( G - H ) Cell migration tracks where each coloured track depicts the distance ( µm ) travelled over 3 . 5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast ( PHS - L - derived FLS tip ( blue cells ) to VA - L ( F , red cells ; n = 5 / 5 ) ) or by the VA - L to the PHS - L - derived FLS tip ( F , n = 5 / 5 ) .

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

(G-H) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3.5 hours by the leading tip cell in a separate animal before and after fusion with another lymphangioblast (PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (F, n = 5/5).


( A - B ' ) Lateral ( A ) and ventrolateral ( B , B ' ) images of the cranial vessels in kdrl : nlsKaede ; kdrl : EGFP embryos , with unconverted tissue shown in green ( A - B ) while photoconverted tissue is shown in red ( B ' ) . The early kdrl - positive VA - L was photoconverted in 34 hpf embryos ( A ) , and followed through to 54 hpf ( B , B ' ) . B ' is a higher magnification image of the box in B and is accompanied by a red channel only ( traced photoconverted cells ) image with the VA - L ( dotted line ) demarcated from the adjacent VA . The VA - L photoconverted cells are indicated by arrowheads .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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') Lateral (A) and ventrolateral (B, B') images of the cranial vessels in kdrl:nlsKaede;kdrl:EGFP embryos, with unconverted tissue shown in green (A-B) while photoconverted tissue is shown in red (B'). The early kdrl-positive VA-L was photoconverted in 34 hpf embryos (A), and followed through to 54 hpf (B, B'). B' is a higher magnification image of the box in B and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA. The VA-L photoconverted cells are indicated by arrowheads.


( C ) Still images from Movie EV5 of VA - L development in a kdrl : EGFP ; kdrl : nlsmCherry compound transgenic from 31 - 35 . 5 hpf . This movie shows that the VA - L begins expressing kdrl just after 32 hpf , in a manner that is isolated and independent of any neighbouring kdrl - positive tissue or vessel . Putative kdrl - positive primitive myeloid cells ( light blue asterisks ) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA - L . ( D ) Still images from Movie EV6 depicting a 3D reconstruction of the 35 . 5 hpf timepoint from Movie EV5 . A dorsal view that scrolls through the Y - stack of the VA - L is shown , which illustrates that the VA - L is closely associated with , but not connected to the VA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Still images from Movie EV5 of VA-L development in a kdrl:EGFP;kdrl:nlsmCherry compound transgenic from 31-35.5 hpf. This movie shows that the VA-L begins expressing kdrl just after 32 hpf, in a manner that is isolated and independent of any neighbouring kdrl-positive tissue or vessel. Putative kdrl-positive primitive myeloid cells (light blue asterisks) can also be seen migrating along the surface of the yolk in the immediate vicinity of the VA and VA-L. (D) Still images from Movie EV6 depicting a 3D reconstruction of the 35.5 hpf timepoint from Movie EV5. A dorsal view that scrolls through the Y-stack of the VA-L is shown, which illustrates that the VA-L is closely associated with, but not connected to the VA.


( E - E ' ) Still images from Movie EV7 of VA - L development in an etv2 : EGFP ( E ) , kdrl : nlsmCherry ( E ' ) compound transgenic from 28 - 35 . 2 hpf . This movie shows that the VA - L begins expressing etv2 ( E ) at 29 hpf and is independent of any neighbouring vessel . kdrl expression ( red arrowheads ) occurs 4 hours later in the VA - L ( demarcated by the green dotted line ) ( E ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E') Still images from Movie EV7 of VA-L development in an etv2:EGFP (E), kdrl:nlsmCherry (E') compound transgenic from 28-35.2 hpf. This movie shows that the VA-L begins expressing etv2 (E) at 29 hpf and is independent of any neighbouring vessel. kdrl expression (red arrowheads) occurs 4 hours later in the VA-L (demarcated by the green dotted line) (E').


