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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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

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

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

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

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

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

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

(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 .

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(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 .

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(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 .

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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 .

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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 .

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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.


L . Relative luciferase activity of the wild - type and - 15 binding site mutated promoter in NIH3T3 cells co - transfected with c - Myc or the blank vector ( NC ) . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


M . Relative luciferase activity of wild - type and - 15 binding site mutated promoter in V6 . 5 ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


Validation of the effect of miRNA inhibition in MEFs by q - PCR . Error bars indicate s . d . ( n = 3 ) .

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Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).


AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control , inhibitors of miR - 23a , miR - 27a , miR - 24 and let - 7c respectively .

AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.


Fold increase in Oct4 - GFP - positive cell colonies in the above cells . Data are shown as mean + s . d . ( n = 5 ) ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Morphology of typical Oct4 - GFP - positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors .

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Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.


Detection of endogenous and exogenous Oct4 , Sox2 , Klf4 and Nanog expression in iPSCs , ESCs and MEFs by RT - PCR .

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Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.


Immunofluorescence staining of Oct4 , Nanog and SSEA1 of the iPSC colonies .

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Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.


Q - PCR analysis of the expression of self - renewal ( Oct4 , Sox2 , Nanog ) and differentiation ( Cdx2 , Foxa2 , Brachyury , Hand1 , Nestin , FGF5 ) relative markers in EBs derived from the iPSCs . Error bars indicate s . d . ( n = 3 ) .

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Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).


Hematoxylin and eosin staining of teratoma derived from the iPSC colonies . Neuronal rosette ( Ectoderm ) , Cartilage ( Mesoderm ) and respiratory epithelium ( Endoderm ) are marked with black arrows .

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Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.


Genomic PCR of the specific fragments spanning miR - 23 ~ 27 ~ 24 clusters . WT indicate wild - type ESCs ; DKO indicate miR - 23a ~ 27a ~ 24 - 2 - / - , miR - 23b ~ 27b ~ 24 - 1 - / - ESCs ; KO indicate miR - 23a ~ 27a ~ 24 - 2 - / - ESCs .

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Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.


Real - time PCR analysis of mature miR - 23a / b , miR - 27a / b and miR - 24 expression in wild - type , DKO and KO ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).


AP staining of wild - type and miR - 23 ~ 27 ~ 24 cluster knockout ESCs .

AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.


Real - time PCR analysis of Oct4 , Sox2 and Nanog expression in wild - type and miR - 23 ~ 27 ~ 24 cluster knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).


Oct4 , Sox2 and Nanog immunofluorescence staining of wild - type and the knockout ESCs .

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Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.


Real - time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild - type and knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Beating EB incidence during cardiac differentiation of wild - type and knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Real - time PCR analysis of several cardiac markers expression during cardiac differentiation of wild - type and knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Actinin , ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild - type and knockout ESCs .

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Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.


Volumes of teratomas at different days after injection . Data are shown as mean + s . d . ( n = 4 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Weight of teratomas derived from wild - type and knockout ESCs . Data are shown as mean ± s . d . * * indicates P - value < 0 . 01 .

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Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01.


Oct4 immunohistochemistry staining of teratomas derived from wild - type and knockout ESCs . Representative Oct4 positive regions are shown .

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Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown.


Hematoxylin and eosin staining of teratomas derived from wild - type and knockout ESCs . Ectoderm , Mesoderm and Endoderm are marked with different color arrows .

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Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.


Orthotopic MPNST xenografts at passages 1 ( OT P1 ) and 4 ( OT P4 ) were histopathologically similar to their corresponding primary MPNST ( PT ) in hematoxylin - eosin staining of paraffin - embedded tumor sections from patients MPNST - NF1 - 001 and MPNST - SP - 002 . Main panels show a general view of the tumors at low magnification ( 40 × ) ; inset pictures were taken at higher magnification ( 400 × ) .

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Orthotopic MPNST xenografts at passages 1 (OT P1) and 4 (OT P4) were histopathologically similar to their corresponding primary MPNST (PT) in hematoxylin-eosin staining of paraffin-embedded tumor sections from patients MPNST-NF1-001 and MPNST-SP-002. Main panels show a general view of the tumors at low magnification (40×); inset pictures were taken at higher magnification (400×).


Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features . A representative immunostained section of vimentin , CD34 , S100 , and Ki - 67 is shown for primary tumors ( PT ) and orthotopic tumors ( OT ) from patientsMPNST - NF1 - 001 and MPNST - SP - 002 . Positive antibody signals are shown in brown , and the hematoxylin counterstain in blue . Main panels show pictures at high magnification ( 400 × ) ; inset pictures show mitotic cells present in these tumors .

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Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patientsMPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.


Stromal elements of the primary tumors were labeled with anti - humanCD34 but not anti - mouseCD34 ; patient - derived xenografts are labeled with anti - mouseCD34 only and no anti - human marker . Representative sections ( at 40 × and 400 × magnification ) of the primary tumors ( PT ) and the orthoxenograft tumors at passages 1 ( OT P1 ) and 4 ( OT P4 ) were labeled with anti - humanCD34 ( H ) and anti - mouseCD34 ( M ) .

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Stromal elements of the primary tumors were labeled with anti-humanCD34 but not anti-mouseCD34; patient-derived xenografts are labeled with anti-mouseCD34 only and no anti-human marker. Representative sections (at 40× and 400× magnification) of the primary tumors (PT) and the orthoxenograft tumors at passages 1 (OT P1) and 4 (OT P4) were labeled with anti-humanCD34 (H) and anti-mouseCD34 (M).


Sanger sequencing of the germline NF1 mutation c . 350T > A present in the patient that developed two different tumors ( MPNST - NF1 - 001 and MPNST - NF1 - 002 ) . Sequencing results from normal tissue , primary tumor ( PT ) , and orthotopic xenograft tumors ( OT ) are shown . The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples , and not in the corresponding derived orthoxenografts , indicating probable loss of the human stromal cells .

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Sanger sequencing of the germline NF1 mutation c.350T>A present in the patient that developed two different tumors (MPNST-NF1-001 and MPNST-NF1-002). Sequencing results from normal tissue, primary tumor (PT), and orthotopic xenograft tumors (OT) are shown. The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples, and not in the corresponding derived orthoxenografts, indicating probable loss of the human stromal cells.


SNP array of primary tumor ( PT ) and orthotopic xenograft passages 1 ( OT P1 ) and 4 ( OT P4 ) for MPNST - NF1 - 001 and MPNST - NF1 - 002 tumors . Results correspond to chromosome 17 . Images show the B - allele frequency ( BAF ) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines : 2n regions are shown in gray , gained regions in orange , lost regions in green , and LOH regions are represented in blue . The vertical red line indicates the location of the NF1 locus . In addition , the percentage of tumor versus stromal cells for each sample is represented in a pie chart ( blue , stromal cells ; red , tumor cells ) .

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SNP array of primary tumor (PT) and orthotopic xenograft passages 1 (OT P1) and 4 (OT P4) for MPNST-NF1-001 and MPNST-NF1-002 tumors. Results correspond to chromosome 17. Images show the B-allele frequency (BAF) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines: 2n regions are shown in gray, gained regions in orange, lost regions in green, and LOH regions are represented in blue. The vertical red line indicates the location of the NF1 locus. In addition, the percentage of tumor versus stromal cells for each sample is represented in a pie chart (blue, stromal cells; red, tumor cells).


Tumor growth effects of treatment with doxorubicin , sorafenib , rapamycin and combinations thereof in the five MPNST xenograft models . Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value . Statistically significant differences are shown as * P < 0 . 05 and * * P < 0 . 001 versus control group by the Bonferroni test .

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Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test.


For long - term studies , a subgroup of treated mice ( n = 3 - 5 mice / group ) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses ( usually 1 , 500 - 2 , 000 mm3 ) . The graph illustrates differences in the time delay ( in days ) of relapsed tumor masses for the different treatments .

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For long-term studies, a subgroup of treated mice (n = 3-5 mice/group) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses (usually 1,500-2,000 mm3). The graph illustrates differences in the time delay (in days) of relapsed tumor masses for the different treatments.


D Distributions of pairwise Ka / Ks ( nonsynonymous to synonymous nucleotide substitution rates ) ratios between PIWI duplicates in arthropods ( n = 17 in Drosophila and n = 37 in others , Mann - Whitney U test , * P < 0 . 05 ) . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value .

