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 .

B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O

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 .

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O

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 .

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

(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 .

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

(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 .

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

(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 O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

(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.


( 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 .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

(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 .

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

(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 .

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

(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 .

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

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 .

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

(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 ) .

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

(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 O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

(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.


( 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 .

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

(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 .

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

(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 .

O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

(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 .

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

(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 .

O O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

(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 .

O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

(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 O B-CELL_LINE O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O

(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.


( 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 .

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

(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 .

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

(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 .

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

(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 .

O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

(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 .

(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 O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

(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.


( 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 .

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

(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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O

(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 .

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

(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 .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O

(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 .

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

(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 O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(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.


( 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 .

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

(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 .

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

(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 .

(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 .

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

(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 .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(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 .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

(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 O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(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 .

O O O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(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 .

O O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(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 .

O O O B-CELL_LINE O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O

(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 .

O O O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(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 O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

(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.


( 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 .

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

(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 .

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


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

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

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


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

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

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


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

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

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


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

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

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


Confocal microscopy of IBA - 1 ( green staining ) immunohistochemistry of RPE flatmounts ( RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel ) from a healthy donor ( A ) , a geographic atrophy lesion ( B ) , and large drusen ( C and D ) . ( A , B , D ) : orthogonal Z - stack projection ; ( C ) : oblique Z - stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina ( inset ) .

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

Confocal microscopy of IBA-1 (green staining) immunohistochemistry of RPE flatmounts (RPE autofluorescence visible as orange due to its autofluorescence in the red and green channel) from a healthy donor (A), a geographic atrophy lesion (B), and large drusen (C and D). (A, B, D): orthogonal Z-stack projection; (C): oblique Z-stack projection and dissecting microscope appearance of postmortem large drusen after removal of the overlaying retina (inset).


Double - labeling on the subretinal side of the retina ( to avoid masking by RPE autofluorescence ) of IBA - 1 + ( E , green fluorescence ) and CD18 ( F , red fluorescence ; G , merge ) .

O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O

Double-labeling on the subretinal side of the retina (to avoid masking by RPE autofluorescence) of IBA-1+ (E, green fluorescence) and CD18 (F, red fluorescence; G, merge).


Orthogonal and lateral Z - stack of a subretinal IBA - 1 + ( green fluorescence ) MPs adjacent to the RPE ( orange autofluorescence ) in the vicinity of a large drusen .

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

Orthogonal and lateral Z-stack of a subretinal IBA-1+ (green fluorescence) MPs adjacent to the RPE (orange autofluorescence) in the vicinity of a large drusen.


APOE ( A ) and IBA - 1 ( B , merge in C ) double - labeling on paraffin sections of surgical specimens of human tonsils .

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

APOE (A) and IBA-1 (B, merge in C) double-labeling on paraffin sections of surgical specimens of human tonsils.


APOE ( D ) and IBA - 1 ( E , merge in F ) double - labeling on the subretinal aspect of a large drusen overlaying retina ( to avoid specific signal masking by the RPE orange autofluorescence ) .

B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O O O B-TISSUE O O O O

APOE (D) and IBA-1 (E, merge in F) double-labeling on the subretinal aspect of a large drusen overlaying retina (to avoid specific signal masking by the RPE orange autofluorescence).


APOE ( red staining ) immunohistochemistry of control donor tissue ( G ) and adjacent to GA lesion ( H ) .

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APOE (red staining) immunohistochemistry of control donor tissue (G) and adjacent to GA lesion (H).


IBA - 1 ( green staining , RPE far - red autofluorescence ) double - labeling of the GA lesion shown in ( H ) . Representative images from five donor eyes with large drusen , five donor eyes with GA , and three healthy donors between 70 and 89 years old .

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IBA-1 (green staining, RPE far-red autofluorescence) double-labeling of the GA lesion shown in (H). Representative images from five donor eyes with large drusen, five donor eyes with GA, and three healthy donors between 70 and 89 years old.