( F - G ' ) Lateral images of the cranial vessels in etv2 : Kaede embryos , with unconverted tissue shown in green ( F - G ) while photoconverted tissue is shown in red ( G ' ) . The earliest visible form of the VA - L was photoconverted at 29 hpf ( F ) , then followed through to 54 ( G ) . G ' is a higher magnification image of the box in G and is accompanied by a red channel only ( traced photoconverted cells ) image with the VA - L ( dotted line ) demarcated from the adjacent VA , and VA - L photoconverted cells indicated ( arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G') Lateral images of the cranial vessels in etv2:Kaede embryos, with unconverted tissue shown in green (F-G) while photoconverted tissue is shown in red (G'). The earliest visible form of the VA-L was photoconverted at 29 hpf (F), then followed through to 54 (G). G' is a higher magnification image of the box in G and is accompanied by a red channel only (traced photoconverted cells) image with the VA-L (dotted line) demarcated from the adjacent VA, and VA-L photoconverted cells indicated (arrowheads).


( A - G ) Lateral ( A - B ) , ventrolateral ( C - F ) and ventral ( G ) images of the cranial vessels in an etv2 : EGFP ; kdrl : nlsmCherry compound transgenic embryo , which has had the etv2 - expressing angioblast population ( early VA - L ) on the left lateral side of the yolk sac ablated . Images were taken at 30 hpf before ( A ) and after ( B ) ablation of the early VA - L , with the site of ablation indicated ( B ; blue dotted circle ) . Note that the LDA and VA adjacent to the VA - L become photobleached during the ablation process ( B ) , however these were not ablated as they can be seen developing normally at 54 hpf ( D ) . Follow - up images were taken of both the contralateral ( C , E ) and ablated ( D , F ) sides of the embryo ' s head at 54 hpf ( C - D ) and 78 hpf ( E - G ) . Although the VA - L ( light blue dotted line ) is seen migrating towards the FLS ( white dotted lined ) on the contralateral side at 54 hpf ( C ) , it is missing from the ablated side ( D ) . Subsequently , the LFL ( white dotted line ) of the ablated side is short ( F ) compared to that of the contralateral side ( E ) . In addition , the HA ( pink dotted line ) is underdeveloped on the ablated side compared to the contralateral side , as it no longer extends towards nor connects to the left AA1 ( G ) . ( H ) Quantitation of the length of the LFL ( µm ) on the contralateral ( control ) side and on the ablated side at 78 hpf ( n = 16 ) . ( I ) Quantitation of the total number of cells within the LFL on the contralateral ( control ) side and from the ablated side at 78 hpf ( n = 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Lateral (A-B), ventrolateral (C-F) and ventral (G) images of the cranial vessels in an etv2:EGFP;kdrl:nlsmCherry compound transgenic embryo, which has had the etv2-expressing angioblast population (early VA-L) on the left lateral side of the yolk sac ablated. Images were taken at 30 hpf before (A) and after (B) ablation of the early VA-L, with the site of ablation indicated (B; blue dotted circle). Note that the LDA and VA adjacent to the VA-L become photobleached during the ablation process (B), however these were not ablated as they can be seen developing normally at 54 hpf (D). Follow-up images were taken of both the contralateral (C, E) and ablated (D, F) sides of the embryo's head at 54 hpf (C-D) and 78 hpf (E-G). Although the VA-L (light blue dotted line) is seen migrating towards the FLS (white dotted lined) on the contralateral side at 54 hpf (C), it is missing from the ablated side (D). Subsequently, the LFL (white dotted line) of the ablated side is short (F) compared to that of the contralateral side (E). In addition, the HA (pink dotted line) is underdeveloped on the ablated side compared to the contralateral side, as it no longer extends towards nor connects to the left AA1 (G). (H) Quantitation of the length of the LFL (µm) on the contralateral (control) side and on the ablated side at 78 hpf (n = 16). (I) Quantitation of the total number of cells within the LFL on the contralateral (control) side and from the ablated side at 78 hpf (n = 16).