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D Distributions of pairwise Ka/Ks (nonsynonymous to synonymous nucleotide substitution rates) ratios between PIWI duplicates in arthropods (n = 17 in Drosophila and n = 37 in others, Mann-Whitney U test, *P<0.05). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.


A PIWI genes are present in the locust brain . Rp49 served as a loading control ( n = 4 biological replicates ) . Data information : data are presented as mean ± SEM .

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A PIWI genes are present in the locust brain. Rp49 served as a loading control (n=4 biological replicates). Data information: data are presented as mean ± SEM.


C The fluorescence signals of PIWI proteins were determined in locust brains . Hochest33342 was used to label cell nuclei in locust brains . The scale bars represent 20 µm .

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C The fluorescence signals of PIWI proteins were determined in locust brains. Hochest33342 was used to label cell nuclei in locust brains. The scale bars represent 20 µm.


G Food intake by dsPiwi1 - treated locusts and dsGFP - treated control locusts within 1 h ( n = 5 biological replicates ) , * * * P < 0 . 001 ( Student ' s t - test ) . Data information : All assays were performed on fourth - instar locusts .

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G Food intake by dsPiwi1-treated locusts and dsGFP-treated control locusts within 1 h (n=5 biological replicates) , ***P<0.001 (Student's t-test). Data information: All assays were performed on fourth-instar locusts.


C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . The scale bars represent 50 µm . D Quantification of lipid droplet staining in dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . E Data information : All data are presented as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . All assays were performed on four - day - old fourth - instar locusts .

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C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). The scale bars represent 50 µm. D Quantification of lipid droplet staining in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). E Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.


E , F Measurements of glycogen ( n = 5 biological replicates ) and TAGs ( n = 10 biological replicates ) in fat bodies . Data information : All data are presented as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . All assays were performed on four - day - old fourth - instar locusts .

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E, F Measurements of glycogen (n=5 biological replicates) and TAGs (n=10 biological replicates) in fat bodies. Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.


B Food intake within 1 h following treatment with candidate genes ( dsNPF1 - treated , n = 4 biological replicates ; dsCG10621 - treated , n = 5 biological replicates ; dsMFS3 - treated , n = 5 biological replicates ) , the data are shown as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 ) .

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B Food intake within 1 h following treatment with candidate genes (dsNPF1-treated, n=4 biological replicates; dsCG10621-treated, n=5 biological replicates; dsMFS3-treated, n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, **P<0.01).


D , E Representative images ( D ) and quantification ( E ) of Nile red staining of fat body lipid droplets in dsNPF1 - treated locusts and dsGFP - treated controls ( n = 5 biological replicates ) . The scale bars represent 50 µm , the data are shown as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 ) .

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D, E Representative images (D) and quantification (E) of Nile red staining of fat body lipid droplets in dsNPF1-treated locusts and dsGFP-treated controls (n=5 biological replicates). The scale bars represent 50 µm, the data are shown as the mean ± SEM (Student's t-test, **P<0.01).


G Food intake within 1 h in the food instake assays ( n = 5 biological replicates ) , the data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * * P < 0 . 001 ) . H Food intake within 2 h in the food instake assays ( n = 5 biological replicates ) . Five locusts were included in each replicate , the data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 ) .

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G Food intake within 1 h in the food instake assays (n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, *P<0.05, ***P<0.001) . H Food intake within 2 h in the food instake assays (n=5 biological replicates). Five locusts were included in each replicate, the data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01).


B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs - 3 - I2 and piRs - 3 - I3 was injected ( n = 5 biological replicates ) . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs-3-I2 and piRs-3-I3 was injected (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


E Body weight measurement of locusts administered inhibitors ( NC , n = 25 locusts ; Inhibitors - piRs - 3 - I2 + I3 , n = 23 locusts ) and mimics ( NC , n = 37 locusts ; Inhibitors - piRs - 3 - I2 + I3 , n = 39 locusts ) of piRs - 3 - I2 and piRs - 3 - I3 . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value .

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E Body weight measurement of locusts administered inhibitors (NC, n=25 locusts; Inhibitors- piRs-3-I2+I3, n=23 locusts) and mimics (NC, n=37 locusts; Inhibitors-piRs-3-I2+I3, n=39 locusts) of piRs-3-I2 and piRs-3-I3. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.