Immunohistochemistry of APOE ( red ) and IBA - 1 ( green ) on a section ( A , blue Hoechst ) and the subretinal side of a retinal flatmount ( B and C ) from a 12 - month - old Cx3cr1GFP / GFP mouse ( representative of 3 independent experiments , experiments omitting the primary antibody immunostaining served as negative controls ) . ONL : outer nuclear layer ; RPE : retinal pigment epithelium .

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Immunohistochemistry of APOE (red) and IBA-1 (green) on a section (A, blue Hoechst) and the subretinal side of a retinal flatmount (B and C) from a 12-month-old Cx3cr1GFP/GFP mouse (representative of 3 independent experiments, experiments omitting the primary antibody immunostaining served as negative controls). ONL: outer nuclear layer; RPE: retinal pigment epithelium.


Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 , the experiment was repeated twice with similar results ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP FACS - sorted microglial cells , freshly extracted from adult brain ( n = 4 - 5 / group ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0159 ) . Quantitative RT - PCR of ApoEmRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages cultured for 24 h with CX3CL1 . ( n = 4 / group per experiment ; Mann - Whitney U - test , * P = 0 . 0286 . The experiment was repeated twice with similar results ) .

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

Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP monocytes cultured for 24 h in contact with POS of an overlaying retinal explant (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286, the experiment was repeated twice with similar results).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP FACS-sorted microglial cells, freshly extracted from adult brain (n = 4-5/group; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0159).Quantitative RT-PCR of ApoEmRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages cultured for 24 h with CX3CL1. (n = 4/group per experiment; Mann-Whitney U-test, *P = 0.0286. The experiment was repeated twice with similar results).


APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1 - exposed C57BL / 6J and Cx3cr1GFP / GFP peritoneal macrophages at 24 h . Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control . The experiment was repeated twice with similar results .

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APOE Western blot analysis of equivalent amounts of supernatant protein from CX3CL1-exposed C57BL/6J and Cx3cr1GFP/GFP peritoneal macrophages at 24 h. Soluble Mer receptor tyrosine kinase that is released constitutively from cultured macrophages served as a loading control. The experiment was repeated twice with similar results.


12 - month - old IBA - 1 stained RPE flatmounts of Cx3cr1GFP / GFP ( H ) and Cx3cr1GFP / GFPApoE − / − ( I ) mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes in 2 - month - old ( left ) and 12 - month - old ( right ) mice of the indicated strains ( n = 9 - 20 / group ANOVA / Dunnett test at 12 months : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test at 12 months of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

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

12-month-old IBA-1 stained RPE flatmounts of Cx3cr1GFP/GFP (H) and Cx3cr1GFP/GFPApoE−/− (I) mice.Quantification of subretinal IBA-1+ mononuclear phagocytes in 2-month-old (left) and 12-month-old (right) mice of the indicated strains (n = 9-20/group ANOVA/Dunnett test at 12 months: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test at 12 months of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


Quantification of subretinal IBA - 1 + mononuclear phagocytes after 4 days of light challenge of 2 - month - old mice of the indicated strains ( n = 10 - 25 / group ANOVA / Dunnett test : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

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Quantification of subretinal IBA-1+ mononuclear phagocytes after 4 days of light challenge of 2-month-old mice of the indicated strains (n = 10-25/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


IBA - 1 - ( green ) and CD102 - ( red ) stained RPE flatmounts 7 days after laser injury of 2 - month - old Cx3cr1GFP / GFP ( E ) and Cx3cr1GFP / GFPApoE − / − ( F ) mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes on the RPE counted on the RPE at a distance of 0 - 500 μm to CD102 + CNV 7 days after the laser injury of 2 - month - old mice of the indicated strains ( n = 8 - 10 eyes / group ANOVA / Dunnett test : Cx3cr1GFP / GFP versus any other group * P < 0 . 0001 ; Mann - Whitney U - test of Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P < 0 . 0001 ) .

