Figure 7A - C TEM analysis of eVLP morphology . A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG - eVP40 and eGP following 48 hours of DMSO ( left panel ) or 5 µM fendiline treatment ( right panel ) . B Quantification of eVLP length ( µm ) of DMSO - derived eVLPs ( black ) and fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - tailed t - test was performed ( * * p = 0 . 0139 ) . C Quantification of eVLP diameter ( nm ) of DMSO - derived eVLPs ( black ) and fendiline - derived eVLPs ( blue ) . N > 50 VLPs per biological replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A two - tailed t - test was performed ( * p = 0 . 0430 ) . Data information : TEM : transmission electron microscopy ; eVLPs : entry - competent VLPs ; eGP : Ebola virus glycoproteinD , E Fluorescence based DiI TIM - 1 dependent entry assay . D Representative confocal images from the DiI - entry assay comparing entry of eVLPs produced from DMSO ( top panel ) and fendiline - treated HEK293 cells ( bottom panel ) into target cells ( HEK293 cells transiently expressing increasing amounts of TIM - 1 ; 0 . 0 µg , 0 . 5 µg , 1 . 0 µg ) . A stack of 10 frames was acquired for each image . DiI ( initially red ) was recolored to yellow for easier observation in print ; blue ( Hoechst 3342 stain ) ; scale bar = 10 µm . E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI - positive cells . Three images from each z - stack were quantified . N = 9 cell per replicate , n = 3 biological replicates . Values are reported as mean ± s . d . A one - way ANOVA was performed with multiple comparisons against the 0 . 0 µg TIM - 1 condition for both DMSO - and fendiline derived eVLPs . ( * * * * p < 0 . 0001 ; * * p = 0 . 0093 ) . Data information : TIM - 1 : t - cell immunoglobulin receptor - 1 ; eVLPs : entry - competent VLPs ; DiI : 1 , 1 ' - Dioctadecyl - 3 , 3 , 3 ' , 3 ' - Tetramethylindocarbocyanine Perchlorate .

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

Figure 7A-C TEM analysis of eVLP morphology. A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG-eVP40 and eGP following 48 hours of DMSO (left panel) or 5 µM fendiline treatment (right panel). B Quantification of eVLP length (µm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (**p=0.0139). C Quantification of eVLP diameter (nm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (*p=0.0430). Data information: TEM: transmission electron microscopy; eVLPs: entry-competent VLPs; eGP: Ebola virus glycoproteinD, E Fluorescence based DiI TIM-1 dependent entry assay. D Representative confocal images from the DiI-entry assay comparing entry of eVLPs produced from DMSO (top panel) and fendiline-treated HEK293 cells (bottom panel) into target cells (HEK293 cells transiently expressing increasing amounts of TIM-1; 0.0 µg, 0.5 µg, 1.0 µg). A stack of 10 frames was acquired for each image. DiI (initially red) was recolored to yellow for easier observation in print; blue (Hoechst 3342 stain); scale bar = 10 µm. E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI-positive cells. Three images from each z-stack were quantified. N=9 cell per replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons against the 0.0 µg TIM-1 condition for both DMSO- and fendiline derived eVLPs.(****p<0.0001; **p=0.0093). Data information: TIM-1: t-cell immunoglobulin receptor-1; eVLPs: entry-competent VLPs; DiI: 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate.


figf1a , Protein extracts from drpr null ( w ; drpr ∆ 5 / drpr ∆ 5 ) pupae at puparium formation ( 0 h ) and wild - type ( Canton - S ) salivary glands6 h , 12 h and 14 h after puparium formation , analysed by western blotting with anti - Drpr antibody . b , Samples from control animals ( + / w ; + / drpr ∆ 5 ) , n = 12 , and drpr null mutants ( w ; drpr ∆ 5 / drpr ∆ 5 ) , n = 47 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . c , Quantification of data from b and d . d , Samples from control animals ( + / w ; + / UAS - drprIR ) , n = 11 , and those with salivary gland - specific knockdown of drpr ( fkh - Gal4 / w ; UAS - drprIR / + ) , n = 19 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . e , Control animals ( + / w ; + / UAS - drpr - IIR ) , n = 9 , and those with salivary gland - specific knockdown of drpr - I ( fkh - Gal4 / w ; UAS - drpr - IIR / + ) , n = 20 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . f , Quantification of data from e . g , Samples from drpr null animals ( + / w ; + / UAS - Drpr - I ; drpr ∆ 5 / drpr ∆ 5 ) , n = 9 , and those with salivary gland - specific expression of Drpr - I ( fkh - Gal4 / w ; UAS - Drpr - I / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 20 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . h , Quantification of data from g .

