( C ) Met5 - A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( D ) The percentage of BrdU positive cells from ( C ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

(C) Met5-A cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (D) The percentage of BrdU positive cells from (C) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.01, ***P<0.001.


( E ) Meso - 33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( F ) The percentage of BrdU positive cells from ( E ) was quantified . Mean ± s . e . m , N = 3 biological repeats , ordinary one - way ANOVA . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Meso-33 cells transduced with vector or the indicated Merlin forms were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (F) The percentage of BrdU positive cells from (E) was quantified. Mean±s.e.m, N=3 biological repeats, ordinary one-way ANOVA. *P<0.05, **P<0.01.


( G ) Meso - 33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay . The percentage of BrdU positive cells was quantified . Mean ± s . e . m , N = 3 biological repeats , two - 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 O 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) Meso-33 cells stably transduced with the indicated shRNAs targeting NEDD4L or a scrambled shRNA were then transduced with vector or Merlin prior to the BrdU incorporation assay. The percentage of BrdU positive cells was quantified. Mean±s.e.m, N=3 biological repeats, two-way ANOVA.


( H ) FC - 1801 cells stably transduced with vector or the indicated Merlin forms were subjected to the sphere formation assay . Scale bar = 500 μm . ( I ) The number of spheres in each well from ( H ) was quantified . Mean ± s . e . m , N = 3 biological repeats , paired t - test . * * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subjected to the sphere formation assay. Scale bar = 500 μm. (I) The number of spheres in each well from (H) was quantified. Mean±s.e.m, N=3 biological repeats, paired t-test. **P<0.05.


( J ) FC - 1801 cells stably transduced with vector or the indicated Merlin forms were subcutaneously injected into nude mice . Tumor sizes in each group were quantified and plotted . Mean ± s . e . m , NVector = 8 mice , NWT = 10 mice , NK396R = 10 mice , NS518D = 8 mice , two - way ANOVA . P values of the last measurements ( day 37 after injection ) are shown . * P < 0 . 05 , * * P < 0 . 01 . ( K ) Tumors were collected when the vector group in ( J ) reached the end point ( day 37 after injection ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FC-1801 cells stably transduced with vector or the indicated Merlin forms were subcutaneously injected into nude mice. Tumor sizes in each group were quantified and plotted. Mean±s.e.m, NVector=8 mice, NWT=10 mice, NK396R=10 mice, NS518D=8 mice, two-way ANOVA. P values of the last measurements (day 37 after injection) are shown. *P<0.05, **P<0.01. (K) Tumors were collected when the vector group in (J) reached the end point (day 37 after injection).


( B ) Representative large image of a brain slice from R6 / 2 mice after 4 - week striatal infusion of fluorescent cholesterol ( bodipy - chol , green ) . In the inlet , infused striatum is shown . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bar : 1000 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-MEASURED_VAR 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

(B) Representative large image of a brain slice from R6/2 mice after 4-week striatal infusion of fluorescent cholesterol (bodipy-chol, green). In the inlet, infused striatum is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar: 1000 µm.


( C ) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt ( N = 7 ) and R6 / 2 ( N = 4 ) , R6 / 2 ACSF ( N = 5 ) , R6 / 2 chol - low ( N = 5 ) , R6 / 2 chol - medium ( N = 5 ) , and R6 / 2 chol - high ( N = 5 ) mice at 12 weeks of age after 4 - week striatal cholesterol infusion . The low , medium , and high doses respectively correspond to 15 µg , 185 µg , and 369 µg of cholesterol infused in the striatum after 4 weeks . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(C) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt (N=7) and R6/2 (N=4), R6/2 ACSF (N=5), R6/2 chol-low (N=5), R6/2 chol-medium (N=5), and R6/2 chol-high (N=5) mice at 12 weeks of age after 4-week striatal cholesterol infusion. The low, medium, and high doses respectively correspond to 15 µg, 185 µg, and 369 µg of cholesterol infused in the striatum after 4 weeks. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( D ) Latency to fall ( seconds ) from an accelerating rotarod from 5 weeks ( pre - surgery ; i . e . , before pump implantation ) to 10 weeks of age in wt ( N = 23 at 5 weeks , N = 28 at 8 and 10 weeks ) , R6 / 2 ACSF ( N = 35 at 5 weeks , N = 31 at 8 weeks and N = 30 at 10 weeks ) , R6 / 2 chol - low ( N = 22 at 5 weeks , N = 14 at 8 weeks and N = 13 ) , R6 / 2 chol - medium ( N = 13 at 5 weeks , N = 9 at 8 weeks and N = 8 at 10 weeks ) , and R6 / 2 chol - high ( N = 19 at 5 weeks , N = 17 at 8 and 10 weeks ) mice . The graph shows means ± standard error for each time point . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Latency to fall (seconds) from an accelerating rotarod from 5 weeks (pre-surgery; i.e., before pump implantation) to 10 weeks of age in wt (N=23 at 5 weeks, N=28 at 8 and 10 weeks), R6/2 ACSF (N=35 at 5 weeks, N=31 at 8 weeks and N=30 at 10 weeks), R6/2 chol-low (N=22 at 5 weeks, N=14 at 8 weeks and N=13), R6/2 chol-medium (N=13 at 5 weeks, N=9 at 8 weeks and N=8 at 10 weeks), and R6/2 chol-high (N=19 at 5 weeks, N=17 at 8 and 10 weeks) mice. The graph shows means±standard error for each time point. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


Global motor activity ( E ) , in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

Global motor activity (E), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


total distance traveled ( F ) , mean velocity ( G ) , stereotyped movements ( H ) , in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

total distance traveled (F), mean velocity (G), stereotyped movements (H), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


number of rearings ( I ) in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

number of rearings (I) in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( J , K ) Representative track plots ( J ) generated from the open - field test from wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 26 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 11 at 9 weeks , N = 7 at 11 weeks ) , R6 / 2 chol - medium ( N = 11 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high mice ( N = 18 at 9 weeks , N = 16 at 11 weeks ) and relative quantification ( K ) of the time spent in the center and in the periphery ( % ) of the arena . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 track plots (J) generated from the open-field test from wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=26 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=11 at 9 weeks, N=7 at 11 weeks), R6/2 chol-medium (N=11 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks) and relative quantification (K) of the time spent in the center and in the periphery (%) of the arena. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( L ) Discrimination index ( DI ; % ) in the novel object recognition test at 6 weeks of age ( before pump implantation ) and 11 weeks of age ( 4 weeks after cholesterol infusion ) of wt ( N = 16 at 9 weeks , N = 21 at 11 weeks ) , R6 / 2 ACSF ( N = 28 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 21 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 10 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high mice ( N = 18 at 9 weeks , N = 16 at 11 weeks ) . DI above zero indicates a preference for the novel object ; DI below zero indicates a preference for the familiar object . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(L) Discrimination index (DI; %) in the novel object recognition test at 6 weeks of age (before pump implantation) and 11 weeks of age (4 weeks after cholesterol infusion) of wt (N=16 at 9 weeks, N=21 at 11 weeks), R6/2 ACSF (N=28 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=21 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=10 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks). DI above zero indicates a preference for the novel object; DI below zero indicates a preference for the familiar object. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( B ) Spontaneous EPSCs were recorded from striatal MSNs ( wt = 6 ; R6 / 2 ACSF = 5 ; R6 / 2 chol - high = 5 ) at a holding potential of - 70 mV .

O 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

(B) Spontaneous EPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of -70 mV.