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IBA-1- (green) and CD102- (red) stained RPE flatmounts 7 days after laser injury of 2-month-old Cx3cr1GFP/GFP (E) and Cx3cr1GFP/GFPApoE−/− (F) mice.Quantification of subretinal IBA-1+mononuclear phagocytes on the RPE counted on the RPE at a distance of 0-500 μm to CD102+CNV 7 days after the laser injury of 2-month-old mice of the indicated strains (n = 8-10 eyes/group ANOVA/Dunnett test: Cx3cr1GFP/GFP versus any other group *P < 0.0001; Mann-Whitney U-test of Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P < 0.0001).


Representative image of a RPE flatmount 12 h after the subretinal injection ( red marking ) of 4 μl PBS with 12 , 000 CFSE - stained thioglycollate - elicited peritoneal cells that contain 70 % macrophages ( inset close - up view ) . Quantifications of CFSE + F4 / 80 + macrophages at different time points after subretinal injections of C57BL / 6J and Cx3cr1GFP / GFP CFSE + macrophages ( n = 5 / per group ( 12 h ) and n = 6 / per group thereafter ; Mann - Whitney U - test , C57BL / 6J versus Cx3cr1GFP / GFP : 1 day n = 20 / group * P < 0 . 0001 ; 2 day n = 6 / group * P = 0 . 0317 ) .

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

Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view).Quantifications of CFSE+F4/80+ macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1GFP/GFP CFSE+ macrophages (n = 5/per group (12 h) and n = 6/per group thereafter; Mann-Whitney U-test, C57BL/6J versus Cx3cr1GFP/GFP: 1 day n = 20/group *P < 0.0001; 2 day n = 6/group *P = 0.0317).


Representative image of TUNEL / Hoechst double - staining 12 h after subretinal injection of C57BL / 6J CFSE + macrophages ( experiment repeated three times , inset close - up view ) .

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Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE+ macrophages (experiment repeated three times, inset close-up view).


Representative cytometry images of SSC - A / CFSE and CD11b / F4 / 80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP / GFP CFSE + macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL / 6J and Cx3cr1GFP / GFP macrophages into C57BL / 6J ( n = 16 - 20 / group ; Mann - Whitney U - test , * P = 0 . 0024 ) .

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Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1GFP/GFP CFSE+ macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1GFP/GFP macrophages into C57BL/6J (n = 16-20/group; Mann-Whitney U-test, *P = 0.0024).


Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + magnetic - bead - sorted bone marrow - derived monocytes ( Mo ) from C57BL / 6J and Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 8 - 12 / group ; Mann - Whitney U - test , * P = 0 . 0006 ) . Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + CD11bFACS - sorted brainmicroglial cells from C57BL / 6J and Cx3cr1GFP / GFPmice into C57BL / 6Jmice ( n = 9 - 12 / group ; Mann - Whitney U - test , * P = 0 . 0087 ) .

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

Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+ magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 8-12/group; Mann-Whitney U-test, *P = 0.0006).Quantification of subretinal CFSE+ cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+CD11bFACS-sorted brainmicroglial cells from C57BL/6J and Cx3cr1GFP/GFPmice into C57BL/6Jmice (n = 9-12/group; Mann-Whitney U-test, *P = 0.0087).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + macrophages from C57BL / 6J , Cx3cr1GFP / GFP , Cx3cr1GFP / GFPApoE − / − , and ApoE − / − mice into C57BL / 6Jmice ( n = 8 - 12 / group ; one - way ANOVA / Dunnett test of Cx3cr1GFP / GFP versus any other group * P ≤ 0 . 0001 ; Mann - Whitney U - test , Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − * P = 0 . 0006 ) .

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Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE+macrophages from C57BL/6J, Cx3cr1GFP/GFP, Cx3cr1GFP/GFPApoE−/−, and ApoE−/−mice into C57BL/6Jmice (n = 8-12/group; one-way ANOVA/Dunnett test of Cx3cr1GFP/GFP versus any other group *P ≤ 0.0001; Mann-Whitney U-test, Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/− *P = 0.0006).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL / 6J CFSE + macrophages into C57BL / 6J and with exogenously added APOE3 at 1 , 10 , or 100 μg / ml calculated intraocular concentrations ( n = 6 - 7 / group ; one - way ANOVA / Dunnett test : C57BL / 6J versus 10 μg * P = 0 . 0488 ; C57BL / 6J versus 100 μg ‡ P = 0 . 006 . Mann - Whitney U - test : C57BL / 6J versus 10 μg * P = 0 . 0012 ; C57BL / 6J versus 100 μg ‡ P = 0 . 0013 ) .