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

figf1a, Protein extracts from drpr null (w; drpr∆5/drpr∆5) pupae at puparium formation (0 h) and wild-type (Canton-S) salivary glands6 h, 12 h and 14 h after puparium formation, analysed by western blotting with anti-Drpr antibody.b, Samples from control animals (+/w; +/drpr∆5), n = 12, and drpr null mutants (w; drpr∆5/drpr∆5), n = 47, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. c, Quantification of data from b and d. d, Samples from control animals (+/w; +/UAS-drprIR), n = 11, and those with salivary gland-specific knockdown of drpr (fkh-Gal4/w; UAS-drprIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation.e, Control animals (+/w; +/UAS-drpr-IIR), n = 9, and those with salivary gland-specific knockdown of drpr-I (fkh-Gal4/w; UAS-drpr-IIR/+), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. f, Quantification of data from e.g, Samples from drpr null animals (+/w; +/UAS-Drpr-I; drpr∆5/drpr∆5), n = 9, and those with salivary gland-specific expression of Drpr-I (fkh-Gal4/w; UAS-Drpr-I/+; drpr∆5/drpr∆5), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.


figf2a , Samples from animals with salivary gland - specific expression of p35 ( fkh - Gal4 / + ; UAS - p35 / + ) , n = 18 ( left ) , drpr null animals ( + / w ; + / UAS - p35 ; drpr ∆ 5 / drpr ∆ 5 ) , n = 10 ( middle ) , and drpr null animals with salivary gland - specific expression of p35 ( fkh - Gal4 / w ; UAS - p35 / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 16 ( right ) , analysed by histology for the presence of salivary gland material 24 h after puparium formation . Red circles , cell fragments ; yellow circle , gland fragments . a , Samples from animals with salivary gland - specific expression of p35 ( fkh - Gal4 / + ; UAS - p35 / + ) , n = 18 ( left ) , drpr null animals ( + / w ; + / UAS - p35 ; drpr ∆ 5 / drpr ∆ 5 ) , n = 10 ( middle ) , and drpr null animals with salivary gland - specific expression of p35 ( fkh - Gal4 / w ; UAS - p35 / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 16 ( right ) , analysed by histology for the presence of salivary gland material 24 h after puparium formation . Red circles , cell fragments ; yellow circle , gland fragments . b , Quantification of data from a . c , Salivary glands dissected from animals expressing drprIR specifically in GFP - marked clone cells ( heat shock ( hs ) flp / w ; UAS - drprIR / + ; act , cd2 , FRT > Gal4 , UAS - GFP / + ) 6 h and 14 h after puparium formation . Stained with GFP antibody ( green ) , to label cells expressing drpr IR , and Lamin antibody ( red ) .

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

figf2a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments.a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments. b, Quantification of data from a.c, Salivary glands dissected from animals expressing drprIR specifically in GFP-marked clone cells (heat shock (hs)flp/w; UAS-drprIR/+; act,cd2, FRT > Gal4, UAS-GFP/+) 6 h and 14 h after puparium formation. Stained with GFP antibody (green), to label cells expressing drpr IR, and Lamin antibody (red).