( A ) Top panel displays structure of each CDK4 / 6 inhibitor tested . Bottom panel shows dose response curves with these inhibitors displaying percentage of G1 - arrested RPE1 - FUCCI cells . To obtain dose response curves , the number of mKO - Cdt1 positive ( G1 - arrested ) cells were calculated following 24hr drug addition ( dark blue solid lines ) or 24h after subsequent drug washout ( light blue dotted lines ) . Cmax values observed in patients ( taken from ( He et al . , 2017 ; Klein et al . , 2018 ) ) are represented on each graph with red dotted lines . Graphs display mean data - / + SEM from 3 experiments , with at least 500 cells counted per condition per experiment . ( B ) Percentage of G1 - arrested RPE1 - FUCCI cells , calculated as in panel A , but using a fixed concentration of CDK4 / 6 inhibitor for different durations of time , as indicated . Note , this is a fixed assay that quantifies the percentage of G1 - arrested cells prior to , or 24 hrs following , CDK4 / 6 inhibitor washout . Each bar displays mean data + SEM from 3 experiments , with at least 500 cells counted per condition per experiment . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top panel displays structure of each CDK4/6 inhibitor tested. Bottom panel shows dose response curves with these inhibitors displaying percentage of G1-arrested RPE1-FUCCI cells. To obtain dose response curves, the number of mKO-Cdt1 positive (G1-arrested) cells were calculated following 24hr drug addition (dark blue solid lines) or 24h after subsequent drug washout (light blue dotted lines). Cmax values observed in patients (taken from (He et al., 2017; Klein et al., 2018)) are represented on each graph with red dotted lines. Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (B) Percentage of G1-arrested RPE1-FUCCI cells, calculated as in panel A, but using a fixed concentration of CDK4/6 inhibitor for different durations of time, as indicated. Note, this is a fixed assay that quantifies the percentage of G1-arrested cells prior to, or 24 hrs following, CDK4/6 inhibitor washout. Each bar displays mean data + SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (


( A ) Dose response curves displaying the percentage of G1 - arrested p53 - WT ( blue ) or KO ( green ) RPE1 - FUCCI cells following 24hr incubation with palbociclib ( dark solid lines ) or 24h after subsequent washout ( light dotted lines ) . Graphs display mean data - / + SEM from 3 experiments , with at least 500 cells counted per condition per experiment .

O O O O O O O O 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

(A) Dose response curves displaying the percentage of G1-arrested p53-WT (blue) or KO (green) RPE1-FUCCI cells following 24hr incubation with palbociclib (dark solid lines) or 24h after subsequent washout (light dotted lines). Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.


( C ) Representative images and quantifications of colony forming assays in p53 - WT or KO RPE1 cells treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days and then grown at low density without inhibitor for 10 days . Each bar displays mean data + SEM from 3 experiments .

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

(C) Representative images and quantifications of colony forming assays in p53-WT or KO RPE1 cells treated with palbociclib (1.25μM) for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Each bar displays mean data + SEM from 3 experiments.


( E ) Quantification of cell cycle profiles from cells treated as in D . Graph shows the mean percentage of cells + SD that divide at each round of division , with 150 cells analysed in total from 3 experimental repeats .

O O O O O O O O O O O O O O O O O O O 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) Quantification of cell cycle profiles from cells treated as in D. Graph shows the mean percentage of cells + SD that divide at each round of division, with 150 cells analysed in total from 3 experimental repeats.


( A ) Representative immunofluorescence images of p21 levels in p53 - WT or KO RPE1 cells , 48 hours after release from 1 , 4 or 7 days palbociclib ( 1 . 25μM ) treatment . Zoom inserts are 3x magnification of the indicated regions . Scale bars = 250 μM . ( B ) Quantification of p21 intensities in cells treated as in panel A . At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats . Violin plots display the variation in intensities between individual cells . Horizontal lines display the median , and error bars show 95 % confidence intervals . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM. (B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals. (


( D - E ) Quantification of nuclear morphologies ( D ) and γH2AX - positive DNA damage foci ( E ) following palbociclib ( 1 . 25μM ) treatment in p53 WT and KO RPE1 cells . Cells were treated for 1 , 4 or 7 days and then analysed before or after drug washout for 48 hours . 100 cells ( nuclear morphology ) or 50 cells ( γH2AX foci ) were scored per condition per experiment , and bar graphs represent mean data + SEM from 6 experiments .

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

(D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments.


( J - K ) Representative immunofluorescence images ( J ) and quantifications ( K ) of mitotic DNA replication assays ( MiDAS ) in p53 - KO RPE1 cells released from 7 days of palbociclib ( 1 . 25μM ) treatment or following 0 . 4uM aphidicolin treatment for 40Hrs . EdU foci were quantified in nocodazole - arrested cells . Scale bar = 5 μM , zoom inserts = 3x magnification of highlighted areas . 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.