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

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE+ macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations (n = 6-7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg *P = 0.0488; C57BL/6J versus 100 μg ‡P = 0.006. Mann-Whitney U-test: C57BL/6J versus 10 μg *P = 0.0012; C57BL/6J versus 100 μg ‡P = 0.0013).


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 2 - month - , 12 - month - , and 2 - month - old mice after 4 days of light challenge of C57BL / 6 and Cx3cr1GFP / GFP mouse RPE / choroid plexus ( aging : n = 5 - 6 / group ; Mann - Whitney U - test : 12 months * P = 0 . 0129 ; 4 days light challenge : n = 7 - 8 / group ; * P = 0 . 029 ) .

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Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 2-month-, 12-month-, and 2-month-old mice after 4 days of light challenge of C57BL/6 and Cx3cr1GFP/GFP mouse RPE/choroid plexus (aging: n = 5-6/group; Mann-Whitney U-test: 12 months *P = 0.0129; 4 days light challenge: n = 7-8/group; *P = 0.029).


Immunohistochemistry of FASL ( red ) and IBA - 1 ( green ) of a RPE flatmounts and sections ( insets , Hoechst staining blue ) from a 12 - month - old WT ( B ) and Cx3cr1GFP / GFP ( C ) mouse ( representative of three independent experiments , immunostainings omitting the primary antibody served as negative controls ) .

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Immunohistochemistry of FASL (red) and IBA-1 (green) of a RPE flatmounts and sections (insets, Hoechst staining blue) from a 12-month-old WT (B) and Cx3cr1GFP/GFP(C) mouse (representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus extracts 3 h after subretinal injection of C57BL / 6J macrophages or Cx3cr1GFP / GFP macrophages ( n = 6 per group ) , Mann - Whitney U - test , * P = 0 . 0087 .

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Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus extracts 3 h after subretinal injection of C57BL/6J macrophages or Cx3cr1GFP/GFP macrophages (n = 6 per group), Mann-Whitney U-test, *P = 0.0087.


IBA - 1 - stained RPE flatmounts of 2 - month - old C57BL / 6J , Faslgld / gld , and Faslpr / lpr mice after 4 days of light challenge . Quantification of subretinal IBA - 1 + mononuclear phagocytes in control ( left ) and 4 - day light - challenged ( right ) 2 - month - old mice of the indicated strains ( n = 6 - 10 / group ANOVA / Dunnett test at 4 - day light challenge : C57BL / 6J versus FasLgld / gld and C57BL / 6J versus Faslpr / lpr both * P < 0 . 0001 ; Mann - Whitney U - test at 4 - day light challenge : C57BL / 6J versus FasLgld / gld * P < 0 . 0001 ; C57BL / 6J versus Faslpr / lpr * P < 0 . 0001 ) .

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

IBA-1-stained RPE flatmounts of 2-month-old C57BL/6J, Faslgld/gld, and Faslpr/lpr mice after 4 days of light challenge.Quantification of subretinal IBA-1+ mononuclear phagocytes in control (left) and 4-day light-challenged (right) 2-month-old mice of the indicated strains (n = 6-10/group ANOVA/Dunnett test at 4-day light challenge: C57BL/6J versus FasLgld/gld and C57BL/6J versus Faslpr/lpr both *P < 0.0001; Mann-Whitney U-test at 4-day light challenge: C57BL/6J versus FasLgld/gld *P < 0.0001; C57BL/6J versus Faslpr/lpr *P < 0.0001).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL / 6JCFSE + macrophages into C57BL / 6J and FasLgld / gldmice and Faslpr / lprmacrophages into C57BL / 6Jmice ( n = 10 - 15 / group , one - way ANOVA / Dunnett test : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into Fasgld / gldmice * P = 0 . 0002 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 . Mann - Whitney U - test : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into FasLgld / gldmice * P < 0 . 0001 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 ) .