( C - D ) Average amplitude ( C ) and average frequency ( D ) of EPSCs from wt , R6 / 2 ACSF , and R6 / 2 chol - high MSNs . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of each cell recorded Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(C-D) Average amplitude (C) and average frequency (D) of EPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( E - G ) Number of excitatory synapses per volume of striatum by using FIB / SEM followed by 3D reconstruction . ( E ) Representative tissue block of striatum ( 10 µm x 15 µm x 10 µm ) used for the 3D analysis . ( F ) Representative FIB - SEM segmentation and reconstruction of excitatory synapses ( yellow ) in 200 serial sections of striatum for a total volume of 500 µm3 , of wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice . ( G ) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 animals / group ) . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of , the number of synapses counted / µm3 in different blocks of striatal tissue ( G ) Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(E-G) Number of excitatory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (E) Representative tissue block of striatum (10 µm x 15 µm x 10 µm) used for the 3D analysis. (F) Representative FIB-SEM segmentation and reconstruction of excitatory synapses (yellow) in 200 serial sections of striatum for a total volume of 500 µm3, of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (G) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 animals/group). Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of , the number of synapses counted/µm3 in different blocks of striatal tissue (G) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( H - I ) TEM images ( H ) and 2D profile ( I ) of pre - synaptic terminal sections contain SVs and mitochondria ( m ) . Docked vesicles are indicated by a white arrowhead ( H ) and black circle ( I ) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane . Scale bar : 100 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) TEM images (H) and 2D profile (I) of pre-synaptic terminal sections contain SVs and mitochondria (m). Docked vesicles are indicated by a white arrowhead (H) and black circle (I) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane. Scale bar: 100 nm.


( J , K ) Quantification showing the total SVs / µm2 ( J ) and the docked SVs / µm ( K ) in R6 / 2 , R6 / 2 ACSF , and wt striatal synapses ( N = 3 animals / group ) . N = 60 excitatory synapses / group were counted . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the number of total SVs / µm2 ( J ) and docked SVs / µm of active zone ( K ) counted in each synapse . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification showing the total SVs/µm2 (J) and the docked SVs/µm (K) in R6/2, R6/2 ACSF, and wt striatal synapses (N=3 animals/group). N=60 excitatory synapses/group were counted. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of total SVs/µm2 (J) and docked SVs/µm of active zone (K) counted in each synapse. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( A ) Spontaneous IPSCs were recorded from striatal MSNs ( wt = 6 ; R6 / 2 ACSF = 5 ; R6 / 2 chol - high = 5 ) at a holding potential of 0 mV .

O 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

(A) Spontaneous IPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of 0 mV.


( B - C ) Average amplitude ( B ) and average frequency ( C ) of IPSCs from wt , R6 / 2 ACSF , and R6 / 2 chol - high MSNs . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of each cell recorded ( B , C ) Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) .

O O O O O O 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

(B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).


( D - E ) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB / SEM followed by 3D reconstruction . ( D ) Representative FIB - SEM segmentation and reconstruction of inhibitory synapses ( green ) in 200 serial sections of striatum for a total volume of 500 µm3 in wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice . ( E ) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 mice / group ) . Data information : The data E ) are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the number of synapses counted / µm3 in different blocks of tissue for each group of animals ( N = 3 animals / group ) ( E ) . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

(D-E) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (D) Representative FIB-SEM segmentation and reconstruction of inhibitory synapses (green) in 200 serial sections of striatum for a total volume of 500 µm3 in wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (E) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 mice/ group). Data information: The data E) are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of synapses counted /µm3 in different blocks of tissue for each group of animals (N=3 animals/group) (E). Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).


( B ) 24S - OHC level measured by mass spectrometry in the infused and contralateral striatum of wt , R6 / 2 ‐ ACSF , and R6 / 2 chol - high mice at 12 weeks of age after a 4 - week striatal infusion of cholesterol ( N = 4 / group ) . All values are expressed as % above the mean of wt , and these data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each animal .

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

(B) 24S-OHC level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2‐ACSF, and R6/2 chol-high mice at 12 weeks of age after a 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.


( C - E ) Lanosterol ( C ) , lathosterol ( D ) , and desmosterol ( E ) level measured by mass spectrometry in the infused and contralateral striatum of wt , R6 / 2 ACSF , and R6 / 2 chol - high mice at 12 weeks of age after 4 - week striatal infusion of cholesterol ( N = 4 / group ) . All values are expressed as % above the mean of wt , and these data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each animal .

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

(C-E) Lanosterol (C), lathosterol (D), and desmosterol (E) level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2 ACSF, and R6/2 chol-high mice at 12 weeks of age after 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.


( F - I ) Nuclear ( white arrows ) and perinuclear ( yellow arrows ) localization of endogenous SREBP2 in the striatum of wt , R6 / 2 and R6 / 2 - chol mice . Representative confocal image ( F and H ) and relative quantification ( G and I ) of SREBP2 ( red ) in wt and R6 / 2 mice and in the infused and contralateral striatum of R6 / 2 chol - high mice ( N = 4 ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bar in F and H : 10 µm . Graphs in ( G and I ) represents the intensity of SREBP2 normalized on nuclei ( % ) . Data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each image . Statistics : Student ' s t - test ( * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

O O O O O O O O O O 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 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

(F-I) Nuclear (white arrows) and perinuclear (yellow arrows) localization of endogenous SREBP2 in the striatum of wt, R6/2 and R6/2-chol mice. Representative confocal image (F and H) and relative quantification (G and I) of SREBP2 (red) in wt and R6/2 mice and in the infused and contralateral striatum of R6/2 chol-high mice (N=4). Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar in F and H: 10 µm. Graphs in (G and I) represents the intensity of SREBP2 normalized on nuclei (%). Data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each image. Statistics: Student's t-test (**p<0.01; ****p<0.0001).


J , K Representative high - magnification confocal images of immunostaining against SREBP2 ( red ) and NeuN ( J ) or GFAP ( K ) ( green ) on coronal sections of brains from R6 / 2 chol - high mice . A cell in the infused striatum , positive for NeuN and GFAP respectively , is shown . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bars : 1 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 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

J, K Representative high-magnification confocal images of immunostaining against SREBP2 (red) and NeuN (J) or GFAP (K) (green) on coronal sections of brains from R6/2 chol-high mice. A cell in the infused striatum, positive for NeuN and GFAP respectively, is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bars: 1 µm.


Immunolabeling of muHTT aggregates ( red ) in R6 / 2 ACSF and R6 / 2 chol - high mice ( N = 3 / group ) . Zoom of representative confocal images of immunostaining against muHTT aggregates ( red ) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum ( A ) and relative quantification of number ( B ) and size ( C ) of aggregates . Hoechst ( Ho , blue ) was used to counterstain nuclei . 18 images / animal were analyzed from 9 sections throughout the entire striatum . The data are shown as scatterplots with means ± standard error . Each dot corresponds to aggregates counted in all the images from 3 animals . Scale bars : 10 µm ( A ) Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

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

Immunolabeling of muHTT aggregates (red) in R6/2 ACSF and R6/2 chol-high mice (N=3/group). Zoom of representative confocal images of immunostaining against muHTT aggregates (red) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum (A) and relative quantification of number (B) and size (C) of aggregates. Hoechst (Ho, blue) was used to counterstain nuclei. 18 images/animal were analyzed from 9 sections throughout the entire striatum. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 10 µm (A) Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).