figf3a , Samples from animals with salivary gland - specific knockdown of Atg12 ( fkh - Gal4 / w ; UAS - Atg12IR / + ) , n = 21 , and those with salivary gland - specific knockdown of both Atg12 and drpr - I ( fkh - Gal4 / w ; UAS - Atg12IR / + ; UAS - drpr - IIR / + ) , n = 19 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . b , Quantification of data from a . c , GFP - LC3 was expressed in salivary glands of control animals ( + / w ; UAS - GFP - LC3 / + ; fkh - Gal4 / + ) and those with salivary gland - specific knockdown of drpr ( w ; UAS - drprIR / UAS - GFP - LC3 ; fkh - Gal4 / + ) . Salivary glands were dissected 6 h and 14 h after puparium formation , imaged for GFP - LC3 , and LC3 puncta were quantified using Zeiss Automeasure software . d , Quantification of data from c . Error bars represent s . e . m . ; n ≥ 10 ; p 0 . 0000001 . e , LAMP1 - GFP was expressed in control animals ( tub - LAMP - GFP / w ; + / fkh - Gal4 ) and those with salivary gland - specific knockdown of drpr ( tub - LAMP - GFP / w ; UAS - drprIR / + ; fkh - Gal4 / + ) . Salivary glands were dissected 14 h after puparium formation , imaged for LAMP - GFP , and LAMP puncta were quantified using Zeiss Automeasure software . f , Quantification of data from e . Error bars represent s . e . m . ; n ≥ 10 ; p 0 . 05 . g , Samples from drpr mutant animals ( + / w ; + / UAS - Atg16A ; drprDgr ; 5 / drprDgr ; 5 ) , n = 10 , and those with salivary gland - specific expression of Atg16A ( fkh - Gal4 / w ; UAS - Atg16A / + ; drprDgr ; 5 / drprDgr ; 5 ) , n = 16 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . h , Quantification of data from g .

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

figf3a, Samples from animals with salivary gland-specific knockdown of Atg12 (fkh-Gal4/w; UAS-Atg12IR/+), n = 21, and those with salivary gland-specific knockdown of both Atg12 and drpr-I (fkh-Gal4/w; UAS-Atg12IR/+; UAS-drpr-IIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. b, Quantification of data from a.c, GFP-LC3 was expressed in salivary glands of control animals (+/w; UAS-GFP-LC3/+; fkh-Gal4/+) and those with salivary gland-specific knockdown of drpr (w; UAS-drprIR/UAS-GFP-LC3; fkh-Gal4/+). Salivary glands were dissected 6 h and 14 h after puparium formation, imaged for GFP-LC3, and LC3 puncta were quantified using Zeiss Automeasure software. d, Quantification of data from c. Error bars represent s.e.m.; n ≥ 10; p  0.0000001.e, LAMP1-GFP was expressed in control animals (tub-LAMP-GFP/w; +/fkh-Gal4) and those with salivary gland-specific knockdown of drpr (tub-LAMP-GFP/w; UAS-drprIR/+; fkh-Gal4/+). Salivary glands were dissected 14 h after puparium formation, imaged for LAMP-GFP, and LAMP puncta were quantified using Zeiss Automeasure software. f, Quantification of data from e. Error bars represent s.e.m.; n ≥ 10; p  0.05.g, Samples from drpr mutant animals (+/w; +/UAS-Atg16A; drprDgr;5/drprDgr;5), n = 10, and those with salivary gland-specific expression of Atg16A (fkh-Gal4/w; UAS-Atg16A/+; drprDgr;5/drprDgr;5), n = 16, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.


figf4a , Protein extracts from drpr null ( w ; drprDgr ; 5 / drprDgr ; 5 ) pupae at puparium formation ( 0 h ) and from the fat bodies of wild - type ( Canton - S ) third - instar larvae were analysed by western blotting with anti - Drpr antibody . Third - instar larvae were either fed or starved for 4 h . b , We either fed Wild - type ( Canton - S ) third - instar larvae or starved them for 4 h , and dissected fat bodies , stained them with anti - Drpr antibody , and imaged them for Drpr ( green ) . We stained nuclei with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( blue ) . c , We starved third - instar larvae expressing GFP - Atg8a in all cells , and drprIR specifically in dsRed - marked clone cells ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , for 4 h . We dissected larval fat bodies and imaged them for GFP - Atg8a ( green ) and dsRed ( red ) . d , Salivary glands of animals expressing GFP - Atg8a in all cells , and drprIR specifically in dsRed - marked clone cells ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , dissected 14 h after puparium formation . Imaged for GFP - Atg8a ( green ) and dsRed ( red ) . We stained nuclei with Hoechst ( blue ) .