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

Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6JCFSE+macrophages into C57BL/6J and FasLgld/gldmice and Faslpr/lprmacrophages into C57BL/6Jmice (n = 10-15/group, one-way ANOVA/Dunnett test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into Fasgld/gldmice *P = 0.0002; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001. Mann-Whitney U-test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into FasLgld/gldmice *P < 0.0001; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFP CFSE + macrophages into C57BL / 6J with or without the Fas agonist MegaFasL ( calculated intraocular concentrations 1 ng / ml ; n = 7 - 8 ; Mann - Whitney U - test : * P = 0 . 014 ) .

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

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFP CFSE+ macrophages into C57BL/6J with or without the Fas agonist MegaFasL (calculated intraocular concentrations 1 ng/ml; n = 7-8; Mann-Whitney U-test: *P = 0.014).


In vitro MegaFasL - induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h . TUNEL + nucleus quantification expressed as percentage of non - MegaFasL - exposed control .

O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O

In vitro MegaFasL-induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h. TUNEL+ nucleus quantification expressed as percentage of non-MegaFasL-exposed control.


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus 3 h after subretinal injection of PBS ( PBS ; n = 36 ) , IL - 6 ( n = 17 ) , and APOE3 ( n = 21 ) ; calculated intraocular concentrations : 5 ng / ml and 10 μg / ml , respectively ; one - way ANOVA / Dunnett post hoc test PBS versus IL - 6 * P = 0 . 038 ; t - test PBS versus IL - 6 * P = 0 . 043 .

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

Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus 3 h after subretinal injection of PBS (PBS; n = 36), IL-6 (n = 17), and APOE3 (n = 21); calculated intraocular concentrations: 5 ng/ml and 10 μg/ml, respectively; one-way ANOVA/Dunnett post hoc test PBS versus IL-6 *P = 0.038; t-test PBS versus IL-6 *P = 0.043.


Mouse IL - 6 ELISA of supernatants from C57BL / 6J peritoneal macrophages incubated for 24 h in control medium , lipid - free APOE3 ( 5 μg / ml ) , APOE3 ( 5 μg / ml ) , and polymyxin B ( 25 μg / ml ) , heat - denatured APOE3 ( dAPOE3 , 5 μg / ml ) , APOE3 ( 5 μg / ml ) , and rat IgG1 isotype control ( IgG , 100 μg / ml ) or APOE3 ( 5 μg / ml ) and rat anti - CD14 antibody ( aCD14 Ab , 100 μg / ml ) or APOE3 ( 5 μg / ml ) and rat anti - TLR2 antibody ( aTLR2 Ab , 100 μg / ml ) ( n = 5 - 6 / group ; one - way ANOVA / Bonferroni multi - comparison tests : APOE3 versus CTL * P < 0 . 0001 ; dAPOE3 versus APOE3 # P < 0 . 0001 ; APOE3 IgG versus CTL P < 0 . 0001 ; APOE3 IgG versus APOE3 aCD14 Ab § P < 0 . 0001 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P < 0 . 0001 . Mann - Whitney U - test : APOE3 versus CTL * P = 0 . 0043 ; dAPOE3 versus APOE3 # P = 0 . 0117 ; APOE3 IgG versus CTL P = 0 . 0080 ; APOE3 IgG versus APOE3 aCD14 Ab § P = 0 . 0117 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P = 0 . 0079 . The experiment was repeated twice with similar results ) .