Representative confocal images of immunostaining against muHTT ( red ) and DARPP32 ( D ) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification ( E Hoechst ( Ho , blue ) was used to counterstain nuclei . All values are expressed as % above the mean of aggregates in the contralateral striatum of R6 / 2 chol - high . The data are shown as scatterplots with means ± standard error . Each dot corresponds to aggregates counted in all the images from 3 animals . Scale bars : 5 µm Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

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

Representative confocal images of immunostaining against muHTT (red) and DARPP32 (D) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification (E Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).


Representative confocal images of immunostaining against muHTT ( red ) and GFAP ( F ) ( green ) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification G ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . All values are expressed as % above the mean of aggregates in the contralateral striatum of R6 / 2 chol - high . The data are shown as scatterplots with means ± standard error . Each dot corresponds to aggregates counted in all the images from 3 animals . Scale bars : 5 µm Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

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

Representative confocal images of immunostaining against muHTT (red) and GFAP (F) (green) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification G). Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).


( H ) TEM images of EM48 pre - embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6 / 2 ACSF mice and R6 / 2 chol - high mice ( upper panels ) . In the lower panels , muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images . Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus , and arrowheads indicate single 10 - nm gold particles in MSN . Nucleus ( N ) , nucleolus ( n ) , mitochondrion ( m ) , rough endoplasmic reticulum ( rer ) , Golgi apparatus ( g ) , and white arrows indicate the nuclear envelope ( N = 3 animals / group ) . Scale bars : 700 nm and 300 nm .

O O O O O O O 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

(H) TEM images of EM48 pre-embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6/2 ACSF mice and R6/2 chol-high mice (upper panels). In the lower panels, muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images. Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus, and arrowheads indicate single 10-nm gold particles in MSN. Nucleus (N), nucleolus (n), mitochondrion (m), rough endoplasmic reticulum (rer), Golgi apparatus (g), and white arrows indicate the nuclear envelope (N=3 animals/group). Scale bars: 700 nm and 300 nm.


Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4 - week cholesterol infusion by TR - FRET analysis using different antibody pairs . Preliminary assessment of the sustainability of the assay in wt and R6 / 2 striata ( N = 5 / group ) using 4C9 - 4C9 and 2B7 - MW1 antibodies in combination to detect , respectively , muHTT aggregates ( J ) and total muHTT ( K ) . Quantification of muHTT aggregates ( L ) and soluble and other muHTT species ( M ) in the infused striata of R6 / 2 ACSF and R6 / 2 chol - high mice . Data in ( J - M ) are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from one striatum . Statistics : student ' s t - test ( * p < 0 . 05 ) .

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 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

Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4-week cholesterol infusion by TR-FRET analysis using different antibody pairs. Preliminary assessment of the sustainability of the assay in wt and R6/2 striata (N=5/group) using 4C9-4C9 and 2B7-MW1 antibodies in combination to detect, respectively, muHTT aggregates (J) and total muHTT (K). Quantification of muHTT aggregates (L) and soluble and other muHTT species (M) in the infused striata of R6/2 ACSF and R6/2 chol-high mice. Data in (J-M) are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from one striatum. Statistics: student's t-test (*p<0.05).


Representative confocal images showing p62 ( A , red ) in the infused and contralateral striatum on brain coronal sections from wt , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 / group ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Quantification of dots for p62 ( B ) in the contralateral and infused striatum of R6 / 2 ACSF and R6 / 2 chol - high mice . 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed . The data in ( B are shown as scatterplots with means ± standard error , and each dot corresponds to the value obtained from each image . Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . Scale bars in ( A 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images showing p62 (A, red) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of dots for p62 (B) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in (B are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in (A 10 µm.


Representative confocal images showing LAMP1 ( C , green ) in the infused and contralateral striatum on brain coronal sections from wt , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 / group ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Quantification of density for LAMP1 ( D ) in the contralateral and infused striatum of R6 / 2 ACSF and R6 / 2 chol - high mice . 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed . The data in D ) are shown as scatterplots with means ± standard error , and each dot corresponds to the value obtained from each image . Statistics : one ‐ way ANOVA followed by Newman - Keuls multiple comparison tests ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . Scale bars in C ) : 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images showing LAMP1 (C, green) in the infused and contralateral striatum on brain coronal sections from wt, R6/2 ACSF, and R6/2 chol-high mice (N=3/group). Hoechst (Ho, blue) was used to counterstain nuclei. Quantification of density for LAMP1 (D) in the contralateral and infused striatum of R6/2 ACSF and R6/2 chol-high mice. 20 images from 3 sections in the middle of the striatum for each animal were acquired and analyzed. The data in D) are shown as scatterplots with means±standard error, and each dot corresponds to the value obtained from each image. Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (***p<0.001; ****p<0.0001). Scale bars in C): 10 µm.


VERO AT2 cells were infected with WT and alpha ( α ) SARS - CoV - 2 live virus variants , and heat inactivated α variant at MOI 0 . 1 . ( B ) Cells were lysed 48h post - infection and viral protein as well as cell cycle associated protein expression was analysed by western blot . N , nucleocapsid .

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VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (B) Cells were lysed 48h post-infection and viral protein as well as cell cycle associated protein expression was analysed by western blot. N, nucleocapsid.


VERO AT2 cells were infected with WT and alpha ( α ) SARS - CoV - 2 live virus variants , and heat inactivated α variant at MOI 0 . 1 . ( C ) VERO AT2 Cells were fixed 24h post - infection and stained for viral proteins and cyclins . Arrowheads highlight un - infected cells and cyclin D / A nuclear localization . Arrowheads : Nuclear cyclin staining in uninfected cells . Scale bars : 20μm . ( D ) VERO AT2 cells . Quantification of cyclin A2 , D1 and D3 relocalization from nucleus after infection . Uninfected ( - ) and SARS - CoV - 2 infected cells were identified by negative / positive nucleocapsid or Spike staining . Ratio between nuclear and cytoplasm staining intensity of cyclins was measured using ImageJ and Harmony ( PerkinElmer ) . At least 50 cells were counted . Bars indicate mean with SD . Statistical analysis was performed using two - sided unpaired Student ' s t - tests ; ns , non - significant ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

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

VERO AT2 cells were infected with WT and alpha (α) SARS-CoV-2 live virus variants, and heat inactivated α variant at MOI 0.1. (C) VERO AT2 Cells were fixed 24h post-infection and stained for viral proteins and cyclins. Arrowheads highlight un-infected cells and cyclin D/A nuclear localization. Arrowheads: Nuclear cyclin staining in uninfected cells. Scale bars: 20μm. (D) VERO AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗∗p < 0.0001.


( E ) A549 AT2 cells were infected with WT , alpha ( α ) , and delta ( ∆ ) SARS - CoV - 2 variants . Cells were lysed 48h post - infection and viral , cyclin proteins expression was analysed by western blot . N , nucleocapsid .

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(E) A549 AT2 cells were infected with WT, alpha (α), and delta (∆) SARS-CoV-2 variants. Cells were lysed 48h post-infection and viral, cyclin proteins expression was analysed by western blot. N, nucleocapsid.


( F ) A549 AT2 cells . Quantification of cyclin A2 , D1 and D3 relocalization from nucleus after infection . Uninfected ( - ) and SARS - CoV - 2 infected cells were identified by negative / positive nucleocapsid or Spike staining . Ratio between nuclear and cytoplasm ( N / C ratio ) staining intensity of cyclins was measured using ImageJ and Harmony ( PerkinElmer ) . At least 50 cells were counted . Bars indicate mean with SD . Statistical analysis was performed using two - sided unpaired Student ' s t - tests ; ns , non - significant ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ ∗ ∗ p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A549 AT2 cells. Quantification of cyclin A2, D1 and D3 relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid or Spike staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells were counted. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ns, non-significant; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.


