( D and E ) In vitro palmitoylation assay for the indicated proteins . NBD - Palm - CoA : ( N - [ ( 7 - nitro - 2 - 1 , 3 - benzoxadiazol - 4 - yl ) - methyl ] amino ) palmitoyl - CoA . The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining .

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(D and E) In vitro palmitoylation assay for the indicated proteins. NBD-Palm-CoA: (N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA. The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining.


( F ) LC - MS / MS analysis of palmitoylated peptides of cGAS .

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(F) LC-MS/MS analysis of palmitoylated peptides of cGAS.


( G ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : HAM :

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(G) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: HAM: hydroxylamine


( H - I ) THP - 1 cells were treated with DMSO or 2 - BP ( 50 μM ) ( H ) or palmitic acid ( 100 μM ) ( I ) for 12 h . Six hours after transfection with HT - DNA ( 2 μg / mL ) , cGAMP was extracted and quantified by cGAMP ELISA . Data information : 2 - BP : 2 - bromopalmitate Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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(H-I) THP-1 cells were treated with DMSO or 2-BP (50 μM) (H) or palmitic acid (100 μM) (I) for 12 h. Six hours after transfection with HT-DNA (2 μg/mL), cGAMP was extracted and quantified by cGAMP ELISA. Data information: 2-BP: 2-bromopalmitate Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


( J ) THP - 1 cells were treated with palmitic acid ( 0 , 50 μM , or 100 μM ) for 12 h before a cGAMP bioassay . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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(J) THP-1 cells were treated with palmitic acid (0, 50 μM, or 100 μM) for 12 h before a cGAMP bioassay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h and transfected with HT - DNA ( 2 μg / mL ) for 6 h before RT - qPCR analysis of Ifna4 ( K ) , IFNb1 ( L ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of Ifna4 (K), IFNb1 (L) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h and transfected with HT - DNA ( 2 μg / mL ) for 6 h before RT - qPCR analysis of , Cxcl10 ( M ) and Rantes ( N ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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

BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of , Cxcl10 (M) and Rantes (N) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


( O ) L929 cells were treated with BSA and palmitic acid ( 100 μM ) and transfected with HT - DNA ( 2 μg / mL ) for the indicated times before immunoblotting analysis with the indicated antibodies .

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(O) L929 cells were treated with BSA and palmitic acid (100 μM) and transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies.


( A ) Acyl - RAC assay of HEK293T cells transfected with Flag - cGAS 24 h after transfection with the indicated shRNA . ( B ) Quantification of palmitoylation levels of Flag - cGAS in ( A ) . Data information :

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

(A) Acyl-RAC assay of HEK293T cells transfected with Flag-cGAS 24 h after transfection with the indicated shRNA. (B) Quantification of palmitoylation levels of Flag-cGAS in (A). Data information:


( C ) Acyl - RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h . HAM : hydroxylamine .

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(C) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine.


( D ) HEK293T cells were transfected with HA - cGAS ( WT or C474S mutant ) ( 200 ng ) , Flag - STING ( 200 ng ) and Myc - ZDHHC18 ( 200 ng ) expression plasmids for 24 h before luciferase reporter assays . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 (

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(D) HEK293T cells were transfected with HA-cGAS (WT or C474S mutant) (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (200 ng) expression plasmids for 24 h before luciferase reporter assays. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 (D


( E and F ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; FL : full

O O O O O O O O O O O O O O O O O O O O O O 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 and F) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length


( H ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA - cGAS and Flag - ZDHHC18 truncations or GFP for 24 h . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; FL : full

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(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-cGAS and Flag-ZDHHC18 truncations or GFP for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length


( J ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA - ZDHHC18 and Flag - cGAS truncates or GFP for 24 h . Data information : WCL : whole - cell

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(J) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-ZDHHC18 and Flag-cGAS truncates or GFP for 24 h. Data information: WCL: whole-cell lysate


( K ) Immunofluorescence analysis of HA - cGAS ( green ) and Flag - ZDHHC18 ( red ) in HT - DNA - stimulated ( or not ) HeLa cells . Scale bars : 7 μm . ( L ) Colocalization ( merged volume of total cGAS signal ) of cGAS and ZDHHC18 in ( K ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 L ) .

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(K) Immunofluorescence analysis of HA-cGAS (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa cells. Scale bars: 7 μm. (L) Colocalization (merged volume of total cGAS signal) of cGAS and ZDHHC18 in (K). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 L).


( M - N ) Comparison of the binding free energy ( M ) and binding energy decomposition of a per - residue to the binding affinity between ZDHHC18 and cGAS ( N ) in different complexes .

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(M-N) Comparison of the binding free energy (M) and binding energy decomposition of a per-residue to the binding affinity between ZDHHC18 and cGAS (N) in different complexes.


( A ) Immunofluorescence analysis of GFP - cGAS , the nucleus ( DAPI ) , the ER ( calnexin ) , the Golgi apparatus ( GM130 ) , and early endosomes ( EEA1 ) in HeLa cells . 2 - BP : 2 - bromopalmitate ; ISD : IFN - stimulatory DNA . Arrows indicate cGAS puncta . Scale bars : 10 μm .

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(A) Immunofluorescence analysis of GFP-cGAS, the nucleus (DAPI), the ER (calnexin), the Golgi apparatus (GM130), and early endosomes (EEA1) in HeLa cells. 2-BP: 2-bromopalmitate; ISD: IFN-stimulatory DNA. Arrows indicate cGAS puncta. Scale bars: 10 μm.


( F HEK293T cells were transfected with the indicated plasmids . Biotin - conjugated ISD was transfected into cells 6 h before harvesting . Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation . Data are representative of at least two independent experiments .

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(F HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; IP: immunoprecipitation. Data are representative of at least two independent experiments.


G ) HEK293T cells were transfected with the indicated plasmids . Biotin - conjugated ISD was transfected into cells 6 h before harvesting . Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown . Data information : WCL : whole - cell lysate ; Data are representative of at least two independent experiments .

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G) HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; Data are representative of at least two independent experiments.


( H ) Confocal microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 − / − ) transfected with FITC - conjugated ISD for 6 h ( left ) . Scale bar : 20 μm .

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(H) Confocal microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18−/−) transfected with FITC-conjugated ISD for 6 h (left). Scale bar: 20 μm.


( C ) Cross - correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins .

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(C) Cross-correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins.


( D ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with plasmids encoding Flag - cGAS , HA - cGAS , and Myc - ZDHHC18 ( WT or CS mutant ) for 24 h . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; Data are representative of at least two independent experiments .

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

(D) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding Flag-cGAS, HA-cGAS, and Myc-ZDHHC18 (WT or CS mutant) for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; Data are representative of at least two independent experiments.


( E ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids followed by the addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation ; 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments .

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(E) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids followed by the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Data information: WCL: whole-cell lysate; IP: immunoprecipitation; 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments.


( F ) FRET assay of HeLa cells transfected with the indicated plasmids with the addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Scale bar : 8 μm . ( G ) FRET efficiency of cGAS - GFP and cGAS - mCherry in ( F ) . Data information : 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 (

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

(F) FRET assay of HeLa cells transfected with the indicated plasmids with the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Scale bar: 8 μm. (G) FRET efficiency of cGAS-GFP and cGAS-mCherry in (F). Data information: 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 (G


( H ) Immunoprecipitation ( with an anti - Flag antibody ) and immunoblot analysis of HEK293T cells transfected with Flag - cGAS and HA - cGAS ( WT , C405S , C409S and C474S ) expression plasmids . Data information : WCL : whole - cell lysate ; IP : immunoprecipitation Data are representative of at least two independent experiments .

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

(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with Flag-cGAS and HA-cGAS (WT, C405S, C409S and C474S) expression plasmids. Data information: WCL: whole-cell lysate; IP: immunoprecipitation Data are representative of at least two independent experiments.


( I ) FRET assay of HeLa cells transfected with the indicated plasmids . Scale bar : 8 μm . ( J ) FRET efficiency of cGAS - GFP and cGAS - mCherry in ( I ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 J ) .

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(I) FRET assay of HeLa cells transfected with the indicated plasmids. Scale bar: 8 μm. (J) FRET efficiency of cGAS-GFP and cGAS-mCherry in (I). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 J).


( A ) HEK293T cells ( 5 × 105 ) were transfected with HA - cGAS ( 200 ng ) , Flag - STING ( 200 ng ) and Myc - ZDHHC18 ( WT or CS mutant ) expression plasmids ( 0 , 50 , 100 , or 200 ng ) for 24 h before luciferase reporter assays were conducted . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 (

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(A) HEK293T cells (5×105) were transfected with HA-cGAS (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (WT or CS mutant) expression plasmids (0, 50, 100, or 200 ng) for 24 h before luciferase reporter assays were conducted. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (A


( B ) HEK293T cells ( 1 × 106 ) were transfected with HA - cGAS ( 500 ng ) , Flag - STING ( 500 ng ) and Myc - ZDHHC18 ( 500 ng ) expression plasmids for 24 h . TBK1 phosphorylation was detected by immunoblotting . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in the DHHC motif of

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

(B) HEK293T cells (1×106) were transfected with HA-cGAS (500 ng), Flag-STING (500 ng) and Myc-ZDHHC18 (500 ng) expression plasmids for 24 h. TBK1 phosphorylation was detected by immunoblotting. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18


( C and D ) HEK293T cells were transfected with the indicated plasmids , and the amount of cGAMP in the lysates was quantified by LC - MS / MS . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in the DHHC motif of ZDHHC18 ; LC - MS / MS : liquid chromatography - tandem mass spectrometry ;

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

(C and D) HEK293T cells were transfected with the indicated plasmids, and the amount of cGAMP in the lysates was quantified by LC-MS/MS. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18; LC-MS/MS: liquid chromatography-tandem mass spectrometry;


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT - qPCR ( E ) results . Data information : NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) results. Data information: NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting ( F ) results . Data information : NC : negative control .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control.


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before RT - qPCR analysis of Ifna4 ( G ) , expression . Data information : HT - DNA : herring testis DNA ; NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of Ifna4 (G), expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before RT - qPCR analysis of IFNb1 ( H ) , and Cxcl10 ( I ) expression . Data information : HT - DNA : herring testis DNA ; NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of IFNb1 (H), and Cxcl10 (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


( J ) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before immunoblotting analysis with the indicated antibodies . Data information : HT - DNA : herring testis DNA ; NC : negative control .

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(J) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.


( A ) Survival analysis of Zdhhc18 + / + and Zdhhc18 - / - mice ( n = 8 each , 6 - 8 weeks old ) intranasally injected with VACV ( 5 × 107 PFU per mouse ) and monitored daily for survival for 13 days using Kaplan - Meier curves and a log - rank ( Mantel - Cox ) test . * * * , P ˂ 0 . 001 .

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

(A) Survival analysis of Zdhhc18+/+ and Zdhhc18-/- mice (n = 8 each, 6-8 weeks old) intranasally injected with VACV (5 × 107 PFU per mouse) and monitored daily for survival for 13 days using Kaplan-Meier curves and a log-rank (Mantel-Cox) test. ***, P˂0.001.


( B ) Representative photomicrographs of H & E - stained liver and kidney tissue sections from mice ( Zdhhc18 + / + or Zdhhc18 - / - ) intravenously injected with HSV - 1 - FS ( 5 × 107 PFU per mouse ) or not after 2 days . Scale bars : 100 μm ( liver ) , 200 μm ( kidney ) . Data information : PFU : plaque - forming units ; H & E : hematoxylin - eosin . Data are representative of at least two independent experiments .

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

(B) Representative photomicrographs of H&E-stained liver and kidney tissue sections from mice (Zdhhc18+/+ or Zdhhc18-/-) intravenously injected with HSV-1-FS (5 × 107 PFU per mouse) or not after 2 days. Scale bars: 100 μm (liver), 200 μm (kidney). Data information: PFU: plaque-forming units; H&E: hematoxylin-eosin. Data are representative of at least two independent experiments.


( C BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments .

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

(C BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments.


D ) BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001 (

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

D) BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001 (D


( E Frequency of GFP + cells among infected cells ( E ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Frequency of GFP+ cells among infected cells (E). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


F ) Microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) infected with GFP - HSV - 1 ( MOI = 1 ) for 10 h . Scale bar : 200 μm . Data information : Data are representative of at least two independent experiments .

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

F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Data information: Data are representative of at least two independent experiments.


( G ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before immunoblot analysis .