( A ) Volcano plot of proteins up or downregulated following prolonged palbociclib ( 1 . 25μM ) treatment in RPE1 cells . The top 10 significantly upregulated and downregulated proteins are shown in blue and red , respectively .

O O O O O O O O O O O O O O O O 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) Volcano plot of proteins up or downregulated following prolonged palbociclib (1.25μM) treatment in RPE1 cells. The top 10 significantly upregulated and downregulated proteins are shown in blue and red, respectively.


( D ) Representative western blots of whole cell lysates from RPE1 - WT cells treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days , or treated identically , and then washed out for the indicated times to reflect when the majority of cells are in S - phase ( see Figure 1C ) .

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

(D) Representative western blots of whole cell lysates from RPE1-WT cells treated with palbociclib (1.25μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times to reflect when the majority of cells are in S-phase (see Figure 1C).


( F - G ) Representative images ( F ) and quantifications ( G ) of colony forming assays with RPE1 cells treated with palbociclib ( 1 . 25μM ) for indicated times , and then grown at low density without palbociclib for 10 days . DMSO ( control ) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout . Each bar displays mean data + SD from 4 experiments .

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 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

(F-G) Representative images (F) and quantifications (G) of colony forming assays with RPE1 cells treated with palbociclib (1.25μM) for indicated times, and then grown at low density without palbociclib for 10 days. DMSO (control) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout. Each bar displays mean data + SD from 4 experiments.


( A ) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib ( 1 μM ) for 1 , 4 or 7 days , or treated identically , and then washed out for the indicated times ( see Figure 1C ) . ( B ) Analysis of adjusted relative density from 3 independent western blot experiments . Bars display mean values - / + SD . Significance determined by unpaired Student ' s T test comparing treated target protein to asynchronous target control . ( * < 0 . 01 , * * < 0 . 001 , * * * < 0 . 0001 ) .

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

(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C). (B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001).


( C , D ) Quantification of the nuclear morphologies ( C ) or γH2AX - positive DNA damage foci ( D ) from MCF7 , MCF7 p53 KO or T47D cells that were treated with palbociclib ( 1μM ) for 0 , 1 , 4 , or 7 days and then analysed 48hr after drug washout . Either 100 cells ( nuclear morphology ) or 50 cells ( γH2AX ) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments .

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

(C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( F ) Quantification of colony forming assays of MCF7 , MCF7 p53 KO and T47D cells treated with CDK4 / 6 inhibitor for 0 , 1 , 4 or 7 days and then grown at a low density without palbociclib for 14 - 21 days . Bar graphs displays mean data + SEM from 4 - 5 experiments .

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

(F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.


( B , C ) Quantification of the nuclear morphologies ( B ) or γH2AX - positive DNA damage foci ( C ) from different cancer lines , as indicated , treated continuously with palbociclib ( 1μM ) for 0 , 1 , 2 , or 3 weeks . Either 100 cells ( nuclear morphology ) or 50 cells ( γH2AX ) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments .

O O O O O O O O O O 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

(B,C) Quantification of the nuclear morphologies (B) or γH2AX-positive DNA damage foci (C) from different cancer lines, as indicated, treated continuously with palbociclib (1μM) for 0, 1, 2, or 3 weeks. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( D ) Quantification of weekly proliferation rate in cells treated as in B . A total of 60 , 000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks . Every 7 days , cells were trypsinised and total cell counts were determined . The 0 - week timepoint shows the 7 - day fold increase of untreated cells . The data points show fold increase in cell count over each 7 - day period , and bars represent the mean from 2 experiments .

O O O O O O O O O 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

(D) Quantification of weekly proliferation rate in cells treated as in B. A total of 60,000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks. Every 7 days, cells were trypsinised and total cell counts were determined. The 0-week timepoint shows the 7-day fold increase of untreated cells. The data points show fold increase in cell count over each 7-day period, and bars represent the mean from 2 experiments.


Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM - KO mice ( n = 5 mice per group ) .

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

Mean ± SEM CB2 mRNA levels in cerebellar extracts of WT and ASM-KO mice (n = 5 mice per group).


Western blot against CB2 and PSD - 95 ( used as loading control ) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM - KO mice ( n = 6 mice per group ) .