A549 AT2 cells were infected with Delta SARS - CoV - 2 variant . Proteasome inhibitor Bortezomib ( BZ , 1μM ) was added to cells 18h post - infection . Cells were lysed 24h post - addition of inhibitor and cyclins and viral proteins were detected by western blot . ( N ) nucleocapsid . Densitometry analysis of western blots for D - cyclins ( normalized to actin ) in A549 AT2 cells . Plots are average of 2 ( cyclin D1 ) , 3 ( cyclin D3 ) biological replicates . Bars indicate mean with SD . Statistical analysis was performed using ordinary two way ANOVA ; ns , non - significant ; ∗ p < 0 . 1 .

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

A549 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 18h post-infection. Cells were lysed 24h post-addition of inhibitor and cyclins and viral proteins were detected by western blot. (N) nucleocapsid. Densitometry analysis of western blots for D-cyclins (normalized to actin) in A549 AT2 cells. Plots are average of 2 (cyclin D1), 3 (cyclin D3) biological replicates. Bars indicate mean with SD. Statistical analysis was performed using ordinary two way ANOVA; ns, non-significant; ∗p < 0.1.


A459 AT2 cells were infected with Delta SARS - CoV - 2 variant . Proteasome inhibitor Bortezomib ( BZ , 1μM ) was added to cells 8h post - infection . Cells were fixed and stained 24h post - addition of inhibitor . ( N ) nucleocapsid . Scale bars : 40μm . Contour lines represent outlines of infected cells .

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

A459 AT2 cells were infected with Delta SARS-CoV-2 variant. Proteasome inhibitor Bortezomib (BZ, 1μM) was added to cells 8h post-infection. Cells were fixed and stained 24h post-addition of inhibitor. (N) nucleocapsid.Scale bars: 40μm. Contour lines represent outlines of infected cells.


Quantification of D3 cyclin relocalization from nucleus after infection . Uninfected ( - ) and SARS - CoV - 2 infected cells were identified by negative / positive nucleocapsid staining . Ratio between nuclear and cytoplasm ( N / C ratio ) staining intensity of cyclins was measured using ImageJ and Harmony ( PerkinElmer ) . At least 50 cells have been counted . Bars indicate mean with SD . Statistical analysis was performed using ordinary two - way ANOVA ; ∗ ∗ ∗ p < 0 . 001 .

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

Quantification of D3 cyclin relocalization from nucleus after infection. Uninfected (-) and SARS-CoV-2 infected cells were identified by negative/positive nucleocapsid staining. Ratio between nuclear and cytoplasm (N/C ratio) staining intensity of cyclins was measured using ImageJ and Harmony (PerkinElmer). At least 50 cells have been counted. Bars indicate mean with SD. Statistical analysis was performed using ordinary two-way ANOVA; ∗∗∗p < 0.001.


( A , D and A - cyclins were depleted using siRNA in A549 AT2 cells . ( A , Representative example of western blot from uninfected cell lysates collected at 72h post - knockdown .

O O O O O O O O O O O O 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, D and A-cyclins were depleted using siRNA in A549 AT2 cells. (A, Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.


B ) D and A - cyclins were depleted using siRNA in A549 AT2 cells . Cells were infected 18h later with Delta ( ∆ ) , Alpha ( α ) or wild type ( WT ) SARS - CoV - 2 variants at MOI 0 . 001 , 0 . 1 , 0 . 1 respectively . Cells were washed 4h post - infection and new media were added . Supernatants and cells were collected 48h later for TCID50 ( B ) . ( B , Virus titres in cell culture supernatants were determined as TCID50 Data information : Graphs represent average of n = 3 ( B , Statistical analysis was performed using one - way ANOVA with Dunnett ' s multiple comparisons test . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D and A-cyclins were depleted using siRNA in A549 AT2 cells. Cells were infected 18h later with Delta (∆), Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.001, 0.1, 0.1 respectively. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for TCID50 (B). (B, Virus titres in cell culture supernatants were determined as TCID50 Data information: Graphs represent average of n=3 (B, Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


( C , D and A - cyclins were depleted using siRNA in VERO AT2 cells . C ) Representative example of western blot from uninfected cell lysates collected at 72h post - knockdown .

O O O O O O O O O O O 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 and A-cyclins were depleted using siRNA in VERO AT2 cells. C) Representative example of western blot from uninfected cell lysates collected at 72h post-knockdown.


D ) D and A - cyclins were depleted using siRNA in VERO AT2 cells . Cells were infected 18h later with Alpha ( α ) or wild type ( WT ) SARS - CoV - 2 variants at MOI 0 . 1 . Cells were washed 4h post - infection and new media added . Supernatants and cells were collected at 24h and 48h later . D ) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells . Data information : Graphs represent average of n = 3 D ) ; Statistical analysis was performed using one - way ANOVA with Dunnett ' s multiple comparisons test . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D and A-cyclins were depleted using siRNA in VERO AT2 cells. Cells were infected 18h later with Alpha (α) or wild type (WT) SARS-CoV-2 variants at MOI 0.1. Cells were washed 4h post-infection and new media added. Supernatants and cells were collected at 24h and 48h later. D) Virus titres in cell culture supernatants were determined as TCID50 in VERO AT2 cells. Data information: Graphs represent average of n=3 D); Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


Cyclin D3 was depleted using siRNA in Calu3 cells . Cells were infected 48h later with Delta SARS - CoV - 2 at MOI 0 . 01 . Cells were washed 4h post - infection and new media were added . Supernatants and cells were collected 48h later for western blot and TCID50 . Uninfected cell lysates were used for western blot . Data information : Graphs represent average of n = 4 ( E ) biological replicates . Statistical analysis was performed using one - way ANOVA with Dunnett ' s multiple comparisons test . NT , non - targeting control . ns , non - significant ; ∗ p < 0 . 1 ; ∗ ∗ p < 0 . 01 ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD .

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

Cyclin D3 was depleted using siRNA in Calu3 cells. Cells were infected 48h later with Delta SARS-CoV-2 at MOI 0.01. Cells were washed 4h post-infection and new media were added. Supernatants and cells were collected 48h later for western blot and TCID50. Uninfected cell lysates were used for western blot. Data information: Graphs represent average of n=4 (E) biological replicates. Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test. NT, non-targeting control. ns, non-significant; ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Bars indicate mean with SD.


A549 AT2 cells were depleted for D and A2 cyclins ( A ) A representative western blot from lysates of uninfected knock - down cells .

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

A549 AT2 cells were depleted for D and A2 cyclins (A) A representative western blot from lysates of uninfected knock-down cells.


( E ) A549 AT2 cells were transduced with VSV - G pseudotyped Fucci containing lentiviral particles and VSV - G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 ( mutant not degraded by proteasome ) . Cells were infected 24h later and collected 48h post - infection for flow cytometry analysis of early S and western blot . n = 2 biological replicates ; Ordinary two - way ANOVA with Sidak ' s multiple comparisons test : ns , non - significant ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A549 AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and VSV-G pseudotyped lentiviral particles containing WT cyclin D3 or T283A mutant cyclin D3 (mutant not degraded by proteasome). Cells were infected 24h later and collected 48h post-infection for flow cytometry analysis of early S and western blot. n=2 biological replicates; Ordinary two-way ANOVA with Sidak's multiple comparisons test: ns, non-significant; ∗∗p < 0.01. Bars indicate mean with SD.