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

(G) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before immunoblot analysis.


( H and I ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of Cxcl10 ( H ) and Ifnb1 ( I ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(H and I) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before RT-qPCR analysis of Cxcl10 (H) and Ifnb1 (I) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


( J ) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA ( cocktail ) for 48 h and then infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of IFNB1 expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(J) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA (cocktail) for 48 h and then infected with VACV (1:200) for the indicated times before RT-qPCR analysis of IFNB1 expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


( C ) Quantitative real time PCR analysis showing head enrichment of dEsyt ; the X - axis indicates the wild type tissues from which RNA was isolated and Y - axis indicates the dEsyt transcript level expression normalized to the loading control ( RP49 - Ribosomal protein 49 ) , n = 3 replicates ( biological ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative real time PCR analysis showing head enrichment of dEsyt; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


( D ) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD . Y - axis indicates the dEsyt transcript level expression normalized to the loading control ( RP49 - Ribosomal protein 49 ) , n = 3 replicates ( biological ) .

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

(D) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD. Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


( A ) Representative ERG trace from dark reared 0 - 1 day old flies of the indicated genotype stimulated by 2s flash of green light . The duration of the stimulating light is shown . Y - axis shows the amplitude of the ERG response . X - axis shows the duration of the recording .

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

(A) Representative ERG trace from dark reared 0-1 day old flies of the indicated genotype stimulated by 2s flash of green light. The duration of the stimulating light is shown. Y-axis shows the amplitude of the ERG response. X-axis shows the duration of the recording.


( B ) Graph showing ERG amplitude normalized to body weight . Y - axis shows the ratio of ERG amplitude of individual flies to their body weight . X - axis indicates genotypes . Each data point represents an individual fly tested . Error bar represents s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph showing ERG amplitude normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.


( C ) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH - GFP in the one day old flies of the indicated genotypes ( n = 10 ) .

O O O O O O O O O O O O O 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) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH-GFP in the one day old flies of the indicated genotypes (n=10).


( D ) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors . The fluorescence of the PH - GFP probe is depicted . The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light ( a - f ) used for image capture depicted . Representative deep pseudopupil images acquired at specified time points are depicted . Genotypes as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.


( E ) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time , X - axis represents the genotypes , Y - axis represent intensity normalised to the intensity of the first image . ( n = 10 biological replicates ) . Error bars represent s . e . m .

(E) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalised to the intensity of the first image. (n=10 biological replicates). Error bars represent s.e.m.


( F ) Quantification of the time course of retinal degeneration . 10 ommatidia from 5 separate flies of each genotype were scored and plotted . Y - axis is the number of intact rhabdomeres / ommatidium . The maximum value possible is 7 . X - axis is the age of the flies post eclosion .

(F) Quantification of the time course of retinal degeneration. 10 ommatidia from 5 separate flies of each genotype were scored and plotted. Y-axis is the number of intact rhabdomeres/ommatidium. The maximum value possible is 7. X-axis is the age of the flies post eclosion.


( G ) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes . Rearing conditions and the age of the flies are indicated on top . ( * CL - Constant Light )

O O O O O O O O O O O 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) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CL- Constant Light)


( H ) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt : : mCherry transgene . Genotypes of the flies are indicated on the left . Rearing conditions and the age of the flies are indicated on top .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt::mCherry transgene. Genotypes of the flies are indicated on the left. Rearing conditions and the age of the flies are indicated on top.


( A ) ( i ) Representative optical neutralization ( ON ) images showing rhabdomere structure of the indicated genotypes . Rearing conditions and the age of the flies are indicated on top . ( * CD - Constant Dark ) . ( ii ) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9 ; dEsytKO mutants reared in 12 hr L / D cycle ( / ) for 2 days . Scale bar : 1µm ( asterisk indicates degenerated rhabdomere ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (i) Representative optical neutralization (ON) images showing rhabdomere structure of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CD- Constant Dark). (ii) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9;dEsytKO mutants reared in 12 hr L/D cycle ( / ) for 2 days. Scale bar: 1µm (asterisk indicates degenerated rhabdomere).


( B ) Quantification of the time course taken for retinal degeneration . 10 ommatidia were scored from 5 flies of each genotype and plotted .

O O 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) Quantification of the time course taken for retinal degeneration. 10 ommatidia were scored from 5 flies of each genotype and plotted.


( C ) Representative ERG traces showing the duration of light pulse , X - axis indicates time in msec and Y - axis indicates the average ERG amplitude in mV .

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

(C) Representative ERG traces showing the duration of light pulse, X-axis indicates time in msec and Y-axis indicates the average ERG amplitude in mV.


( D ) Graph showing ERG amplitude of rdgB9 and rdgB9 ; dEsytKO normalized to body weight . Y - axis shows the ratio of ERG amplitude of individual flies to their body weight . X - axis indicates genotypes . Each data point represents an individual fly tested . Error bar represents s . e . m .

(D) Graph showing ERG amplitude of rdgB9 and rdgB9;dEsytKO normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.


( E ) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors . The fluorescence of the PH - GFP probe is depicted . The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light ( a - f ) used for image capture depicted . Representative deep pseudopupil images acquired at specified time points are depicted . Genotypes as indicated .

(E) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.


( F ) Quantification of the mean fluorescence intensity of the PIP2 probe PH - GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes ( n = 10 biological replicates ) .

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

(F) Quantification of the mean fluorescence intensity of the PIP2 probe PH-GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes (n=10 biological replicates).


( G ) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time , X - axis represents the genotypes , Y - axis represent intensity normalized to the intensity of the first image . ( n = 10 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O 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) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalized to the intensity of the first image. (n=10 biological replicates).


( A ) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt . Scale bar : 5µm . dEsytKO shows no staining when probed with dEsyt antibody .

O O O O O O O O O O O O O O O O O O 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) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt. Scale bar: 5µm. dEsytKO shows no staining when probed with dEsyt antibody.


( B ) Confocal images showing the localization of exogenously expressed dEsyt : : mCherry protein expressed using the eye specific Rh1 - Gal4 in one day old dark reared flies . Rh1 - Gal4 is shown as a control . A single ommatidium is shown . Scale bar : 5 µm . Phalloidin marks F - actin staining and highlights rhabdomeres R1 - R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7.


( C ) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB . Rearing conditions , age of the flies and genotype is indicated on top of the blot . Tubulin was used as the loading control .