F RNA - binding protein immunoprecipitation assay of piRs - 3 - I2 and piRs - 3 - I3 in Piwi1 immunoprecipitates from brain tissue extracts ( n = 6 biological replicates ) . Normal mouse IgG was used as a negative control .

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F RNA-binding protein immunoprecipitation assay of piRs-3-I2 and piRs-3-I3 in Piwi1 immunoprecipitates from brain tissue extracts (n=6 biological replicates). Normal mouse IgG was used as a negative control.


A U6 RNA was used as a nuclear RNA control ( relative value set to 1 in the nuclear fraction ) , and glycine tRNA was used as the cytoplasmic RNA control ( relative value set to 1 in the cytoplasmic fraction ) ( n = 4 biological replicates ) , U6 and tRNA - Gly was used as an endogenous control .

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A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear fraction), and glycine tRNA was used as the cytoplasmic RNA control (relative value set to 1 in the cytoplasmic fraction) (n=4 biological replicates), U6 and tRNA-Gly was used as an endogenous control.


B Relative expressions of piRs - 3 - I2 and piRs - 3 - I3 determined by qPCR and high - throughput sequencing in the nucleus ( n = 3 biological replicates ) . The brain tissues were homogenized and then divided into two parts of equal volume ; one was prepared for high - throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation . The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high - throughput sequencing libraries . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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B Relative expressions of piRs-3-I2 and piRs-3-I3 determined by qPCR and high-throughput sequencing in the nucleus (n=3 biological replicates). The brain tissues were homogenized and then divided into two parts of equal volume; one was prepared for high-throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation. The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high-throughput sequencing libraries. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


C Expression level of pre - NPF1 in the nucleus after a mixture of inhibitors ( piRs - 3 - I2 and piRs - 3 - I3 ) was injected ( n = 4 biological replicates ) . D qPCR expression analyses of pre - NPF1 . The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments ( n = 6 biological replicates ) . E Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C Expression level of pre-NPF1 in the nucleus after a mixture of inhibitors (piRs-3-I2 and piRs-3-I3) was injected (n=4 biological replicates). D qPCR expression analyses of pre-NPF1. The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments (n=6 biological replicates). E Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


F Analysis was performed to amplify pre - NPF1 and piRs - 3 - I3 in hnRNP F / H immunoprecipitates from extracts of brain tissue treated with piRs - 3 - I3 mimics compared to the negative controls ( pre - NPF1 n = 3 biological replicates ; piRs - 3 - I3 n = 4 biological replicates ) . G Analysis was performed to amplify pre - NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs - 3 - I3 mimics compared to the negative controls ( pre - NPF1 n = 3 biological replicates ) .

F Analysis was performed to amplify pre-NPF1 and piRs-3-I3 in hnRNP F/H immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates; piRs-3-I3 n=4 biological replicates). G Analysis was performed to amplify pre-NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates). H


J Expression level of the mature NPF1 gene and pre - NPF1 in 293T cells transfected with pre - NPF1 and mutated pre - NPF1 ( n = 5 biological replicates ) . NC means negative control . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

J Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


NE activates mTORC2 in vitro via cAMP , PI3K and Epac1 . ( A ) Immunoblot analysis of BAT cells stimulated with norepinephrine ( NE ) for the indicated proteins .

NE activates mTORC2 in vitro via cAMP, PI3K and Epac1. (A) Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins.


( B ) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin ( Rapa ) , Torin or Wortmannin ( Wrtm ) for the indicated proteins .

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(B) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin (Rapa), Torin or Wortmannin (Wrtm) for the indicated proteins.


( C ) Immunoblot analysis of BAT cells stimulated with 8 - Br - cAMP for 5 minutes in the presence of Rapa , Torin or Wrtm for the indicated proteins .

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(C) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Rapa, Torin or Wrtm for the indicated proteins.


( D ) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm , H89 or ESI - 09 for the indicated proteins .

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(D) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins.


( E ) Immunoblot analysis of BAT cells stimulated with 8 - Br - cAMP for 5 minutes in the presence of Wrtm , H89 or ESI - 09 for the indicated proteins . All experiments were performed in triplicates , a representative replicate is presented .

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(E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.