( F , G ) VERO AT2 cells were transduced with VSV - G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS - CoV - 2 . Cells were fixed and stained for D - cyclins 24h later . ( F ) Example of acquisition using automated microscopic platform . Cells are identified for infection , expression of cyclin D3 , and cell cycle ( Red / arrow = G1phase ; Green / arrowhead = S / G2 / M ; Red + Green / arrowhead = early S ) . Scale bar : 20μm . ( G ) Quantification of D - cyclins re - localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony ( PerkinElmer ) . At least 50 - 200 cells were analysed in each condition . Bars indicate mean with SD . Statistical analysis was performed using two - sided unpaired Student ' s t - tests ; ∗ p < 0 . 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) VERO AT2 cells were transduced with VSV-G pseudotyped Fucci containing lentiviral particles and 18h later infected with Delta variant SARS-CoV-2. Cells were fixed and stained for D-cyclins 24h later. (F) Example of acquisition using automated microscopic platform. Cells are identified for infection, expression of cyclin D3, and cell cycle (Red/arrow=G1phase; Green/arrowhead=S/G2/M; Red+Green/arrowhead=early S). Scale bar: 20μm. (G) Quantification of D-cyclins re-localization from nucleus to cytoplasm and correlation with cell cycle phases using ImageJ and Harmony (PerkinElmer). At least 50-200 cells were analysed in each condition. Bars indicate mean with SD. Statistical analysis was performed using two-sided unpaired Student's t-tests; ∗p < 0.1.


( A ) 293T cells were contransfected with HA - cyclin D3 and Strep - tag - SARS - CoV - 2 E or nsp9 , and control plasmid ( EV ) . Immunoprecipitation was performed using anti - HA antibody . The immunoprecipitates were blotted with anti - Strep , anti - HA , and cyclin D3 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E or nsp9, and control plasmid (EV). Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA, and cyclin D3 antibodies.


( B ) 293T cells were contransfected with HA - cyclin D3 and Strep - tag - SARS - CoV - 2 E , M , both E and M or nsp9 . Immunoprecipitation was performed using anti - HA antibody . The immunoprecipitates were blotted with anti - Strep , anti - HA antibodies . WCL , whole cell lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were contransfected with HA - cyclin D3 and Strep-tag-SARS-CoV-2 E, M, both E and M or nsp9. Immunoprecipitation was performed using anti-HA antibody. The immunoprecipitates were blotted with anti-Strep, anti-HA antibodies. WCL, whole cell lysate.


( C - G ) 293T GFP11 cells were transfected with Spike , and / or with Envelope , Membrane , and cyclin D3 . 24h post - transfection cells were seeded at a 1 : 1 ratio with Vero - GFP10 cells and percentage of GFP + ve area ( syncytia ) were determined 18h later . S , Spike ; E , Envelope ; M , Membrane ; D3 , cyclin D3 . ( C , D ) Representative images of GFP + syncytia . Scale bars : 40μm . ( E - G ) Quantification of cell - to - cell fusion showing percentage of the GFP + ve area to the acquired total cell area . ( E ) n = 4 biological replicates . ( F , G ) n = 4 biological replicates ; technical triplicates shown ; one - way ANOVA with Dunnett ' s multiple comparisons test : ns , non - significant ; ∗ ∗ ∗ ∗ p < 0 . 0001 ; ∗ ∗ ∗ p < 0 . 001 ; ∗ ∗ p < 0 . 01 . Bars indicate mean with SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) 293T GFP11 cells were transfected with Spike, and/or with Envelope, Membrane, and cyclin D3. 24h post-transfection cells were seeded at a 1:1 ratio with Vero-GFP10 cells and percentage of GFP+ve area (syncytia) were determined 18h later. S, Spike; E, Envelope; M, Membrane; D3, cyclin D3. (C,D) Representative images of GFP+ syncytia. Scale bars: 40μm. (E-G) Quantification of cell-to-cell fusion showing percentage of the GFP+ve area to the acquired total cell area. (E) n=4 biological replicates. (F,G) n = 4 biological replicates; technical triplicates shown; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01 . Bars indicate mean with SD.


( I ) Representative example of western blot from purified SARS - CoV - 2 Virus Like Particles ( VLPs ) and whole cell lysates ( WCL ) . MCM2 was used as loading control . ( J ) Quantification of protein incorporation ( into VLPs ) or expression ( in WCL ) . n = 4 biological replicates ; one - way ANOVA with Dunnett ' s multiple comparisons test : ns , non - significant ; ∗ ∗ ∗ p < 0 . 001 . Bars indicate mean with SD . N , nucleocapsid ; M , membrane ; E , envelope ; S , spike .

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

(I) Representative example of western blot from purified SARS-CoV-2 Virus Like Particles (VLPs) and whole cell lysates (WCL). MCM2 was used as loading control. (J) Quantification of protein incorporation (into VLPs) or expression (in WCL). n = 4 biological replicates; one-way ANOVA with Dunnett's multiple comparisons test: ns, non-significant; ∗∗∗p < 0.001. Bars indicate mean with SD. N, nucleocapsid; M, membrane; E, envelope; S, spike.


( A ) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1 , 5 g / L of D - glucose . Glucose was added either at the beginning of growth ( + glucose , left panel ) or after 3h of growth ( right panel ) .

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

(A) Northern blot experiments show the expression of RsaI during growth phase in the HG001 strain in MHB medium with or without the addition of 1,5 g/L of D-glucose. Glucose was added either at the beginning of growth (+ glucose, left panel) or after 3h of growth (right panel).


( B ) Northern blot experiment shows the expression of RsaI during growth phase in the HG001 , ∆ ccpA mutant strain , and ∆ codY mutant strain , in MHB medium with ( + ) or without ( - ) the addition of 1 , 5 g / L of D - glucose .

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

(B) Northern blot experiment shows the expression of RsaI during growth phase in the HG001, ∆ccpA mutant strain, and ∆codY mutant strain, in MHB medium with (+) or without (-) the addition of 1,5 g/L of D-glucose.


( C ) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ ccpA mutant strains . Total RNA was prepared after 2 , 3 , 4 , 5 and 6h of culture in BHI medium at 37 ° C .

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

(C) Northern blot experiment shows the expression of RsaI in the HG001 and ∆ccpA mutant strains. Total RNA was prepared after 2, 3, 4, 5 and 6h of culture in BHI medium at 37°C.


( D ) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g / L of glucose , fructose or xylose . For all the experiments , loading controls were done using the expression of 5S rRNA ( 5S ) as revealed after hybridization of the membranes with a specific probe . However for these controls , we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Northern blot analysis of RsaI in the HG001 strain grown in MHB medium with or without the addition of 1 g/L of glucose, fructose or xylose. For all the experiments, loading controls were done using the expression of 5S rRNA (5S) as revealed after hybridization of the membranes with a specific probe. However for these controls, we used aliquots of the same RNA preparations but the migration of the samples was performed in parallel to the experiments on a separate agarose gel because RsaI and 5S rRNA have very similar sizes.