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

(C) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB. Rearing conditions, age of the flies and genotype is indicated on top of the blot. Tubulin was used as the loading control.


( D , E ) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are ( D ) 1 day old - dark reared and ( E ) 6 days old - exposed to constant illumination . For ( D ) and ( E ) RDGB visualized using an antibody against the endogenous protein . Rhabdomeres are outlined using phalloidin which marks F - actin . Scale bar : 5 µm .

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

(D, E) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.


( A ) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark ( i ) day 1 ( D1 ) and ( iii ) day 14 ( D14 ) , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from the same ommatidium ( ii ) day 1 ( iv ) day 14 . Scale bar : 200 nm . Origin boxes for magnification shown . Arrow in ( Aiv ) indicates the SMC forming an MCS with the microvillar PM . M - Microvillar plasma membrane , S - Sub microvillar cisternae , MCS - Membrane Contact Site , P - Pigment granules . ( B ) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark ( from figure 6A ) , Y - axis indicates the number of MCS / photoreceptor . n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) for ( i ) . n = 9 photoreceptors from 3 separate flies ( R7 ) in ( ii ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark (i) day 1 (D1) and (iii) day 14 (D14), scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day 1 (iv) day 14. Scale bar: 200 nm. Origin boxes for magnification shown. Arrow in (Aiv) indicates the SMC forming an MCS with the microvillar PM. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark (from figure 6A), Y-axis indicates the number of MCS/photoreceptor. n=30 photoreceptors from 3 separate flies (R1-R6) for (i). n=9 photoreceptors from 3 separate flies (R7) in (ii).


( C ) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark ( i ) day 1 and ( iii ) day 14 , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from the same ommatidium ( ii ) day1 ( iv ) day 14 . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( D ) Quantification of the number of MCSs per photoreceptor ( from figure 6C ) , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 )

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(C) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark (i) day 1 and (iii) day 14, scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day1 (iv) day 14. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCSs per photoreceptor (from figure 6C), X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7)


( E ) TEM images of a single ommatidium from ( i ) wild type and ( iii ) dEsytKO photoreceptors of 0 - 1 day old flies reared in dark , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) ( ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( F ) Quantification of the number of MCS per photoreceptor of wild type day 1 v / s dEsytKO day 1 old flies reared in dark , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from (i) wild type and (iii) dEsytKO photoreceptors of 0-1 day old flies reared in dark, scale bar: 1µm (red asterisk indicates R7 photoreceptor) (ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (F) Quantification of the number of MCS per photoreceptor of wild type day 1 v/s dEsytKO day 1 old flies reared in dark, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7).


( A ) TEM images of a single ommatidium from photoreceptors of ( i ) dEsytKO - day 1 ( iii ) dEsytKO - day 14 flies reared in dark , scale bar : 1 µm except for ( Ai ) : 2 µm ( red asterisk indicates R7 photoreceptor ) . ( A - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Boxes represent the origin boxes for magnification . M - Microvillar plasma membrane , S - Sub microvillar cisternae , MCS - Membrane Contact Site , P - Pigment granules . ( B ) Quantification of the number of MCS per photoreceptor , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor . n = 9 photoreceptors from 3 separate flies for R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from photoreceptors of (i) dEsytKO- day 1 (iii) dEsytKO- day 14 flies reared in dark, scale bar: 1 µm except for (Ai): 2 µm (red asterisk indicates R7 photoreceptor). (A-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Boxes represent the origin boxes for magnification. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. n=9 photoreceptors from 3 separate flies for R7.


( C ) TEM images of a single ommatidium from photoreceptors of ( i ) norpAP24 ; dEsytKO - day 1 ( iii ) norpAP24 ; dEsytKO - day 14 flies reared in dark , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( C - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( D ) Quantification of the number of MCS per photoreceptor , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor . ( i ) n = 30 photoreceptors from 3 separate flies for R1 - R6 . ( ii ) n = 9 photoreceptors from 3 separate flies for R7 .

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(C) TEM images of a single ommatidium from photoreceptors of (i) norpAP24;dEsytKO- day 1 (iii) norpAP24;dEsytKO- day 14 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (C-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. (i) n=30 photoreceptors from 3 separate flies for R1-R6. (ii) n=9 photoreceptors from 3 separate flies for R7.


( E ) TEM images of a single ommatidium from photoreceptors of ( i ) rdgB9 - day1 and ( iii ) rdgB9 ; dEsytKO - day 1 flies reared in dark , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( E - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . ( F ) Quantification of the number of MCS per photoreceptor for R1 - R6 in 1 day old dark reared wild type ( from Figure 5B i ) , rdgB9 and rdgB9 ; dEsytKO flies . n = 30 photoreceptors from 3 separate flies .

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

(E) TEM images of a single ommatidium from photoreceptors of (i) rdgB9-day1 and (iii) rdgB9;dEsytKO - day 1 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (E-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. (F) Quantification of the number of MCS per photoreceptor for R1-R6 in 1 day old dark reared wild type (from Figure 5B i), rdgB9 and rdgB9;dEsytKO flies. n=30 photoreceptors from 3 separate flies.


Example of a 384 - well plate treated with compounds . The panel shows the staining with filipin ( free cholesterol in green pseudo - colour ) and anti - LBPA antibodies ( red ) . The leftmost column shows control wells treated with DMSO only , and the right penultimate column shows wells treated with U18666A .

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

Example of a 384-well plate treated with compounds. The panel shows the staining with filipin (free cholesterol in green pseudo-colour) and anti-LBPA antibodies (red). The leftmost column shows control wells treated with DMSO only, and the right penultimate column shows wells treated with U18666A.


Effects of U18666A , thioperamide and trimeprazine , compared with controls . Cells treated with the indicated compounds or with DMSO alone for 18h at 37 ° C , were processed for immunofluorescence microscopy . Low magnification views are shown after staining with anti - LBPA antibodies ( red ) , filipin ( green pseudo - color ) and DAPI ( blue ) . The scale bar is 20 µm .

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Effects of U18666A, thioperamide and trimeprazine, compared with controls. Cells treated with the indicated compounds or with DMSO alone for 18h at 37°C, were processed for immunofluorescence microscopy. Low magnification views are shown after staining with anti-LBPA antibodies (red), filipin (green pseudo-color) and DAPI (blue). The scale bar is 20 µm.


