Figure 4A , ChIP - seq analysis of BRPF3 binding on the human genome . BRPF3 enrichment signal obtained by ChIP - seq with asynchronous ( AS ) , synchronized ( S ) and released into HU ( S + HU ) RKO cells , on the MCM4 ( left ) and Lamin B2 ( right ) replication origins . Signals were compared to the published enrichment signals for ORC1 in HeLa cells ( ( Dellino et al , 2013 ) , GSE37583 ) , HBO1 in RKO cells ( Avvakumov et al . , 2012 , GSE33221 ) , and H3K14ac in IMR90 cells ( GSM521881 ) . The negative control IgG signal shown is from the S + HUchromatin . B , Venn diagrams illustrating the overlap of genome binding sites between ( top ) BRPF3 , HBO1 and ORC1 , and ( bottom ) BRPF3 , BRPF1 / 2 and ORC1 in human cells . C , Heatmaps of BRPF3 , HBO1 and H3K14ac ChIP - seq signal on ± 5kb surrounding the TSS of genes . Genes were sorted by BRPF3 abundance from high to low . A . U . arbitrary unit . D , Average profiles of BRPF3 and H3K14ac ChIP - seq signal on ± 2 . 5kb around the centre of ( top ) BRPF3 and ( bottom ) ORC1 genome binding sites . E , ChIP - qPCR analysis of H3K14ac on well - characterized replication origins . H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells . Error bars , range ; n = 2 biological replicas . F , Expression levels of DNA replication factors were not altered by the lack of BRPF3 . Each point represents the expression levels of a gene in control and BRPF3 depleted cells ( GSE65065 ) . Replication factors were classified as described previously ( Alabert et al , 2014 ) . Total genes ( grey ) , DNA replication factors ( colors ) and BRPF3 ( black ) expression levels are shown . Continuous black lines mark 1 . 5 fold change in expression levels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, ChIP-seq analysis of BRPF3 binding on the human genome. BRPF3 enrichment signal obtained by ChIP-seq with asynchronous (AS), synchronized (S) and released into HU (S+HU) RKO cells, on the MCM4 (left) and Lamin B2 (right) replication origins. Signals were compared to the published enrichment signals for ORC1 in HeLa cells ((Dellino et al, 2013), GSE37583), HBO1 in RKO cells (Avvakumov et al., 2012, GSE33221), and H3K14ac in IMR90 cells (GSM521881). The negative control IgG signal shown is from the S+HUchromatin.B, Venn diagrams illustrating the overlap of genome binding sites between (top) BRPF3, HBO1 and ORC1, and (bottom) BRPF3, BRPF1/2 and ORC1 in human cells.C, Heatmaps of BRPF3, HBO1 and H3K14ac ChIP-seq signal on ± 5kb surrounding the TSS of genes. Genes were sorted by BRPF3 abundance from high to low. A.U. arbitrary unit.D, Average profiles of BRPF3 and H3K14ac ChIP-seq signal on ± 2.5kb around the centre of (top) BRPF3 and (bottom) ORC1 genome binding sites.E, ChIP-qPCR analysis of H3K14ac on well-characterized replication origins. H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells. Error bars, range; n= 2 biological replicas.F, Expression levels of DNA replication factors were not altered by the lack of BRPF3. Each point represents the expression levels of a gene in control and BRPF3 depleted cells (GSE65065). Replication factors were classified as described previously (Alabert et al, 2014). Total genes (grey), DNA replication factors (colors) and BRPF3 (black) expression levels are shown. Continuous black lines mark 1.5 fold change in expression levels.


Figure 5A , Western blot of DNA damage signalling in BRPF3 depleted cells . siRNA transfected U - 2 - OS cells were treated with HU for 2 h or 24 h . One representative experiment out of two biological experiments is shown . B - C , Recovery from replication stress . siRNA treated cells were treated for 24 hrs with HU ( 0 h ) , and released into normal medium for the indicated times ( see also Fig . S6A , B ) . ( B ) DNA replication rate measured by EdU incorporation . Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells . n > 1000 . Mann - Whitney : * * * * P < 10 - 4 . ( C ) Cell cycle progression measured by FACS analysis of DNA content . One representative experiment out of two independent biological replicas is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown.B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.