( B ) Gel retardation assays show the formation of the complex between RsaI and icaR , glcU _ 2 and fn3K mRNAs . The 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 3 ( RsaI mut3 , deletion of the two G - track motifs ) , and RsaI mutant 4 ( RsaI mut4 , deletion of the C / U rich unpaired interhelical region ) were incubated with increasing concentrations of mRNAs : lane 1 , 0 nM ; lane 2 , 25 nM ; lane 3 , 50 nM ; lane 4 , 100 nM and lane 5 , 200 nM . Below the gels , the predicted interactions between RsaI and its targets are shown . Translation start codons are in green and SD is for Shine Dalgarno sequence . Graphs represented the % of complex formed between either RsaI or its two mutant forms ( RsaI mut3 and RsaI mut4 ) and the target mRNA ( icaR , glcU _ 2 , fn3K ) as the function of mRNA concentrations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gel retardation assays show the formation of the complex between RsaI and icaR, glcU_2 and fn3K mRNAs. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 3 (RsaI mut3, deletion of the two G-track motifs), and RsaI mutant 4 (RsaI mut4, deletion of the C/U rich unpaired interhelical region) were incubated with increasing concentrations of mRNAs: lane 1, 0 nM; lane 2, 25 nM; lane 3, 50 nM; lane 4, 100 nM and lane 5, 200 nM. Below the gels, the predicted interactions between RsaI and its targets are shown. Translation start codons are in green and SD is for Shine Dalgarno sequence. Graphs represented the % of complex formed between either RsaI or its two mutant forms (RsaI mut3 and RsaI mut4) and the target mRNA (icaR, glcU_2, fn3K) as the function of mRNA concentrations.


( C ) Gel retardation assays show the formation of the complex between RsaI and RsaG . The 5 ' end - labeled wild - type RsaI ( RsaI ) , RsaI mutant 2 ( RsaI mut2 ) , RsaI mutant 4 ( RsaI mut4 ) , and RsaI mutant 1 ( RsaI mut1 ) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gel retardation assays show the formation of the complex between RsaI and RsaG. The 5' end-labeled wild-type RsaI (RsaI), RsaI mutant 2 (RsaI mut2), RsaI mutant 4 (RsaI mut4), and RsaI mutant 1 (RsaI mut1) were incubated with increasing concentrations of RsaG given in nM on the top of the autoradiographies.


( A ) Toe - print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU _ 2 and fn3K mRNAs , respectively . Lane 1 : incubation control of mRNA alone ; lane 2 : incubation control of mRNA with 30S subunits ; lane 3 : incubation control of mRNA with RsaI ; lane 4 : formation of the ribosomal initiation complex containing mRNA , 30S and the initiator tRNAfMet ( tRNAi ) ; lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI , respectively : 50 nM ( lane 5 ) , 100 nM ( lane 6 ) , 150 nM ( lane 7 ) , 300 nM ( lane 8 ) , and 400 nM ( lane 9 ) . Lanes T , A , C , G : sequencing ladders . The Shine and Dalgarno ( SD ) sequence , the start site of translation ( START ) and the toe - printing signals ( + 16 ) are indicated . At the bottom of the gels are shown the predicted interactions between RsaI and its targets . Translation start codons are in green , and the Shine and Dalgarno ( SD ) sequence is underlined , the arrowheads depict the toe - printing signals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Toe-print assays showing the effect of RsaI on the formation of the ribosomal initiation complex of glcU_2 and fn3K mRNAs, respectively. Lane 1 : incubation control of mRNA alone; lane 2 : incubation control of mRNA with 30S subunits; lane 3: incubation control of mRNA with RsaI; lane 4 : formation of the ribosomal initiation complex containing mRNA, 30S and the initiator tRNAfMet (tRNAi); lanes 5 to 9 : formation of the initiation complex in the presence of increasing concentrations of RsaI, respectively : 50 nM (lane 5), 100 nM (lane 6), 150 nM (lane 7), 300 nM (lane 8), and 400 nM (lane 9). Lanes T, A, C, G: sequencing ladders. The Shine and Dalgarno (SD) sequence, the start site of translation (START) and the toe-printing signals (+16) are indicated. At the bottom of the gels are shown the predicted interactions between RsaI and its targets. Translation start codons are in green, and the Shine and Dalgarno (SD) sequence is underlined, the arrowheads depict the toe-printing signals.


( B ) The β - galactosidase activity ( Miller Units ) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter : PrpoB : : glcU : : lacZ : : rsaI , PrpoB : : fn3K : : lacZ : : rsaI , PrpoB : : treB : : lacZ : : rsaI , PrpoB : : HG001 _ 1242 : : lacZ : : rsaI , and PrpoB : : HG001 _ 2520 : : lacZ : : rsaI expressed in the mutant strain HG001 - ∆ rsaI . The same constructs were made in the absence of rsaI gene . For the fn3K - lacZ fusion , we also used an additional construct PrpoB : : fn3K : : lacZ : : rsaImut4 expressing both the fusion FN3K - LacZ protein and RsaI mut4 . The β - galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments . The error bars are standard deviations and the statistical significance was determined using the Student ' s t - test . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 , ns is for not significant .

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(B) The β-galactosidase activity (Miller Units) has been measured from various fusions expressed from a plasmid which also carries rsaI gene under its own promoter: PrpoB::glcU::lacZ::rsaI, PrpoB::fn3K::lacZ::rsaI, PrpoB::treB::lacZ::rsaI, PrpoB::HG001_1242::lacZ::rsaI, and PrpoB::HG001_2520::lacZ::rsaI expressed in the mutant strain HG001-∆rsaI. The same constructs were made in the absence of rsaI gene. For the fn3K-lacZ fusion, we also used an additional construct PrpoB::fn3K::lacZ::rsaImut4 expressing both the fusion FN3K-LacZ protein and RsaI mut4. The β-galactosidase activity was normalized for bacterial density and the results represented the mean of four independent experiments. The error bars are standard deviations and the statistical significance was determined using the Student's t-test. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001, ns is for not significant.


( B ) Gel retardation assays show the formation of the complex between RsaI and icaR full length , icaR SUBST , icaR - 5 ' UTR , and icaR - 3 ' UTR . The 5 ' end - labeled of RsaI was incubated with increasing concentrations of the various mRNAs . UTR is for untranslated region , SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC . This mutation , which is located in the 3 ' UTR of icaR , significantly destabilizes the long - range interaction , and enhances icaR translation . Lane C represents binding between radiolabelled RsaI and full - length icaR mRNA ( 50 nM ) . Due to the rather short migration of the gel , the full - length mRNA is still observed in the pocket .

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(B) Gel retardation assays show the formation of the complex between RsaI and icaR full length, icaR SUBST, icaR-5'UTR, and icaR-3'UTR. The 5' end-labeled of RsaI was incubated with increasing concentrations of the various mRNAs. UTR is for untranslated region, SUBST stands for the substitution of UCCCCUG sequence by AGGGGAC. This mutation, which is located in the 3'UTR of icaR, significantly destabilizes the long-range interaction, and enhances icaR translation. Lane C represents binding between radiolabelled RsaI and full-length icaR mRNA (50 nM). Due to the rather short migration of the gel, the full-length mRNA is still observed in the pocket.


( C ) In vivo effect of RsaI on PIA - PNAG synthesis in the S . aureus wild - type ( WT ) 132 strain , the ∆ rsaI mutant , and the strain carrying a deletion of icaR 3 ' UTR ( ∆ 3 ' UTR ) . This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5 . The PIA - PNAG exopolysaccharide biosynthesis was quantified using dot - blot assays . Serial dilutions ( 1 / 5 ) of the samples were spotted onto nitrocellulose membranes and PIA - PNAG production was detected with specific anti - PIA - PNAG antibodies . RsaI was detected in the same samples by Northern blot using a probe directed against RsaI . Ethidium bromide staining of rRNA was used as loading controls of the same gel .

