses ( n = 32 cells , from 2 cultures ) . * * * * P < 0 . 0001 , ns , not significant ( Wilcoxon matched - pairs signed rank test ) . Data information : Data are represented as mean ± SEM .

O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-GENEPROD B-SMALL_MOLECULE O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-SMALL_MOLECULE O B-EXP_ASSAY O O B-GENEPROD B-SMALL_MOLECULE O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs showing puro-PLA staining of newly synthesized SP (green) in neurons transfected with Homer1c-GFP (magenta) and treated with TTX for 24 h or left untreated. Scale bar: 5 µm. Arrowheads indicate puro-PLA+ spines. (F) Percentage of SP puro-PLA+ synapses (UT, n = 26, TTX, n = 33; n indicates the number of cells, from 2 cultures). **P < 0.01, ns, not significant (Mann-Whitney test). (G) Homer1c-GFP integrated fluorescence intensity at SP puro-PLA- vs SP puro-PLA+ synapses (n = 32 cells, from 2 cultures). ****P < 0.0001, ns, not significant (Wilcoxon matched-pairs signed rank test). Data information: Data are represented as mean ± SEM.


( A ) Confocal images showing dendrites from CA3 pyramidal cells transfected with RFP - SP ( green ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR + SP - shRNA - BFP ( magenta ) . Arrowheads indicate SP + spines . Scale bar : 10 µm . ( B ) Graph plot showing spine head volume vs SP integrated intensity ( n = 5 cells ) . Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP + spines in the same neuron . Plots for SP - and SP + spines appear in grey and magenta , respectively . The black dotted line represents linear regression ( R2 = 0 . 25 , P = 0 . 0004 ) . ( C ) Paired data showing spine head volume for SP - vs SP + spines . Each pair of plot represents average values for SP - vs SP + spines for a given neuron . * P = 0 . 0031 ( two - tailed Student t test ) . Data information : Data are represented as mean ± SEM .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_TYPE O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-CELL_TYPE O O O 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 images showing dendrites from CA3 pyramidal cells transfected with RFP-SP (green) containing wild-type (WT) or mutated (MUT) 3'UTR + SP-shRNA-BFP (magenta). Arrowheads indicate SP+ spines. Scale bar: 10 µm. (B) Graph plot showing spine head volume vs SP integrated intensity (n = 5 cells). Each plot represents a single spine for which SP integrated intensity has been normalized to average SP intensity of SP+ spines in the same neuron. Plots for SP- and SP+ spines appear in grey and magenta, respectively. The black dotted line represents linear regression (R2 = 0.25, P = 0.0004). (C) Paired data showing spine head volume for SP- vs SP+ spines. Each pair of plot represents average values for SP- vs SP+ spines for a given neuron. *P = 0.0031 (two-tailed Student t test). Data information: Data are represented as mean ± SEM.


( F ) Higher magnification of dendritic spines from neurons transfected with rescue RFP - SP ( magenta ) containing wild - type ( WT ) or mutated ( MUT ) 3 ' UTR and BFP - shRNA - SP ( grey ) and contacted by GFP - Syp + terminals ( green ) . Arrowheads indicate putative synaptic contacts . Scale bar : 5 μm . ( G ) Percentage of SP + spines in neurons expressing RFP - SP - 3 ' UTR - WT vs - MUT and depending on the apposition or not to a GFP - Syp + terminal ( SP - 3 ' UTR - WT : TetTx - , n = 220 , TetTx + , n = 26 ; SP - 3 ' UTR - MUT : TetTx - , n = 616 , TetTx + , n = 25 ; n indicates the number of spines from 5 - 7 pairs ) . ( H ) Spine head volume in same conditions as in ( G ) ( SP - 3 ' UTR - WT : TetTx - , n = 26 , TetTx + , n = 26 spines from 5 pairs , 4 slices ; SP - 3 ' UTR - MUT : TetTx - , n = 25 , TetTx + , n = 25 ; n indicates the number of spines from 7 pairs , 7 slices ) . * * * P < 0 . 001 , * P < 0 . 05 , ns , not significant ( Kruskall - Wallis test followed by Dunn ' s multiple test ) . ( I ) Cumulative probability distributions of spine head volumes for TetTx - and TetTx + spines from neurons expressing RFP - SP - 3 ' UTR - WT ( SP - WT , light gray and light green ) or RFP - SP - 3 ' UTR - MUT ( SP - MUT , dark gray and dark green ) . Data information : Data are represented as mean ± SEM .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(F) Higher magnification of dendritic spines from neurons transfected with rescue RFP-SP (magenta) containing wild-type (WT) or mutated (MUT) 3'UTR and BFP-shRNA-SP (grey) and contacted by GFP-Syp+ terminals (green). Arrowheads indicate putative synaptic contacts. Scale bar: 5 μm. (G) Percentage of SP+ spines in neurons expressing RFP-SP-3'UTR-WT vs -MUT and depending on the apposition or not to a GFP-Syp+ terminal (SP-3'UTR-WT: TetTx-, n = 220, TetTx+, n = 26; SP-3'UTR-MUT: TetTx-, n = 616, TetTx+, n = 25; n indicates the number of spines from 5-7 pairs). (H) Spine head volume in same conditions as in (G) (SP-3'UTR-WT: TetTx-, n = 26, TetTx+, n = 26 spines from 5 pairs, 4 slices; SP-3'UTR-MUT: TetTx-, n = 25, TetTx+, n = 25; n indicates the number of spines from 7 pairs, 7 slices). ***P < 0.001, *P < 0.05, ns, not significant (Kruskall-Wallis test followed by Dunn's multiple test). (I) Cumulative probability distributions of spine head volumes for TetTx- and TetTx+ spines from neurons expressing RFP-SP-3'UTR-WT (SP-WT, light gray and light green) or RFP-SP-3'UTR-MUT (SP-MUT, dark gray and dark green). Data information: Data are represented as mean ± SEM.