Figure 1 ( A ) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting . s . e . , short exposure ; l . e . , long exposure . The major Merlin band is indicated by an asterisk . The ratio of mono - ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot . The same lysates were also used in Figure EV1A . Asterisks indicate native Merlin , arrows show ubiquitinated Merlin . ( B ) Total lysates from LN229 cells in which Merlin was or was not knocked out ( KO ) were subjected to western blotting . LN229 ( C ) cells were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) and reseeded ( attach ) for two ( LN229 ) hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with arrows . Met5 - A ( D ) cells were detached ( denoted by sus . , referring to suspension ) by trypsinization ( see Methods ) and reseeded ( attach ) for four ( Met5 - A ) hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with arrows . ( E ) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) . Total lysates of these cells were subjected to western blotting . The major Merlin band is indicated by an asterisk . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Ubiquitinated Merlin is marked with an arrow . ( F ) LN229 cells treated with DMSO or thapsigargin ( TG ) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG . The immunoprecipitated products were subjected to western blotting . The number of conjugated ubiquitin ( Ub ) moieties was marked . Tetra - ubiquitinated Merlin is indicated by an arrow . ( G ) LN229 cells stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were treated with DMSO or thapsigargin ( TG ) . These cells were lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Both arrowhead and arrow point to the mono - ubiquitinated Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( H ) LN229 cells stably transduced with 6x - Histidine - tagged ubiquitin ( His - Ubi ) were detached or reseeded as in ( C ) . These cells were lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . Arrowheads and arrows point to the mono - or di - ubiquitinated Merlin ( see a longer exposed blotting results in Figure EV1D ) . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Total lysates from the indicated cell lines cultured in regular conditions at a steady state were subjected to western blotting. s.e., short exposure; l.e., long exposure. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot. The same lysates were also used in Figure EV1A. Asterisks indicate native Merlin, arrows show ubiquitinated Merlin.(B) Total lysates from LN229 cells in which Merlin was or was not knocked out (KO) were subjected to western blotting.LN229 (C) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for two (LN229) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.Met5-A (D) cells were detached (denoted by sus., referring to suspension) by trypsinization (see Methods) and reseeded (attach) for four (Met5-A) hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with arrows.(E) LN229 cells transduced with a Merlin or scrambled shRNA were treated with DMSO, ionomycin (Iono) or thapsigargin (TG). Total lysates of these cells were subjected to western blotting. The major Merlin band is indicated by an asterisk. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Ubiquitinated Merlin is marked with an arrow.(F) LN229 cells treated with DMSO or thapsigargin (TG) were lysed and subjected to immunoprecipitation with a Merlin antibody or IgG. The immunoprecipitated products were subjected to western blotting. The number of conjugated ubiquitin (Ub) moieties was marked. Tetra-ubiquitinated Merlin is indicated by an arrow.(G) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were treated with DMSO or thapsigargin (TG). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Both arrowhead and arrow point to the mono-ubiquitinated Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(H) LN229 cells stably transduced with 6x-Histidine-tagged ubiquitin (His-Ubi) were detached or reseeded as in (C). These cells were lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. Arrowheads and arrows point to the mono- or di-ubiquitinated Merlin (see a longer exposed blotting results in Figure EV1D). The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


Figure 2 ( A ) LN229 cells were detached ( denoted by sus . , referring to suspension ) by trypsinization and reseeded ( denoted by attach ) for two hours . Total lysates from these cells were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( B ) LN229 cells treated with DMSO , ionomycin ( Iono ) or thapsigargin ( TG ) were lysed and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( C ) LN229 cells treated with DMSO or thapsigargin ( TG ) were subjected to active RAC1 pulldown assay followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . LN229 cells transduced with vector or EGFP - RAC1 ( Q61L ) ( D ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( E ) LN229 cells transduced with vector or Myc - PAK1 ( T423E ) ( E ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( F ) LN229 cells stably transduced with wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) and analyzed by western blot . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . Native Merlin ( asterisk ) ; ubiquitinated Merlin ( arrow ) . ( G ) Merlin - depleted LN229 cells stably transduced with Flag - tagged wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) LN229 cells were detached (denoted by sus., referring to suspension) by trypsinization and reseeded (denoted by attach) for two hours. Total lysates from these cells were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(B) LN229 cells treated with DMSO, ionomycin (Iono) or thapsigargin (TG) were lysed and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(C) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to active RAC1 pulldown assay followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.LN229 cells transduced with vector or EGFP-RAC1(Q61L) (D) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(E) LN229 cells transduced with vector or Myc-PAK1(T423E) (E) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(F) LN229 cells stably transduced with wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG) and analyzed by western blot. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. Native Merlin (asterisk); ubiquitinated Merlin (arrow).(G) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