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

Western blot against CB2 and PSD-95 (used as loading control) and graph showing mean ± SEM CB2 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 6 mice per group).


Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM - KO mice ( * * P = 0 . 0013 , n = 6 mice per group , Student ' s t - test ) .

Mean ± SEM CB1 mRNA levels in cerebellar extracts of WT and ASM-KO mice (**P = 0.0013, n = 6 mice per group, Student's t-test).


Western blot against CB1 and GAPDH ( used as loading control ) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM - KO mice ( n = 3 mice per group ) .

Western blot against CB1 and GAPDH (used as loading control) and graph showing mean ± SEM CB1 protein levels in cerebellar extracts of WT and ASM-KO mice (n = 3 mice per group).


Immunofluorescence images against CB1 and the following cellular markers : Calbindin for Purkinje cells ( E ) , GFAP for astrocytes ( F ) and F4 / 80 for microglia ( G ) in the cerebellum of WT and ASM - KO mice . DAPI stains cell nuclei . Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells ( shown by white arrows , E ) , astrocytes ( F ) and microglia ( G ) expressed as percentage of the values obtained in WT mice . Graphs to the right show the Mander ' s coefficient that indicates degree of co - localization between CB1 and the cellular markers for Purkinje cells ( E ) , astrocytes ( F ) and microglia ( G ) ( E : * * PFluorecence intensity = 0 . 0082 , * PMander ' s Coefficient = 0 . 0158 ; F : * PMander ' s Coefficient = 0 . 0115 ) ; n = 5 mice per group , Student ' s t - test ) . Scale bar , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and the following cellular markers: Calbindin for Purkinje cells (E), GFAP for astrocytes (F) and F4/80 for microglia (G) in the cerebellum of WT and ASM-KO mice. DAPI stains cell nuclei. Graphs to the left show mean ± SEM intensity associated to CB1 in the Purkinje cells (shown by white arrows, E), astrocytes (F) and microglia (G) expressed as percentage of the values obtained in WT mice. Graphs to the right show the Mander's coefficient that indicates degree of co-localization between CB1 and the cellular markers for Purkinje cells (E), astrocytes (F) and microglia (G) (E: **PFluorecence intensity = 0.0082, *PMander's Coefficient = 0.0158; F: *PMander's Coefficient = 0.0115); n = 5 mice per group, Student's t-test). Scale bar, 100 μm.


H . Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age - matched control and ASMD - affected children . Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child ( 16 and 15 replicates in control and ASMD , respectively ) . Scale bar , 10 μm .

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

H. Immunofluorescence images against CB1 and the Purkinje cell marker calbindin in the cerebellum of age-matched control and ASMD-affected children. Graph shows mean ± SEM intensity associated to CB1 in the Purkinje cells expressed as percentage of the values obtained in the control child (16 and 15 replicates in control and ASMD, respectively). Scale bar, 10 μm.


Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM - KO mice ( * * * * P < 0 . 0001 , n = 5 independent cultures , Student ' s t - test ) .

Mean ± SEM CB1 mRNA levels in cultured hippocampal neurons from WT and ASM-KO mice (****P < 0.0001, 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 in cultured neurons from WT and ASM - KO mice ( * * * P = 0 . 0009 , n = 5 independent cultures , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 in cultured neurons from WT and ASM-KO mice (***P= 0.0009, n = 5 independent cultures, Student's t-test).


Immunofluorescence images against CB1 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM - KO mice . TOPRO stains cell nuclei . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and the lysosomal marker Lamp1 in cultured neurons from WT and ASM-KO mice. TOPRO stains cell nuclei. 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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-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

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-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 B-CONTROLLED_VAR 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 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

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 )

O O O O O O O O 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

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 .

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

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 ) .

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

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-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

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

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 .

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

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 ) .

O O O O O O O O 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

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

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 .

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

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 O O 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

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 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 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

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 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 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

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 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

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

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-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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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 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 ) .

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

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 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 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

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

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

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 .

O O 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 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

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 .

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

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 .

O O O O O O O O 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

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-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-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

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 .

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

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 ) .

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

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

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

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 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

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

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

D Representative 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 O O 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 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 O O O O O O O O O O O O O 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 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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O 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 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

(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 O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

(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 O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.