Colony formation assay of miRNA mimic transfected - Dgcr8 - / - ESCs . There were 17 miRNAs that decreased the colony forming ability , 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S1 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-CELL_TYPE O O O 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-CELL_TYPE O O O O O O O O O O O O O O O

Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.


Alkaline phosphatase staining of ESCs after miRNA mimic transfection . There were 21 miRNAs that decreased the AP activity , 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S2 .

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Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.


Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8 - / - ESCs . Error bars indicate s . d . ( n = 3 ) . Full data are shown in Supplementary Fig S3A , B .

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Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.


Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs . 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies , 12 miRNAs mildly affected Oct4 expression , and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies . Representative pictures are shown . Full data are shown in Supplementary Fig S3C .

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Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.


AP staining of V6 . 5 ESCs after mimic transfection ( Full data are shown in Supplementary Fig S4B ) .

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AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).


B , C . Northern blot analysis of the expression of miR - 23a , miR - 27a and miR - 24 during EB formation ( B ) and RA induced differentiation of V6 . 5 ESCs ( C ) . The heatmap was representive of the results of quantitative analysis .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O O O O O O O O O

B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.


D , E . The expression of primary miR - 23a ~ 27a ~ 24 - 2 transcript during EB formation ( D ) and RA induced differentiation ( E ) . Error bars indicate s . d . ( n = 3 ) .

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D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).


F . Q - PCR analysis of miR - 23a , miR - 27a and miR - 24 expression in ESCs ( JM8A3 ) and a variety of C57BL / 6J adult tissues . Data are shown as mean + s . d . ( n = 3 ) .

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F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).


G . Relative protein levels of Oct4 , Sox2 and Nanog in miRNA mimic transfected Dgcr8 - / - ESCs and wild - type ESCs .

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G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.


H , I . Oct4 , Nanog , Foxa2 , Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8 - / - ( H ) and wild - type ESCs ( I ) .

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H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs(I).


Western blot analysis of the expression of target genes in miR - 27a or miR - 24 over - expressed V6 . 5 ESCs . Candidate target genes whose expression was decreased were framed .

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Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.


The relative luciferase activity of the reporter constructs co - transfected with scramble or miR - 27a and miR - 24 mimics . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 . Candidate target genes whose luciferase activity was decreased were framed . PC group was positive reporter control which harbored reverse complementary sequence of miR - 27a or miR - 24 .

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The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.


The relative luciferase activity of the wild - type 3 ′ UTR or mutated 3 ′ UTR reporter co - transfected with scramble or miR - 27a and miR - 24 mimics . Data are shown as mean + s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.


F , G RT - PCR and qRT - PCR analysis of in vivo binding of the miRNAs with their targets . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


H . Western blot analysis of c - Myc expression in siRNA treated V6 . 5 ESCs .

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H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.


I . Q - PCR analysis of primary - 23a ~ 27a ~ 24 - 2 , miR - 23a , miR - 27a and miR - 24 expression in c - Myc knocked down ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


K . ChIP - PCR and ChIP - q - PCR analysis of c - Myc binding to the putative sites in the promoter . Error bars indicate s . d . ( n = 3 ) ; * * * indicates P - value < 0 . 001 .

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K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.