Figure 3 ( B ) LN229 cells transduced with a pool of four siRNAs against NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot . ( C ) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( D ) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization ( see Methods ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( E ) LN229 cells stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA . These cells were detached as in ( D ) , lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( F ) HEK293T cells were transfected with indicated genes and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( G ) HEK293T cells were transfected with indicated genes . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( H ) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody , a NEDD4L antibody , or IgG . The immunoprecipitated products were subjected to western blotting .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) LN229 cells transduced with a pool of four siRNAs against NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the short exposure blot was quantified by Image J and is shown under the blot.(C) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(D) LN229 cells stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA were detached by trypsinization (see Methods) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(E) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs targeting NEDD4L or a scrambled shRNA. These cells were detached as in (D), lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(F) HEK293T cells were transfected with indicated genes and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(G) HEK293T cells were transfected with indicated genes. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(H) LN229 cells were lysed and subjected to immunoprecipitation with a Merlin antibody, a NEDD4L antibody, or IgG. The immunoprecipitated products were subjected to western blotting.


Figure 4 ( A ) LN229 cells transduced with a pool of four siRNAs against AMOTL1 ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( B ) LN229 cells stably transduced with 6x - Histidine - tagged Ubiquitin ( His - Ubi ) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control ( sh - Co . ) . These cells were detached , lysed and subjected to nickel - charged affinity purification followed by western blotting for endogenous Merlin . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( D ) Merlin - depleted LN229 cells stably expressing Flag - HA - tagged Merlin were transduced with scrambled shRNA ( sh - Co . ) or two distinct shRNAs targeting AMOTL1 , treated with DMSO or thapsigargin ( TG ) and subjected to Flag immunoprecipitation followed by western blotting . ( E ) HEK293T cells were transfected with the indicated constructs . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . PY : PPEY motif ; LY : LPTY motif . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( F ) Merlin - depleted LN229 cells stably transduced with Flag - tagged wild - type Merlin or the indicated mutants were treated with DMSO or thapsigargin ( TG ) . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( G ) HEK293T cells were transfected with the indicated constructs . The cells were lysed and subjected to immunoprecipitation with a Flag antibody . The lysate and immunoprecipitated products were subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting.(E) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(G) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.


Figure 5 ( A ) LN229 cells stably transduced with Flag - tagged wild - type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS - PAGE . Outlined gel pieces were analyzed by mass spectrometry . ( B ) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in ( A ) are shown . ( D ) and ( E ) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown . ( F ) Merlin - depleted LN229 cells expressing His - Ubiquitin were stably transduced with the indicated HA - tagged Merlin forms or an empty vector . These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA . Arrows indicate mono - ubiquitinated Merlin . The red asterisk indicates native Merlin and black asterisks indicate non - specific signals . The ratio of mono - ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot . ( G ) Merlin - depleted LN229 cells were stably transduced with an empty vector , Flag - tagged wild - type ( WT ) Merlin or its K396R mutant . These cells were detached ( S , suspension ) by trypsinization using the procedure described in the Methods section and reseeded ( A , attached ) for two hours . Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot . ( H ) Merlin - depleted LN229 cells stably transduced with wild - type ( WT ) Merlin or its K396R mutant were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( I ) Merlin - depleted LN229 cells stably transduced with HA - tagged wild - type ( WT ) Merlin or its K396R mutant were treated as described in ( G ) and subjected to western blotting . The ratio of mono - ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) LN229 cells stably transduced with Flag-tagged wild-type Merlin or the S518D mutant were treated with thapsigargin and subjected to immunoprecipitation with a Flag antibody and followed by SDS-PAGE. Outlined gel pieces were analyzed by mass spectrometry. (B) Numbers of unique peptides of Merlin or Ubiquitin from each outlined gel pieces in (A) are shown. (D) and (E) Mass spectrometry spectrums of the indicated peptides from each immunoprecipitated product are shown.(F) Merlin-depleted LN229 cells expressing His-Ubiquitin were stably transduced with the indicated HA-tagged Merlin forms or an empty vector. These cells treated with DMSO or thapsigargin were subjected to nickel affinity purification in a denaturing step followed by western blotting for HA. Arrows indicate mono-ubiquitinated Merlin. The red asterisk indicates native Merlin and black asterisks indicate non-specific signals. The ratio of mono-ubiquitinated to native Merlin in each lane of the input blot was quantified by Image J and is shown under the blot.(G) Merlin-depleted LN229 cells were stably transduced with an empty vector, Flag-tagged wild-type (WT) Merlin or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in the Methods section and reseeded (A, attached) for two hours. Cell lysates were subjected to immunoprecipitation with a Flag antibody followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.(H) Merlin-depleted LN229 cells stably transduced with wild-type (WT) Merlin or its K396R mutant were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(I) Merlin-depleted LN229 cells stably transduced with HA-tagged wild-type (WT) Merlin or its K396R mutant were treated as described in (G) and subjected to western blotting. The ratio of mono-ubiquitinated to native null in each lane was quantified by Image J and is shown under the blot.