Screen data plot . The plot shows the integrated intensity of LBPA vs . filipin ( cholesterol ) staining in all HeLa cells of each well analyzed in the screen ( each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound ) . Only compounds that showed less than 20 % toxicity are plotted . The cells treated with U18666A , thioperamide maleate and trimeprazine tartrate are indicated , as well as the DMSO controls .

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

Screen data plot. The plot shows the integrated intensity of LBPA vs. filipin (cholesterol) staining in all HeLa cells of each well analyzed in the screen (each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound). Only compounds that showed less than 20% toxicity are plotted. The cells treated with U18666A, thioperamide maleate and trimeprazine tartrate are indicated, as well as the DMSO controls.


Integrated intensity of cholesterol and LBPA staining . The integrated intensity of cholesterol ( filipin in green ) and LBPA ( red ) staining in all cells of the indicated samples is quantified after normalization to the DMSO controls . ( n = 3 independent experiments , 64 images analysed per experiments , error bars = SD , two ways ANOVA ; * * = p < 0 . 01 ; * * * = p < 0 . 005 ) .

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Integrated intensity of cholesterol and LBPA staining. The integrated intensity of cholesterol (filipin in green) and LBPA (red) staining in all cells of the indicated samples is quantified after normalization to the DMSO controls. (n=3 independent experiments, 64 images analysed per experiments, error bars = SD, two ways ANOVA; **=p<0.01; ***=p<0.005).


Thioperamide treatment of tissue culture cells . BHK , CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C . Low magnification views are shown after staining with anti - LBPA antibodies ( green ) and DAPI ( blue ) . n = 3 independent experiments with > 200 images acquired and analysed automatically .

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Thioperamide treatment of tissue culture cells. BHK, CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C. Low magnification views are shown after staining with anti-LBPA antibodies (green) and DAPI (blue). n=3 independent experiments with > 200 images acquired and analysed automatically.


Cholesterol quantification by mass spectrometry . A431 cells were treated with DMSO alone , pitolisant 10µM ( Pito ) , thioperamide 10µM ( thio ) or U18666A 10µM ( U18 ) for 18h at 37 ° C . After extraction , free cholesterol , cholesteryl esters and total cholesterol were quantified by mass spectrometry , and normalized to the DMSO controls . ( n = 3 independent experiments , error bars = SD , one - way ANOVA , * * * * = p < 0 .

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Cholesterol quantification by mass spectrometry. A431 cells were treated with DMSO alone, pitolisant 10µM (Pito), thioperamide 10µM (thio) or U18666A 10µM (U18) for 18h at 37°C. After extraction, free cholesterol, cholesteryl esters and total cholesterol were quantified by mass spectrometry, and normalized to the DMSO controls. (n=3 independent experiments, error bars = SD, one-way ANOVA, ****=p<0.0001


Screen data plot of the cholesterol content of LBPA - endosomes . Automated unbiased quantification of the filipin integrated fluorescence signal ( cholesterol content ) in LBPA - containing endosomes , after treatment with each compound of the Prestwick library . As in Fig 1D , each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound - only compounds that showed less than 20 % toxicity are plotted . The fluorescence signal of LBPA - endosomes was used to segment the imaged and generate a mask , which was then applied on the micrographs to quantify the integrated intensity of filipin staining . The samples treated with thioperamide ( pink ) , trimeprazine ( light blue ) , DMSO ( red ) and U18666A ( green ) are indicated .

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

Screen data plot of the cholesterol content of LBPA-endosomes. Automated unbiased quantification of the filipin integrated fluorescence signal (cholesterol content) in LBPA-containing endosomes, after treatment with each compound of the Prestwick library. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The fluorescence signal of LBPA-endosomes was used to segment the imaged and generate a mask, which was then applied on the micrographs to quantify the integrated intensity of filipin staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated.


Immunogold labelling with anti - LBPA antibodies . HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy . Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A - gold . Multivesicular endosomes are pseudocoloured ( see Fig EV1 for uncoloured images ) . Bars , 200nm . The data in ( A ) were quantified in a double - blind analysis of two sets of 16 micrographs for each condition ; the number of gold particles per endosome is shown in a scatter plot ( B ) for each endosome identified without any bias in each micrograph of the two control ( DMSO ) and thioperamide - treated ( THIO ) samples .

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Immunogold labelling with anti-LBPA antibodies. HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy. Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A-gold. Multivesicular endosomes are pseudocoloured (see Fig EV1 for uncoloured images). Bars, 200nm. The data in (A) were quantified in a double-blind analysis of two sets of 16 micrographs for each condition; the number of gold particles per endosome is shown in a scatter plot (B) for each endosome identified without any bias in each micrograph of the two control (DMSO) and thioperamide-treated (THIO) samples.


Distribution of LBPA - containing endosomes in the perinuclear region . Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells , after treatment with each compound of the Prestwick library as in Fig 2C . As in Fig 1D , each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound - only compounds that showed less than 20 % toxicity are plotted . The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell , calculated from the nuclei DAPI - staining . The samples treated with thioperamide ( pink ) , trimeprazine ( light blue ) , DMSO ( red ) and U18666A ( green ) are indicated . Number of perinuclear LBPA endosomes . The number of individual LBPA - positive structures was measured in the perinuclear region as in ( C ) . The z - factors are shown to evaluate the distribution of endosomes containing LBPA ; color code as in ( C ) .

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

Distribution of LBPA-containing endosomes in the perinuclear region. Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells, after treatment with each compound of the Prestwick library as in Fig 2C. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell, calculated from the nuclei DAPI-staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated. Number of perinuclear LBPA endosomes. The number of individual LBPA-positive structures was measured in the perinuclear region as in (C). The z-factors are shown to evaluate the distribution of endosomes containing LBPA; color code as in (C).


Effect of Pitolisant on LBPA . HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37 ° C were labeled with DAPI and anti - LBPA antibodies and analysed by fluorescence microscopy ( quantification in fig EV3A ) .

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Effect of Pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37°C were labeled with DAPI and anti-LBPA antibodies and analysed by fluorescence microscopy (quantification in fig EV3A).