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

Mouse IL-6 ELISA of supernatants from C57BL/6J peritoneal macrophages incubated for 24 h in control medium, lipid-free APOE3 (5 μg/ml), APOE3 (5 μg/ml), and polymyxin B (25 μg/ml), heat-denatured APOE3 (dAPOE3, 5 μg/ml), APOE3 (5 μg/ml), and rat IgG1 isotype control (IgG, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-CD14 antibody (aCD14 Ab, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-TLR2 antibody (aTLR2 Ab, 100 μg/ml) (n = 5-6/group; one-way ANOVA/Bonferroni multi-comparison tests: APOE3 versus CTL *P < 0.0001; dAPOE3 versus APOE3 #P < 0.0001; APOE3 IgG versus CTL P < 0.0001; APOE3 IgG versus APOE3 aCD14 Ab §P < 0.0001; APOE3 IgG versus APOE3 aTLR2 Ab ++P <0.0001. Mann-Whitney U-test: APOE3 versus CTL *P = 0.0043; dAPOE3 versus APOE3 #P = 0.0117; APOE3 IgG versus CTL P = 0.0080; APOE3 IgG versus APOE3 aCD14 Ab §P = 0.0117; APOE3 IgG versus APOE3 aTLR2 Ab ++P = 0.0079. The experiment was repeated twice with similar results).


Quantitative RT - PCR of IL - 6mRNA normalized with S26mRNA of C57BL / 6J and Cx3cr1GFP / GFP , and Cx3cr1GFP / GFP ApoE − / − peritoneal macrophages cultured for 24 h with CX3CL1 ( n = 5 per group , one - way ANOVA / Bonferroni post hoc multi - comparison tests : C57BL / 6J versus Cx3cr1GFP / GFP * P < 0 . 0001 and Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P < 0 . 0001 . Mann - Whitney U - test : C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0079 and Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P = 0 . 0079 . The experiment was repeated twice with similar results . ) .

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

Quantitative RT-PCR of IL-6mRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP, and Cx3cr1GFP/GFP ApoE−/− peritoneal macrophages cultured for 24 h with CX3CL1 (n = 5 per group, one-way ANOVA/Bonferroni post hoc multi-comparison tests: C57BL/6J versus Cx3cr1GFP/GFP *P < 0.0001 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P < 0.0001. Mann-Whitney U-test: C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0079 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P = 0.0079. The experiment was repeated twice with similar results.).


Orthogonal and lateral Z - stack projection of IL - 6 ( D , red ) , IBA - 1 + ( E , green ) , and phalloidin ( E , blue ) of IBA + mononuclear phagocytes adjacent to the phalloidin + RPE of a retinal flatmount from a 12 - month - old Cx3cr1GFP / GFP mouse . ( Representative of three independent experiments , immunostainings omitting the primary antibody served as negative controls ) .

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

Orthogonal and lateral Z-stack projection of IL-6 (D, red), IBA-1+ (E, green), and phalloidin (E, blue) of IBA+ mononuclear phagocytes adjacent to the phalloidin+ RPE of a retinal flatmount from a 12-month-old Cx3cr1GFP/GFP mouse. (Representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).


Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL / 6J CFSE + macrophages into C57BL / 6J with or without IL - 6 ( n = 15 - 20 / group ; calculated intraocular concentrations of 5 ng / ml . Mann - Whitney U - test : * P < 0 . 0001 ) . Quantification of subretinal CFSE + F4 / 80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFPCFSE + Mϕs into C57BL / 6J with control ( IgG , n = 16 ) or anti - IL - 6 antibody ( aIL - 6 Ab , n = 8 ; calculated intraocular concentrations 5 μg / ml ; per group . Mann - Whitney U - test : * P = 0 . 0036 ) .

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

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6J CFSE+ macrophages into C57BL/6J with or without IL-6 (n = 15-20/group; calculated intraocular concentrations of 5 ng/ml. Mann-Whitney U-test: *P < 0.0001).Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFPCFSE+Mϕs into C57BL/6J with control (IgG, n = 16) or anti-IL-6 antibody (aIL-6 Ab, n = 8; calculated intraocular concentrations 5 μg/ml; per group. Mann-Whitney U-test: *P = 0.0036).