Figure 6 ( A ) LN229 cells were treated with DMSO or thapsigargin ( TG ) for the indicated time and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( B ) Merlin - depleted LN229 cells were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( C ) Merlin - depleted LN229 cells were reconstituted with wild type ( WT ) Merlin or its mutants , treated with DMSO or thapsigargin ( TG ) , and subjected to western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot . ( D ) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were treated with DMSO or thapsigargin ( TG ) and subjected to western blotting . ( E ) Merlin - depleted LN229 cells were stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant . These cells were detached ( S , suspension ) by trypsinization using the procedure described in Methods and reseeded ( A , attached ) for two hours . Cell lysates were subjected to western blotting . ( F ) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L ( + ) or a scrambled siRNA ( - ) were detached ( Susp . , suspension ) by trypsinization using the procedure described in Methods and reseeded ( Attach , attached ) for two hours . Total lysates from these cells were subjected to western blotting . ( G ) LN229 cells transfected with indicated siRNAs were seeded for 3 hours . CTGF or CYR61 mRNA in these cells was quantified by q - RT - PCR . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . Mean ± s . e . m , N = 4 biological repeats , unpaired t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) LN229 cells were treated with DMSO or thapsigargin (TG) for the indicated time and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(B) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(C) Merlin-depleted LN229 cells were reconstituted with wild type (WT) Merlin or its mutants, treated with DMSO or thapsigargin (TG), and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.(D) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting.(E) Merlin-depleted LN229 cells were stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant. These cells were detached (S, suspension) by trypsinization using the procedure described in Methods and reseeded (A, attached) for two hours. Cell lysates were subjected to western blotting.(F) LN229 cells transfected with a pool of four siRNAs targeting NEDD4L (+) or a scrambled siRNA (-) were detached (Susp., suspension) by trypsinization using the procedure described in Methods and reseeded (Attach, attached) for two hours. Total lysates from these cells were subjected to western blotting.(G) LN229 cells transfected with indicated siRNAs were seeded for 3 hours. CTGF or CYR61 mRNA in these cells was quantified by q-RT-PCR. *P<0.05, **P<0.01, ****P<0.0001. Mean±s.e.m, N=4 biological repeats, unpaired t-test.