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

figf4a, Protein extracts from drpr null (w; drprDgr;5/drprDgr;5) pupae at puparium formation (0 h) and from the fat bodies of wild-type (Canton-S) third-instar larvae were analysed by western blotting with anti-Drpr antibody. Third-instar larvae were either fed or starved for 4 h.b, We either fed Wild-type (Canton-S) third-instar larvae or starved them for 4 h, and dissected fat bodies, stained them with anti-Drpr antibody, and imaged them for Drpr (green). We stained nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue).c, We starved third-instar larvae expressing GFP-Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), for 4 h. We dissected larval fat bodies and imaged them for GFP-Atg8a (green) and dsRed (red).d, Salivary glands of animals expressing GFP-Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), dissected 14 h after puparium formation. Imaged for GFP-Atg8a (green) and dsRed (red). We stained nuclei with Hoechst (blue).


Figure 1A ) Principal component analysis ( PCA ) of RNA - seq data derived from 54 single embryos in 8 developmental stages . X axis shows the first ( PC1 ) and y axis the second ( PC2 ) principal component . The variance explained by each component is indicated in parenthesis . Morphological stages are differentially coloredB ) Ratio of X - chromosomal over autosomal median transcript abundances ( X / A ) of preblastoderm ( PB ) , female and male embryos in 7 developmental stages . Each data point is a single embryo . A ratio of 1 represents balanced transcript levels between the X chromosome and autosomes ( dotted horizontal line ) . C ) Log2 Fold Change ( log2FC ) of RNA - seq between female ( F ) and male ( M ) embryos of expressed genes on X chromosome ( X , left , total = 1515 ) and on autosomes ( A , right , total = 7451 ) in 7 developmental stages . D ) Log2 Fold Change ( log2FC ) of RNA - seq between female ( F ) and male ( M ) embryos of X - chromosomal genes which are constitutively ( left , total = 303 ) or developmentally expressed ( right , total = 303 ) in 7 developmental stages . P - values ( pval ) are calculated by one - sample Wilcoxon signed rank test to assess the median log2FC values against zero ( balanced expression ) ; ns : non - significant ( p - value > 0 . 001 ) .

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

Figure 1A) Principal component analysis (PCA) of RNA-seq data derived from 54 single embryos in 8 developmental stages. X axis shows the first (PC1) and y axis the second (PC2) principal component. The variance explained by each component is indicated in parenthesis. Morphological stages are differentially coloredB) Ratio of X-chromosomal over autosomal median transcript abundances (X/A) of preblastoderm (PB), female and male embryos in 7 developmental stages. Each data point is a single embryo. A ratio of 1 represents balanced transcript levels between the X chromosome and autosomes (dotted horizontal line).C) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos of expressed genes on X chromosome (X, left, total=1515) and on autosomes (A, right, total=7451) in 7 developmental stages.D) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos of X- chromosomal genes which are constitutively (left, total=303) or developmentally expressed (right, total=303) in 7 developmental stages. P-values (pval) are calculated by one-sample Wilcoxon signed rank test to assess the median log2FC values against zero (balanced expression); ns: non-significant (p-value > 0.001).


Figure 2A ) Example genomic region of MSL2 MNase - ChIP - seq profiles in early ( stage 5 - 8 , orange ) and late ( stage 13 - 15 , purple ) mixed - sex embryos . DNA binding is defined as sub - nucleosomal fragments ( 10 - 130 bp ) , while chromatin binding as mono - nucleosomal fragments ( 130 - 220 bp ) . Called peaks / regions are represented as boxes above the tracks and HAS below . Late - appearing regions are marked by a plus ( + ) sign . Graph represents pooled IP / Input signal of 3 biological replicates . B ) Average composite plots of sub - nucleosomal MSL2 MNase - ChIP - seq ( DNA ) centered at PionX ( left ) or HAS ( right ) in early and late mixed - sex embryos . Total number of PionX = 56 and HAS = 247 . Graph represents average IP / Input signal of 3 biological replicates . Shaded area indicates the 95 % confidence interval across sites . C ) Average composite plots of mono - nucleosomal MSL2 ChIP - seq ( Chromatin ) centered at PionX ( left ) or HAS ( right ) in early and late mixed - sex embryos .

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

Figure 2A) Example genomic region of MSL2 MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. DNA binding is defined as sub-nucleosomal fragments (10 - 130 bp), while chromatin binding as mono-nucleosomal fragments (130 - 220 bp). Called peaks/regions are represented as boxes above the tracks and HAS below. Late-appearing regions are marked by a plus (+) sign. Graph represents pooled IP/Input signal of 3 biological replicates.B) Average composite plots of sub-nucleosomal MSL2 MNase-ChIP-seq (DNA) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos. Total number of PionX = 56 and HAS = 247. Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.C) Average composite plots of mono-nucleosomal MSL2 ChIP-seq (Chromatin) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos.