7 day laser - injured IBA - 1 ( green ) and CD102 ( red ) double - stained RPE flatmounts of control IgG - ( I ) and anti - IL - 6 - treated ( J ) Cx3cr1GFP / GFP mice . Quantification of subretinal IBA - 1 + mononuclear phagocytes / impact localized on the lesion surrounding RPE of Cx3cr1GFP / GFP mice treated with control IgG , IL - 6 - , or CD14 - blocking antibodies ( calculated intraocular concentration 5 μg / ml ; n = 13 - 14 / group , one - way ANOVA / Dunnett ' s post hoc tests of IgG versus any other group * P < 0 . 001 . Mann - Whitney U - test IgG versus anti - IL - 6 * P = 0 . 0021 ; IgG versus anti CD14 * P = 0 . 0028 ) .

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

7 day laser-injured IBA-1 (green) and CD102 (red) double-stained RPE flatmounts of control IgG- (I) and anti-IL-6-treated (J) Cx3cr1GFP/GFP mice.Quantification of subretinal IBA-1+ mononuclear phagocytes/impact localized on the lesion surrounding RPE of Cx3cr1GFP/GFP mice treated with control IgG, IL-6-, or CD14-blocking antibodies (calculated intraocular concentration 5 μg/ml; n = 13-14/group, one-way ANOVA/Dunnett's post hoc tests of IgG versus any other group *P < 0.001. Mann-Whitney U-test IgG versus anti-IL-6 *P = 0.0021; IgG versus anti CD14*P = 0.0028).


heparin . Reducing - end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface . PBS running buffer flow rate was 500 µL . min - 1 . The data for the three sensing channels are reported as an average response . Protein injections are delimited by the red arrows . A Injection of 100 nM FGF2 .

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

heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. A Injection of 100 nM FGF2.


heparin . Reducing - end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface . PBS running buffer flow rate was 500 µL . min - 1 . The data for the three sensing channels are reported as an average response . Protein injections are delimited by the red arrows . B 64 nM S1 RBD protein . C 64 nM S1 RBD protein over a streptavidin surface ( no heparin functionalization ) .

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

heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. B 64 nM S1 RBD protein. C 64 nM S1 RBD protein over a streptavidin surface (no heparin functionalization).


The structural change of the SARS - CoV - 2 S1 RBD observed in the presence of heparin by circular dichroism ( CD ) spectroscopy . ( A ) CD spectra of 200 μg / mL spike 1 alone ( purple ) or with 30 μg heparin ( orange ) in phosphate buffered saline pH 7 . 4 . Theoretical sum of spike 1 alone and 30 μg of heparin ( control ) if no interaction was observed ( black ) . ( B ) Δ secondary structure ( % ) of ( A ) . A 2 . 4 % increase in helix and 2 . 2 % decrease in antiparallel secondary structural features were calculated ( BestSel ) for the observed spectrum compared to that of the theoretical , summative spectrum of the SARS - CoV - 2 S1 RBD in the presence of heparin .

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

The structural change of the SARS-CoV-2 S1 RBD observed in the presence of heparin by circular dichroism (CD) spectroscopy.(A) CD spectra of 200 μg/mL spike 1 alone (purple) or with 30 μg heparin (orange) in phosphate buffered saline pH 7.4. Theoretical sum of spike 1 alone and 30 μg of heparin (control) if no interaction was observed (black). (B) Δ secondary structure (%) of (A). A 2.4% increase in helix and 2.2% decrease in antiparallel secondary structural features were calculated (BestSel) for the observed spectrum compared to that of the theoretical, summative spectrum of the SARS-CoV-2 S1 RBD in the presence of heparin.


D - G Culture dynamics of phage ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Bacterial strains were exposed to phage at time = 0 h , and optical density measurements were read in a 96 ‐ well plate format . Each experiment was performed three times with three technical triplicates for each biological replicate . Error bars represent SEM . Shown are representative results for three of the ten phages tested ; data for the remaining seven phages are found in Supplementary Fig S2 .

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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-G Culture dynamics of phage‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Bacterial strains were exposed to phage at time = 0 h, and optical density measurements were read in a 96‐well plate format. Each experiment was performed three times with three technical triplicates for each biological replicate. Error bars represent SEM. Shown are representative results for three of the ten phages tested; data for the remaining seven phages are found in Supplementary Fig S2.