Figure 7 ( A ) LN229 cells stably transduced withHA - Lats1 ( two leftmost panels ) or vector only ( right panel ) were treated with DMSO or thapsigargin ( TG ) and subjected to PLA using HA and Merlin antibodies . Scale bar = 10 μm . ( B ) PLA signals ( dots ) in each cell from the results in ( A ) were quantified . Mean ± s . e . m , two - way ANOVA . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 cells . N = 4 - 9 images for each condition as indicated . All images were collected from one experiment . Two independent experiments were performed and gave similar results . ( C ) Merlin - depleted LN229 cells were stably transduced with HA - tagged wild - type ( WT ) Merlin or its mutants . These cells were treated with thapsigargin ( TG ) for 15 minutes and subjected to PLA using HA and Lats1 antibodies . Scale bar = 20 μm . ( D ) PLA signals ( dots ) in each cell from the results in ( C ) were quantified . Mean ± s . e . m , Ordinary one - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing an average of 10 cells . NVector = 8 , NWT = 16 , NK396R = 18 , NK159R = 17 images . All images were collected from one experiment . Two independent experiments were performed and gave similar results . ( E ) Merlin - depleted LN229 cells stably transduced with empty vector , Flag - tagged wild - type ( WT ) Merlin , or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting . ( F ) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody . The lysates and immunoprecipitated products were subjected to western blotting . ( G ) Merlin - depleted LN229 cells were stably transduced by HA - tagged Merlin . These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin ( TG ) . PLA was performed in these cells using HA and Lats1 antibodies , and the PLA signals ( dots , showed in Figure EV5E ) in each cell were quantified . Mean ± s . e . m , two - way ANOVA . * * * * P < 0 . 0001 . Each data point represents an image field containing averagely 10 cells . N = 12 images in all conditions , except for Nsi - Control : TG = 13 images . All images were collected from one experiment . Two independent experiments were performed and showed similar results . ( H ) LN229 cells treated with DMSO or thapsigargin ( TG ) were subjected to cytosolic ( C ) / membrane ( M ) fractionation followed by western blotting . The ratio of mono - ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) LN229 cells stably transduced withHA-Lats1 (two leftmost panels) or vector only (right panel) were treated with DMSO or thapsigargin (TG) and subjected to PLA using HA and Merlin antibodies. Scale bar = 10 μm. (B) PLA signals (dots) in each cell from the results in (A) were quantified. Mean±s.e.m, two-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001. Each data point represents an image field containing an average of 10 cells. N=4-9 images for each condition as indicated. All images were collected from one experiment. Two independent experiments were performed and gave similar results. (C) Merlin-depleted LN229 cells were stably transduced with HA-tagged wild-type (WT) Merlin or its mutants. These cells were treated with thapsigargin (TG) for 15 minutes and subjected to PLA using HA and Lats1 antibodies. Scale bar = 20 μm. (D) PLA signals (dots) in each cell from the results in (C) were quantified. Mean±s.e.m, Ordinary one-way ANOVA. ****P<0.0001. Each data point represents an image field containing an average of 10 cells. NVector=8, NWT=16, NK396R=18, NK159R=17 images. All images were collected from one experiment. Two independent experiments were performed and gave similar results. (E) Merlin-depleted LN229 cells stably transduced with empty vector, Flag-tagged wild-type (WT) Merlin, or its K396R mutant were subjected to immunoprecipitation with a Flag antibody followed by western blotting.(F) HEK293T cells were transfected with indicated proteins and subjected to immunoprecipitation with a Flag antibody. The lysates and immunoprecipitated products were subjected to western blotting.(G) Merlin-depleted LN229 cells were stably transduced by HA-tagged Merlin. These cells were then transduced with a pool of four siRNAs against NEDD4L or a scrambled control siRNA and treated with DMSO or thapsigargin (TG). PLA was performed in these cells using HA and Lats1 antibodies, and the PLA signals (dots, showed in Figure EV5E) in each cell were quantified. Mean±s.e.m, two-way ANOVA. ****P<0.0001. Each data point represents an image field containing averagely 10 cells. N=12 images in all conditions, except for Nsi-Control:TG=13 images. All images were collected from one experiment. Two independent experiments were performed and showed similar results.(H) LN229 cells treated with DMSO or thapsigargin (TG) were subjected to cytosolic (C)/membrane (M) fractionation followed by western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot.


Figure 8 ( A ) Met5 - A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay . Scale bar = 40 μm . ( B ) The percentage of BrdU positive cells from ( A ) 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Met5-A cells stably transduced with the indicated shRNAs targeting Merlin or a scrambled shRNA were subjected to the BrdU incorporation assay. Scale bar = 40 μm. (B) The percentage of BrdU positive cells from (A) 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. (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. (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).


Figure 1 ( 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 ) . ( 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 ) . 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).(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).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).(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).


Figure 2 ( 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 ) . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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).(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.(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).


Figure 3 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).(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).


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5Immunolabeling 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 ) . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunolabeling 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).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.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).


Figure 6Representative 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative 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.


Figure 1VERO 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1VERO 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.(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.