Figure 3B ) Average composite plots of MSL2 [ Chromatin ] profiles ( stage 5 - 8 , orange and stage 13 - 15 , purple ) centered at MSL2 DNA peaks which do not overlap ( DNA ) or overlap MSL2 chromatin regions ( DNA + Chromatin ) , and MSL2 chromatin regions which do not overlap MSL2 DNA peaks ( Chromatin ) . Graphs represent average IP / Input signal of 3 biological replicates . Shaded area indicates the 95 % confidence interval across sites . C ) Average composite plots of MSL2 [ Chromatin ] profiles centered at MSL2 chromatin regions called in both early and late ( left ) or only in late stages ( right ) . Graphs represent average IP / Input signal of 3 biological replicates . Shaded area indicates the 95 % confidence interval across regions . Total number of early = 212 and late = 87 . D ) Average composite plots of MOF [ Chromatin ] profiles centered at MSL2 chromatin regions called in both early and late ( left ) or only in late stages ( right ) .

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

Figure 3B) Average composite plots of MSL2 [Chromatin] profiles (stage 5-8, orange and stage 13-15, purple) centered at MSL2 DNA peaks which do not overlap (DNA) or overlap MSL2 chromatin regions (DNA + Chromatin), and MSL2 chromatin regions which do not overlap MSL2 DNA peaks (Chromatin). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.C) Average composite plots of MSL2 [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across regions. Total number of early = 212 and late = 87.D) Average composite plots of MOF [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right).


Figure 4A ) Example genomic region of DNA binding by MSL2 ( DNA ) , and chromatin binding of MSL2 ( Chrom . ) , MOF and H4K16ac MNase - ChIP - seq profiles in early ( stage 5 - 8 , orange ) and late ( stage 13 - 15 , purple ) mixed - sex embryos . Called peaks / regions are represented as boxes above the tracks . Graph represents pooled IP / Input signal of 3 biological replicates . B ) Size distribution of X - chromosomal MOF and H4K16ac regions , which are present in both early and late embryos . P - value ( Wilcoxon rank sum test ) is indicated . Total number of regions are 459 , 443 , 854 and 400 , respectively . C ) Average composite plots of MOF MNase - ChIP - seq centered at Transcription Start Site ( TSS ) or Transcription Termination Site ( TTS ) in early or late embryos for X - chromosomal genes ( total = 1515 ) . Graph represents average IP / Input signal of 3 biological replicates . Shaded area indicates the 95 % confidence interval across sites . D ) Average composite plots of H4K16ac MNase - ChIP - seq centered at TSS or TTS in early or late embryos for X - chromosomal genes ( total = 1515 ) .

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

Figure 4A) Example genomic region of DNA binding by MSL2 (DNA), and chromatin binding of MSL2 (Chrom.), MOF and H4K16ac MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. Called peaks/regions are represented as boxes above the tracks. Graph represents pooled IP/Input signal of 3 biological replicates.B) Size distribution of X-chromosomal MOF and H4K16ac regions, which are present in both early and late embryos. P-value (Wilcoxon rank sum test) is indicated. Total number of regions are 459, 443, 854 and 400, respectively.C) Average composite plots of MOF MNase-ChIP-seq centered at Transcription Start Site (TSS) or Transcription Termination Site (TTS) in early or late embryos for X-chromosomal genes (total=1515). Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.D) Average composite plots of H4K16ac MNase-ChIP-seq centered at TSS or TTS in early or late embryos for X-chromosomal genes (total=1515).


Figure 5A ) Scatterplot comparing X - chromosomal genic H4K16ac MNase - ChIP - seq levels in early ( stage 5 - 8 ) and late ( stage 13 - 15 ) mixed - sex embryos . Genes are grouped by k - means clustering ( k = 3 ) . The numbers of genes are cluster 1 = 979 , cluster 2 = 364 and cluster 3 = 172 . B ) Log2 Fold Change ( log2FC ) of RNA - seq between female ( F ) and male ( M ) embryos at genes grouped as in A ( cluster 1 - 3 ) . C ) Log10 distance between the middle of a gene and its nearest functional MSL2 DNA peak ( cluster 1 - 3 ) . Red line indicates median distance to the nearest peak .