LBPA intensity in a mixed population of cells expressing HRH3 - GFP . After transfection with HRH3 - GFP , uncloned stably expressing cells ( C ) were labelled with anti - LBPA antibodies and analysed by automated microscopy ( C ) . Unbiased quantification ( D ) shows the inverse correlation between HRH3 - GFP expression ( high expressing cells in green ) and the endosome integrated intensity of LBPA staining ( high LBPA labelling in red ) . ( n = 2 independent experiments with 500 , 000 cells analysed automatically , error bars = SD ) .

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

LBPA intensity in a mixed population of cells expressing HRH3-GFP. After transfection with HRH3-GFP, uncloned stably expressing cells (C) were labelled with anti-LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3-GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red). (n=2 independent experiments with 500,000 cells analysed automatically, error bars = SD).


LBPA intensity in cells expressing or not HRH3 - GFP . HeLa MZ cells stably expressing HRH3 - GFP grown in 96 - well plates were separately treated , with 4 different siRNAs that target HRH3 ( siHRH3 # 1 ; siHRH3 # 2 ; siHRH3 # 3 ; siHRH3 # 4 ) or with control non - target ( siNT ) siRNA ( E ) . HRH3 - GFP was analysed by fluorescence microscopy in 4 rows of cells per condition ; micrographs are stitched together in the montage . Panel F shows an example of cells treated with siHRH3 # 2 or siNT , and then labelled with DAPI and antibodies against LBPA . Cells treated as in ( E ) and labelled with DAPI and antibodies against LBPA as in ( F ) , were analysed by automated microscopy and the integrated intensities of LBPA ( left panel ) and HRH3 - GFP ( right panel ) signals are compared ( G ) . ( n = 2 independent experiments with 192 images acquired and analysed automatically , error bars = SD ) .

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

LBPA intensity in cells expressing or not HRH3-GFP. HeLa MZ cells stably expressing HRH3-GFP grown in 96-well plates were separately treated, with 4 different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; siHRH3#4) or with control non-target (siNT) siRNA (E). HRH3-GFP was analysed by fluorescence microscopy in 4 rows of cells per condition; micrographs are stitched together in the montage. Panel F shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F), were analysed by automated microscopy and the integrated intensities of LBPA (left panel) and HRH3-GFP (right panel) signals are compared (G). (n=2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).


Treatment of NPC fibroblasts and NPC null mice with thioperamide . Fibroblast lines obtained from patients with well - established heterozygote mutations in the NPC1 ( GM17912 line : NPC1 P1007A / T1036M ; GM17911 line : NPC1 I1061T / T1036M ) or with a homozygote mutation in the NPC2 gene ( GM17910 line : C93F / C93F ) were treated or not for 72h with thioperamide 10µM , stained with filipin ( cholesterol ) and analysed by fluorescence automated microscopy .

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

Treatment of NPC fibroblasts and NPC null mice with thioperamide. Fibroblast lines obtained from patients with well-established heterozygote mutations in the NPC1 (GM17912 line: NPC1 P1007A / T1036M; GM17911 line: NPC1 I1061T / T1036M) or with a homozygote mutation in the NPC2 gene (GM17910 line: C93F / C93F) were treated or not for 72h with thioperamide 10µM, stained with filipin (cholesterol) and analysed by fluorescence automated microscopy.


Quantification of cholesterol and LBPA staining in NPC fibroblasts . Cells as in ( A ) were treated or not with thioperamide for 48h or 72h , labelled with filipin ( cholesterol ) and anti - LBPA antibodies , and analysed by fluorescence automated microscopy . The panels show the integrated intensity of the filipin ( left ) and LBPA ( right ) signals were quantified after 48 ( top ) and 72h ( bottom ) . The colour code of each fibroblast cell line is as in ( A ) . ( n = 3 independent experiments with 144 images acquired and analysed automatically , > 2000 cells per experiment , error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 ; * * * = p < 0 . 001 ) .

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Quantification of cholesterol and LBPA staining in NPC fibroblasts. Cells as in (A) were treated or not with thioperamide for 48h or 72h, labelled with filipin (cholesterol) and anti-LBPA antibodies, and analysed by fluorescence automated microscopy. The panels show the integrated intensity of the filipin (left) and LBPA (right) signals were quantified after 48 (top) and 72h (bottom). The colour code of each fibroblast cell line is as in (A). (n=3 independent experiments with 144 images acquired and analysed automatically, >2000 cells per experiment, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001).


Quantification of cholesterol in NPC fibroblasts by mass spectrometry . NPC cell lines ( A ) were treated or not with thioperamide or 0 . 1 % ( 2 - Hydroxypropyl ) - ß - cyclodextrin for 72h . After extraction , lipids were analysed and quantified by mass spectrometry . ( n = 3 ; error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 ; * * * = p < 0 . 001 )

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Quantification of cholesterol in NPC fibroblasts by mass spectrometry. NPC cell lines (A) were treated or not with thioperamide or 0.1% (2-Hydroxypropyl)-ß-cyclodextrin for 72h. After extraction, lipids were analysed and quantified by mass spectrometry. (n=3; error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001)


Quantification of cell counts in the 3 NPC cell lines treated with thioperamide . The experiment was as in ( A - C ) except that nuclei were labelled with DAPI and quantified by automated microscopy . Data are normalized to DMSO control ( n = 2 independent experiments , 75 images per experiments )

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Quantification of cell counts in the 3 NPC cell lines treated with thioperamide. The experiment was as in (A-C) except that nuclei were labelled with DAPI and quantified by automated microscopy. Data are normalized to DMSO control (n=2 independent experiments, 75 images per experiments)


Effects of thioperamide on cholesterol - dependent transcriptional regulation . The parental HeLa MZ cells , NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h . Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT - PCR . ( n = 3 independent experiments , error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 )

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Effects of thioperamide on cholesterol-dependent transcriptional regulation. The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h. Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT-PCR. (n=3 independent experiments, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005)


Quantification of cholesterol in NPC null mice by enzymatic analysis . WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed . The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue . The total content of LBPA in the same liver extracts was quantified by mass spectrometry ( see Appendix Figure S1 ) , and is therefore expressed as a percentage of total phospholipids . The total LBPA content of WT mice was only marginally increased by the drug , either because the doses were low , or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level ( despite much effort , we were unable to visualize LBPA in liver samples by immunocytochemistry ) . The relative ratio of LBPA vs . cholesterol values is shown .