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

(C) In vivo effect of RsaI on PIA-PNAG synthesis in the S. aureus wild-type (WT) 132 strain, the ∆rsaI mutant, and the strain carrying a deletion of icaR 3'UTR (∆3'UTR). This last mutant strain has been transformed with the pES plasmids expressing rsaI or rsaI mut5. The PIA-PNAG exopolysaccharide biosynthesis was quantified using dot-blot assays. Serial dilutions (1/5) of the samples were spotted onto nitrocellulose membranes and PIA-PNAG production was detected with specific anti-PIA-PNAG antibodies. RsaI was detected in the same samples by Northern blot using a probe directed against RsaI. Ethidium bromide staining of rRNA was used as loading controls of the same gel.


( A ) Ternary complex formation between RsaI , various mRNAs ( glcU _ 2 , HG001 _ 00942 and HG001 _ 1242 ) , and RsaG . The 5 ' - end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG . The various complexes are notified on the sides of the autoradiographies .

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(A) Ternary complex formation between RsaI, various mRNAs (glcU_2, HG001_00942 and HG001_1242), and RsaG. The 5'-end labeled RsaI was incubated with increasing concentrations of the target mRNA alone or in the presence of 50 nM of RsaG. The various complexes are notified on the sides of the autoradiographies.


( B ) RsaG does not form stable complexes with glcU _ 2 and HG001 _ 01242 . Binding assays were done in the presence of 5 ' end - labeled RsaG and either cold RsaI ( 300 nM ) or increasing concentrations of glcU _ 2 and HG001 _ 01242 .

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(B) RsaG does not form stable complexes with glcU_2 and HG001_01242. Binding assays were done in the presence of 5' end-labeled RsaG and either cold RsaI (300 nM) or increasing concentrations of glcU_2 and HG001_01242.


( C ) Measurements of the half - lives of RsaI and RsaG in HG001 - ∆ rsaG or HG001 - ∆ rsaI mutant strains using Northern blot experiments . The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2 , 4 , 8 , 15 , 30 and 60 min at 37 ° C in BHI . 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples , which were however run on two different gels . Calculated half - lives are shown beneath the autoradiographies and are the average of 2 experiments . The data were normalized to 5S rRNA .

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(C) Measurements of the half-lives of RsaI and RsaG in HG001-∆rsaG or HG001-∆rsaI mutant strains using Northern blot experiments. The cells were treated with rifampicin at 4h of growth and total RNAs were extracted after 2, 4, 8, 15, 30 and 60 min at 37°C in BHI. 5S rRNA was probed to quantify the yield of RNAs in each lane using the same samples, which were however run on two different gels. Calculated half-lives are shown beneath the autoradiographies and are the average of 2 experiments. The data were normalized to 5S rRNA.


factor . a , Duplicate screens depicting HCoV - 229E infection ( 24 hours post - infection ) in Huh7 cells ectopically expressing ISGs .

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factor.a, Duplicate screens depicting HCoV-229E infection (24 hours post-infection) in Huh7 cells ectopically expressing ISGs.


b , HCoV - 229E infection of Huh7 cells expressing LY6E or control vector ( Empty ) . Blue : DAPI , green : HCoV - 229E N protein , red : TagRFP encoded in SCRPSY vector .

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b, HCoV-229E infection of Huh7 cells expressing LY6E or control vector (Empty). Blue: DAPI, green: HCoV-229E N protein, red: TagRFP encoded in SCRPSY vector.


c - h , Effect of LY6E expression on diverse coronaviruses . Stably transduced cells were infected with HCoV - 229E ( c ) , HCoV - OC43 ( d ) , MERS - CoV ( e ) , SARS - CoV ( f ) , SARS - CoV - 2 ( g ) , MHV - Gluc ( h ) . Viral replication readouts included : titer determination by plaque assay ( c ) , infectivity by nucleoprotein staining and quantitation by flow cytometry ( d ) , limiting dilution assay ( e , f , g ) , luciferase activity shown as relative light units ( RLU ) ( h , j , l ) .

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c-h, Effect of LY6E expression on diverse coronaviruses. Stably transduced cells were infected with HCoV-229E (c), HCoV-OC43 (d), MERS-CoV (e), SARS-CoV (f), SARS-CoV-2 (g), MHV-Gluc (h). Viral replication readouts included: titer determination by plaque assay (c), infectivity by nucleoprotein staining and quantitation by flow cytometry (d), limiting dilution assay (e,f,g), luciferase activity shown as relative light units (RLU) (h,j,l).


i , Western blot of WT A549 or two LY6E KO clones ( # 3 , # 4 ) .

i, Western blot of WT A549 or two LY6E KO clones (#3, #4).


j , HCoV - 229E infection of WT or LY6E KO A549 .

j, HCoV-229E infection of WT or LY6E KO A549.


k , Western blot of WT or LY6E KO A549 reconstituted with CRISPR - resistant LY6E ( CR LY6E ) or control vector ( Empty ) .

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k, Western blot of WT or LY6E KO A549 reconstituted with CRISPR-resistant LY6E (CR LY6E) or control vector (Empty).


l , HCoV - 229E infection of CR LY6E - reconstituted WT or LY6E KO A549 .

l, HCoV-229E infection of CR LY6E-reconstituted WT or LY6E KO A549.


formation . a - d , VSV pseudoparticles ( PP ) harboring spike proteins from HCoV - 229E ( a ) , MERS - CoV ( b ) , SARS - CoV ( c ) , SARS - CoV - 2 ( d ) were inoculated on LY6E - or empty vector - expressing cells . Virus entry efficiency was quantified by measuring virus - encoded luciferase reporter activity .

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formation.a-d, VSV pseudoparticles (PP) harboring spike proteins from HCoV-229E (a), MERS-CoV (b), SARS-CoV (c), SARS-CoV-2 (d) were inoculated on LY6E- or empty vector-expressing cells. Virus entry efficiency was quantified by measuring virus-encoded luciferase reporter activity.


e , VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP ( VSV * ΔG ( CoV S ) ) were inoculated with LY6E - or empty vector - expressing Huh7 . Syncytia formation was analyzed by immunofluorescence microscopy . Blue : DAPI , green : GFP , red : TagRFP inserted in SCRPSY vector .

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e, VSV pseudoparticles expressing VSV G protein on their surface and encoding CoV S protein and GFP (VSV*ΔG(CoV S)) were inoculated with LY6E- or empty vector-expressing Huh7. Syncytia formation was analyzed by immunofluorescence microscopy. Blue: DAPI, green: GFP, red: TagRFP inserted in SCRPSY vector.


f , Quantification of VSV * ΔG ( CoV S ) induced syncytia depicted as percentage syncytia area . Three independent areas were analyzed per biological replicate ( circle , square , triangle ) .

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f, Quantification of VSV*ΔG(CoV S) induced syncytia depicted as percentage syncytia area. Three independent areas were analyzed per biological replicate (circle, square, triangle).


h , Cell - cell fusion was measured by co - incubating cells transfected with plasmids encoding CoV S proteins and a split - luciferase construct ( DSP1 - 7 ) together with CoV permissive LY6E - expressing or control cells transfected with the second fragment of split - luciferase ( DSP8 - 11 ) .