A Culture dynamics over an extended period ( > 30 h ) for SPO1 ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Each curve represents a single technical replicate grown in a single well on a 96 ‐ well plate . Culture decline is temporally reproducible for the BREX ‐ lacking strain but occurs later , at apparently stochastic time points , for the BREX ‐ containing strain . Re ‐ growth following culture crash represents phage ‐ resistant mutants .

O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Culture dynamics over an extended period (> 30 h) for SPO1‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Each curve represents a single technical replicate grown in a single well on a 96‐well plate. Culture decline is temporally reproducible for the BREX‐lacking strain but occurs later, at apparently stochastic time points, for the BREX‐containing strain. Re‐growth following culture crash represents phage‐resistant mutants.


B Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with SPO1 . Error bars represent SD . Y ‐ axis represents absolute phage concentrations . Black and red arrows point to the time point of maximal burst for BREX ‐ lacking and BREX ‐ containing strains , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O 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 Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with SPO1. Error bars represent SD. Y‐axis represents absolute phage concentrations. Black and red arrows point to the time point of maximal burst for BREX‐lacking and BREX‐containing strains, respectively.


C Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with Φ3T . Error bars represent SD . Y ‐ axis represents relative phage concentrations normalized to the value at the beginning of the infection .

O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

C Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with Φ3T. Error bars represent SD. Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection.


D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX ( black ) , but not in BREX ‐ containing strain ( red ) , or uninfected ( U ) strains . Amplicons for the bacterial DNA , phage DNA , and lysogen ‐ specific DNA are 293 , 485 , and 1 , 218 bp , respectively .

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D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX‐containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen‐specific DNA are 293, 485, and 1,218 bp, respectively.


A BREX does not work via an abortive infection mechanism . Increasing the MOI of the Φ3T infection from 0 . 05 to 5 shortens the time to culture crash for the BREX ‐ lacking strain , but does not result in culture decline for BREX ‐ containing strain . Error bars represent SD of technical triplicates .

O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A BREX does not work via an abortive infection mechanism. Increasing the MOI of the Φ3T infection from 0.05 to 5 shortens the time to culture crash for the BREX‐lacking strain, but does not result in culture decline for BREX‐containing strain. Error bars represent SD of technical triplicates.


B The system does not interfere with phage adsorption to bacterial cells . Strains either containing ( red ) or lacking ( black ) the BREX system were infected with phage Φ3T and then chloroform ‐ treated 15 min following infection . The culture was plated on Φ3T ‐ sensitive B . subtilis cells and plaques , representing extracellular , unadsorbed phages , were counted .

O O O O O O O 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-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O

B The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform‐treated 15 min following infection. The culture was plated on Φ3T‐sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted.


C Phage DNA replication does not occur in BREX ‐ containing cells ( red ) , but is observed in the BREX ‐ lacking strain ( black ) . Y ‐ axis represents relative phage concentrations normalized to the value at the beginning of the infection , as measured by Illumina sequencing .

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C Phage DNA replication does not occur in BREX‐containing cells (red), but is observed in the BREX‐lacking strain (black). Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection, as measured by Illumina sequencing.


C , D Culture dynamics of non ‐ infected ( C ) or phage ‐ infected ( D ) cultures . Curves depict culture dynamics of strains lacking BREX ( black ) and BREX ‐ containing ( red ) strains of B . subtilis BEST7003 , as well as a BREX ‐ containing strain where the pglX methylase was deleted ( green ) . Axes and error bars are as in Fig .

O O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

C, D Culture dynamics of non‐infected (C) or phage‐infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX‐containing (red) strains of B. subtilis BEST7003, as well as a BREX‐containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig .


E Southern blot analysis of phage Φ3T genome during infection . Numbers indicate time ( in min ) following infection ; U , uninfected . Probe was designed to match positions 94 , 645 - 95 , 416 in the phage genome . Each lane contains 200 ng total DNA .

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E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.