O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cholesterol in NPC null mice by enzymatic analysis. WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed. The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue. The total content of LBPA in the same liver extracts was quantified by mass spectrometry (see Appendix Figure S1), and is therefore expressed as a percentage of total phospholipids. The total LBPA content of WT mice was only marginally increased by the drug, either because the doses were low, or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level (despite much effort, we were unable to visualize LBPA in liver samples by immunocytochemistry). The relative ratio of LBPA vs. cholesterol values is shown.


Silver staining of protein components in the Ago3 complex immunoisolated from BmN4 cells . P150 and P130 co - immunoprecipitated with Ago3 . n . i . : non - immune IgG .

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Silver staining of protein components in the Ago3 complex immunoisolated from BmN4 cells. P150 and P130 co-immunoprecipitated with Ago3. n.i.: non-immune IgG.


Western blots showing the presence of Vret - L ( P150 ) and Vret - S ( P130 ) in the Ago3 complex in A . n . i . : non - immune IgG .

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Western blots showing the presence of Vret-L (P150) and Vret-S (P130) in the Ago3 complex in A. n.i.: non-immune IgG.


Top : western blot showing Vret - L and Vret - S immunoprecipitated from BmN4 cells upon UV irradiation . Bottom : CLIP signals showing that Vret - L , but not Vret - S , binds RNAs in vivo .

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Top: western blot showing Vret-L and Vret-S immunoprecipitated from BmN4 cells upon UV irradiation. Bottom: CLIP signals showing that Vret-L, but not Vret-S, binds RNAs in vivo.


Five upper panels : Northern blots showing the levels of RT3 - piRNA , Bmmar6 - piRNA , R2Bm - piRNA , and R1Bm - piRNA in normal ( Control ) , Vret - L / S - depleted ( Vret KD ) , Vret - L - depleted ( Vret - L KD ) , and Vret - S - depleted ( Vret - S KD ) cells . Bantam ( miRNA ) was detected as a loading control . Two lower panels : western blots showing the efficiency of the Vret knockdown . β - Tub was detected as a loading control .

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Five upper panels: Northern blots showing the levels of RT3-piRNA, Bmmar6-piRNA, R2Bm-piRNA, and R1Bm-piRNA in normal (Control), Vret-L/S-depleted (Vret KD), Vret-L-depleted (Vret-L KD), and Vret-S-depleted (Vret-S KD) cells. Bantam (miRNA) was detected as a loading control. Two lower panels: western blots showing the efficiency of the Vret knockdown. β-Tub was detected as a loading control.


Two upper panels : western blots showing the abundances of Flag - Vret - L and Ago3 in both samples . Note that Flag - Vret - L is present only in the Vret - L complex but not with Ago3 isolated from total cell lysates ( Fig EV1F ) . Bottom panel : Northern blot showing the level of PiggyBac - piRNA associated with Ago3 in total lysate ( Ago3 - IP ) and in the Vret - L complex ( Flag - Vret - IP ) . The ratio of the piRNA signal intensity in the two samples is indicated at the bottom .

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Two upper panels: western blots showing the abundances of Flag-Vret-L and Ago3 in both samples. Note that Flag-Vret-L is present only in the Vret-L complex but not with Ago3 isolated from total cell lysates (Fig EV1F). Bottom panel: Northern blot showing the level of PiggyBac-piRNA associated with Ago3 in total lysate (Ago3-IP) and in the Vret-L complex (Flag-Vret-IP). The ratio of the piRNA signal intensity in the two samples is indicated at the bottom.


Two upper panels : western blots showing the abundances of Flag - Vret - L and Siwi in both samples . Bottom panel : Northern blot showing the level of RT3 - piRNA associated with Siwi in the total lysate ( Siwi - IP ) and in the Vret - L complex ( Flag - Vret - IP ) .

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Two upper panels: western blots showing the abundances of Flag-Vret-L and Siwi in both samples. Bottom panel: Northern blot showing the level of RT3-piRNA associated with Siwi in the total lysate (Siwi-IP) and in the Vret-L complex (Flag-Vret-IP).


Boxplot showing log2 fold - change of Vret - KD ( RPM ) / Control ( RPM ) values of piRNAs in increased , unchanged and decreased groups defined by Ago3 - KD ( RPM ) / Control ( RPM ) values shown in Fig EV1I . Boxplot represents median , first and third quartile , and maximum and minimum values of the log2 fold - change of piRNAs ( N = 2 ) . Outliers are represented as open circles .

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Boxplot showing log2 fold-change of Vret-KD (RPM)/Control (RPM) values of piRNAs in increased, unchanged and decreased groups defined by Ago3-KD (RPM)/Control (RPM) values shown in Fig EV1I. Boxplot represents median, first and third quartile, and maximum and minimum values of the log2 fold-change of piRNAs (N = 2). Outliers are represented as open circles.


Flag - EGFP and - Vret - L WT and mutants were immunoprecipitated using the anti - Flag antibody and then western blotting was performed using anti - Flag , anti - Siwi , anti - Ago3 and anti - Vret antibodies . The domain structures of Tud1mut ( Y753A / Y757A / N759A ) and Tud2mut ( Y973A / Y976A / N978A ) are shown .

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Flag-EGFP and -Vret-L WT and mutants were immunoprecipitated using the anti-Flag antibody and then western blotting was performed using anti-Flag, anti-Siwi, anti-Ago3 and anti-Vret antibodies. The domain structures of Tud1mut (Y753A/Y757A/N759A) and Tud2mut (Y973A/ Y976A/N978A) are shown.


Immunofluorescence shows that the Vret signals ( green ) co - localize with the Ago3 signals ( red ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The three panels on the right - hand side show high magnification images of the part indicated by the white dashed line box in the image on the left - hand side . This panel arrangement applies also for panels B − H . The bar chart shows the percentage of Vret - positive nuage in Ago3 - positive nuage . Immunofluorescence shows that the Vret signals ( green ) partially co - localize with the Siwi signals ( red ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The bar chart shows the percentage of Vret - positive nuage in Siwi - positive nuage .

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Immunofluorescence shows that the Vret signals (green) co-localize with the Ago3 signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the image on the left-hand side. This panel arrangement applies also for panels B−H. The bar chart shows the percentage of Vret-positive nuage in Ago3-positive nuage. Immunofluorescence shows that the Vret signals (green) partially co-localize with the Siwi signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The bar chart shows the percentage of Vret-positive nuage in Siwi-positive nuage.