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h, Cell-cell fusion was measured by co-incubating cells transfected with plasmids encoding CoV S proteins and a split-luciferase construct (DSP1-7) together with CoV permissive LY6E-expressing or control cells transfected with the second fragment of split-luciferase (DSP8-11).


virus . a - b , Female Ly6efl / fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival ( a ) and weight loss ( b ) .

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virus.a-b, Female Ly6efl/fl and Ly6eΔHSC mice were injected with PBS or MHV and monitored for survival (a) and weight loss (b).


c - n , Mice were assessed at 3 ( c - h ) or 5 ( i - n ) days post - infection for serum ALT ( c , i ) , viral titers in liver ( d , j ) and spleen ( e , k ) , and liver necrosis and inflammation ( f - h , l - n ) .

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c-n, Mice were assessed at 3 (c-h) or 5 (i-n) days post-infection for serum ALT (c,i), viral titers in liver (d,j) and spleen (e,k), and liver necrosis and inflammation (f-h, l-n).


mice . a - e , Transcriptomic analysis from Ly6efl / fl ( WT ) and Ly6eΔHSC ( KO ) mice . a - b , Heat maps displaying significant changes ( mean RPKM > 0 . 5 ; fold change > 2 ; FDR ≤ 0 . 05 ) in liver ( a ) and spleen ( b ) . Dendrograms are normalized read data ( row z - score ) clustered with complete linkage method employing Spearman Rank correlation distance measurement .

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mice.a-e, Transcriptomic analysis from Ly6efl/fl (WT) and Ly6eΔHSC (KO) mice. a-b, Heat maps displaying significant changes (mean RPKM > 0.5; fold change > 2; FDR ≤ 0.05) in liver (a) and spleen (b). Dendrograms are normalized read data (row z-score) clustered with complete linkage method employing Spearman Rank correlation distance measurement.


d - e , Expression of select genes in liver ( d ) and spleen ( e ) .

d-e, Expression of select genes in liver (d) and spleen (e).


f - g , Immune cell counts from liver ( f ) and spleen ( g ) .

f-g, Immune cell counts from liver (f) and spleen (g).


h , Infection of cultured splenocytes . For a - e , n = 3 . For f - g , n = 8 MHV - injected , n = 4 PBS - injected from two pooled experiments .

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h, Infection of cultured splenocytes. For a-e, n=3. For f-g, n=8 MHV-injected, n=4 PBS-injected from two pooled experiments.


( A ) Differential intron retention ( IR ) analysis of RNA - seq data from samples under PABPN1 knockdown ( siPABPN1 ) and control ( NC ) conditions . Significantly repressed and induced intron retention events ( retained introns , RIs ) are indicated by blue and red dots , respectively . The threshold of IR difference ( ΔIR , siPABPN1 - siNC ) is 0 . 15 , and the FDR cutoff is 0 . 01 .

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(A) Differential intron retention (IR) analysis of RNA-seq data from samples under PABPN1 knockdown (siPABPN1) and control (NC) conditions. Significantly repressed and induced intron retention events (retained introns, RIs) are indicated by blue and red dots, respectively. The threshold of IR difference (ΔIR, siPABPN1-siNC) is 0.15, and the FDR cutoff is 0.01.


( C ) Cumulative frequency distributions of retained introns ( RIs ) , up - regulated ( red ) , and down - regulated ( blue ) upon PABPN1 knockdown , and of all retained introns in control ( NC , green ) samples by their relative intronic ranks in genes .

(C) Cumulative frequency distributions of retained introns (RIs), up-regulated (red), and down-regulated (blue) upon PABPN1 knockdown, and of all retained introns in control (NC, green) samples by their relative intronic ranks in genes.


Validation of last intron retention of six representative genes in response to PABPN1 knockdown by semi - quantitative PCR in HeLa cells treated with three different NC and PABPN1 siRNAs ( D ) .

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Validation of last intron retention of six representative genes in response to PABPN1 knockdown by semi-quantitative PCR in HeLa cells treated with three different NC and PABPN1 siRNAs (D).


( G ) Western blot validating the degradation effect of PABPN1 under the treatment of dTAG13 for 0 , 3 , and 6 hours .

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(G) Western blot validating the degradation effect of PABPN1 under the treatment of dTAG13 for 0, 3, and 6 hours.


( B ) Western blots of the C1 - C4 reporters for two endogenous regulated genes ( LARP7 and COPS5 ) and three endogenous non - regulated control genes ( TDRD9 , CENPV , and SMTN ) .

(B) Western blots of the C1-C4 reporters for two endogenous regulated genes (LARP7 and COPS5) and three endogenous non-regulated control genes (TDRD9, CENPV, and SMTN).


( C ) RT - PCR analysis of the spliced and unspliced isoforms from the C3 and C4 reporters for two endogenous regulated genes LARP7 and COPS5 .

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(C) RT-PCR analysis of the spliced and unspliced isoforms from the C3 and C4 reporters for two endogenous regulated genes LARP7 and COPS5.


( E ) RT - PCR quantification of MS2 reporters of histone 3 ´ end ( left ) and Malat1 3 ´ end ( right ) for two PABPN1 - regulated genes LARP7 and COPS5 .

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(E) RT-PCR quantification of MS2 reporters of histone 3´end (left) and Malat1 3´end (right) for two PABPN1-regulated genes LARP7 and COPS5.


( D ) RNA quantification by RT - PCR for the spliced and unspliced isoforms ( top ) and calculated IR scores ( bottom ) of the reporters Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) .

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(D) RNA quantification by RT-PCR for the spliced and unspliced isoforms (top) and calculated IR scores (bottom) of the reporters Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).


( E ) Quantification of the spliced ( EEJ ) over unspliced isoforms ( EIJ ) of the reporters in Figure 3F . Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) .

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(E) Quantification of the spliced (EEJ) over unspliced isoforms (EIJ) of the reporters in Figure 3F. Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).


( G ) Splicing analysis of the C3 , C4 , and polyA reporters in Figure 3F for endogenous genes LARP7 , COPS5 , and NSUN5 by RT - PCR upon PABPN1 knockdown ( top ) and the quantified intron retention levels ( IR score ) of these reporters ( bottom ) . Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) .

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(G) Splicing analysis of the C3, C4, and polyA reporters in Figure 3F for endogenous genes LARP7, COPS5, and NSUN5 by RT-PCR upon PABPN1 knockdown (top) and the quantified intron retention levels (IR score) of these reporters (bottom). Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).


( J ) Relative ratios of the unspliced isoforms ( EIJ ) to the spliced isoforms ( EEJ ) of the last introns for LARP7 , DUSP12 , and BRCA1 genes in HeLa cells treated with si - PABPN1 and then rescuing PABPN1 mutants constructed in G . Data information : Data are from n = 3 biologically independent experiments . The error bars represent SD . The P - values are calculated by unpaired t test ( * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ; ns , non - significant ) .

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(J) Relative ratios of the unspliced isoforms (EIJ) to the spliced isoforms (EEJ) of the last introns for LARP7, DUSP12, and BRCA1 genes in HeLa cells treated with si-PABPN1 and then rescuing PABPN1 mutants constructed in G. Data information: Data are from n=3 biologically independent experiments. The error bars represent SD. The P-values are calculated by unpaired t test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, non-significant).


Splicing analysis of NSUN5 and LARP7 reporter mutants by RT - PCR ( E )

Splicing analysis of NSUN5 and LARP7 reporter mutants by RT-PCR (E)


Splicing analysis of NSUN5 and LARP7 reporter mutants by Western blot ( F ) .

Splicing analysis of NSUN5 and LARP7 reporter mutants by Western blot (F).