Immunofluorescence showing the Ago3 signals ( green ) in control and Vret - depleted BmN4 cells ( Vret KD ) . Vret is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing the Siwi signals ( green ) in control and Vret - depleted BmN4 cells ( Vret KD ) . Vret is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing the Ago3 signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing the Vret signals ( red ) in control and Ago3 - depleted BmN4 cells ( Ago3 KD ) . Ago3 is shown in green . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing the Siwi signals ( red ) in control and Ago3 - depleted BmN4 cells ( Ago3 KD ) . Ago3 is shown in green . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

Immunofluorescence showing the Vret signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing Flag - Ago3 WT , KA mutant and DDH mutant signals ( green ) expressed in naïve BmN4 cells ( Control ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing Flag - Ago3 WT , KA mutant and DDH mutant signals ( green ) in Ago3 - depleted cells ( Ago3 KD ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) expressed in naïve BmN4 cells (Control). DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) in Ago3-depleted cells (Ago3 KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing the Ago3 signals ( green ) in control and Siwi - depleted BmN4 cells ( Siwi KD ) . Siwi is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The three panels on the right - hand side show high magnification images of the part indicated by the white dashed line box in the images on the left - hand side . This panel arrangement applies also for panels B , H and I . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 106 ; Small : IntDen value < 106 ) . Immunofluorescence showing the Vret signals ( green ) in control and Siwi - depleted BmN4 cells ( Siwi KD ) . Siwi is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) .

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Immunofluorescence showing the Ago3 signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This panel arrangement applies also for panels B, H and I. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 106; Small: IntDen value < 106). Immunofluorescence showing the Vret signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105).


Western blotting was performed before and after Siwi KD using the anti - Ago3 antibody . Total BmN4 lysate ( Total ) , supernatant ( Sup ) and pellet ( Pellet ) fractions upon centrifugation were used .

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Western blotting was performed before and after Siwi KD using the anti-Ago3 antibody. Total BmN4 lysate (Total), supernatant (Sup) and pellet (Pellet) fractions upon centrifugation were used.


Western blotting was performed using the anti - Ago3 antibody on Ago3 immunoisolated from Siwi - depleted cells . Ago3 immunoprecipitated was untreated ( − ) or treated with phosphatase ( + ) prior to western botting .

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Western blotting was performed using the anti-Ago3 antibody on Ago3 immunoisolated from Siwi-depleted cells. Ago3 immunoprecipitated was untreated (−) or treated with phosphatase (+) prior to western botting.


Western blot showing the degree of phosphorylation of Flag - Ago3 WT and the 8SA mutant in Siwi - depleted cells ( Siwi KD ) .

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Western blot showing the degree of phosphorylation of Flag-Ago3 WT and the 8SA mutant in Siwi-depleted cells (Siwi KD).


Western blots showing the degree of phosphorylation of Flag - Ago3 WT and its DDH and KA mutants in Siwi - depleted cells ( Siwi KD ) . The second panel from the top corresponds to the top panel with a longer exposure time . Siwi and β - Tub as a loading control were also detected also by western blotting .

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Western blots showing the degree of phosphorylation of Flag-Ago3 WT and its DDH and KA mutants in Siwi-depleted cells (Siwi KD). The second panel from the top corresponds to the top panel with a longer exposure time. Siwi and β-Tub as a loading control were also detected also by western blotting.


Western blotting was performed using anti - Ago3 , anti - Vret and anti - Siwi antibodies on lysates of BmN4 cells before ( Control ) and after Siwi ( Siwi KD ) and Siwi / Vret ( Siwi / Vret KD ) depletion . β - Tub was detected as a loading control .

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Western blotting was performed using anti-Ago3, anti-Vret and anti-Siwi antibodies on lysates of BmN4 cells before (Control) and after Siwi (Siwi KD) and Siwi/Vret (Siwi/Vret KD) depletion. β-Tub was detected as a loading control.


Immunofluorescence showing the Flag - Ago3 signals ( red ) in normal ( Control ) and Siwi - depleted BmN4 cells ( Siwi KD ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing the Flag-Ago3 signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing the Flag - Ago3 8SA and 8SE mutant signals ( red ) in normal ( Control ) and Siwi - depleted BmN4 cells ( Siwi KD ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing the Flag-Ago3 8SA and 8SE mutant signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Western blot showing Ago3 immunopurified from BmN4 cells before ( Control ) and after Siwi knockdown ( Siwi KD ) . 32P - labeling of RNAs isolated from Ago3 ( a mixture of unphosphorylated and phosphorylated ) immunopurified from Siwi - depleted cells . 15 - 20 - nt RNAs ( indicated by a black line ) were isolated and sequenced .

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Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). 32P-labeling of RNAs isolated from Ago3 (a mixture of unphosphorylated and phosphorylated) immunopurified from Siwi-depleted cells. 15-20-nt RNAs (indicated by a black line) were isolated and sequenced.


The measurement of distances between the 3 ′ - ends of Target - S ( 15 - 20 - nt RNAs in A ) and the 5 ′ - ends of Ago3 - bound piRNAs .

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The measurement of distances between the 3′-ends of Target-S (15-20-nt RNAs in A) and the 5′-ends of Ago3-bound piRNAs.


32P - labeling of RNAs isolated from Ago3 . Target - Ss are isolated from Siwi - depleted BmN4 cells but are not isolated from Siwi / Vret double knockdown cells .

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32P-labeling of RNAs isolated from Ago3. Target-Ss are isolated from Siwi-depleted BmN4 cells but are not isolated from Siwi/Vret double knockdown cells.


Western blot showing Ago3 immunopurified from BmN4 cells before ( Control ) and after Siwi knockdown ( Siwi KD ) . Northern blot showing Bmmar6 - piRNA precursors in the Ago3 complex . 50 - 100 - nt RNAs ( indicated by a black line ) were isolated and sequenced .

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Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). Northern blot showing Bmmar6-piRNA precursors in the Ago3 complex. 50-100-nt RNAs (indicated by a black line) were isolated and sequenced.


The measurement of distances between the 5 ′ - ends of Target - L and the 5 ′ - ends of Ago3 - bound piRNAs .

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The measurement of distances between the 5′-ends of Target-L and the 5′-ends of Ago3-bound piRNAs.