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

Figure 5A) Scatterplot comparing X-chromosomal genic H4K16ac MNase-ChIP-seq levels in early (stage 5-8) and late (stage 13-15) mixed-sex embryos. Genes are grouped by k-means clustering (k=3). The numbers of genes are cluster 1 = 979, cluster 2 = 364 and cluster 3 = 172. B) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos at genes grouped as in A (cluster 1-3). C) Log10 distance between the middle of a gene and its nearest functional MSL2 DNA peak (cluster 1-3). Red line indicates median distance to the nearest peak.


Figure 3 ( A - C ) Tissue lysates from ( A ) spleen , ( B ) liver and ( C ) kidney from 6 - to 9 - day old indicated mice were immunoblotted to determine cleavage of caspase - 1 . Each lane represents a lysate from a different mouse . Positive control lanes ( + ) are BMDMs stimulated with LPS / ATP ; negative control lanes ( - ) are respective organ homogenates from caspase - 1 - / - mice . ( D - G ) Tissue lysates from ( D , F ) spleen and ( E , G ) liver from 6 - to 9 - day old indicated mice were immunoblotted to determine pro - IL - 1β cleavage to IL - 1β ( D - E ) or GSDMD cleavage to its p30 and p20 fragments ( F - G ) . Each lane represents a lysate from a different mouse . Positive control lanes ( + ) are BMDMs stimulated with LPS / ATP ; negative control lanes ( - ) are respective organ homogenates from ( D - E ) caspase - 1 - / - or ( F - G ) Gsdmd - / - mice .

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

Figure 3(A-C) Tissue lysates from (A) spleen, (B) liver and (C) kidney from 6- to 9-day old indicated mice were immunoblotted to determine cleavage of caspase-1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from caspase-1-/- mice.(D-G) Tissue lysates from (D, F) spleen and (E, G) liver from 6- to 9-day old indicated mice were immunoblotted to determine pro-IL-1β cleavage to IL-1β (D-E) or GSDMD cleavage to its p30 and p20 fragments (F-G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (-) are respective organ homogenates from (D-E) caspase-1-/- or (F-G) Gsdmd-/- mice.


Figure 1 ( B ) Gating strategy . pDC were identified as lineage ( TCRb , CD19 , Ly6G , NK1 . 1 ) - CD11b - CD11clow / int CD317high cells . 5 . 2 vs 5 . 1 cells were finally discriminated within the pDC gate ( F ) Enrichment of the gene modules identified in ( E ) for independent gene sets previously established from the analysis of other microarray datasets ( blue font ) , belonging to specific signaling pathways ( red font ) , associated to specific functions or diseases ( violet font ) or regulated by the indicated upstream regulators ( black font ) , as assessed by using Ingenuity Pathway Analysis ( IPA ) . Data are represented as a heatmap , with gene modules identified in panel E as columns and gene sets as rows . The colored cells indicate the significance of the enrichment of each gene module for each gene set , as computed by IPA analysis and according to the color scale shown below the heatmap

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

Figure 1 (B) Gating strategy. pDC were identified as lineage (TCRb, CD19, Ly6G, NK1.1)- CD11b- CD11clow/int CD317high cells. 5.2 vs 5.1 cells were finally discriminated within the pDC gate  (F) Enrichment of the gene modules identified in (E) for independent gene sets previously established from the analysis of other microarray datasets (blue font), belonging to specific signaling pathways (red font), associated to specific functions or diseases (violet font) or regulated by the indicated upstream regulators (black font), as assessed by using Ingenuity Pathway Analysis (IPA). Data are represented as a heatmap, with gene modules identified in panel E as columns and gene sets as rows. The colored cells indicate the significance of the enrichment of each gene module for each gene set, as computed by IPA analysis and according to the color scale shown below the heatmap


Figure 2Representative genes were selected for modules identified on Venn diagram in Fig 1E and their expression pattern across all samples shown as a heatmap . Red font , pDC ISG ( module A ) ; green font , pDC MSG ( module D ) ; mixed red and green font , pDC ISG and MSG ( module E ) ; black font , non - ISG , non - MSG ( module B ) . The relative mRNA expression levels for each gene ( lanes ) across biological samples ( rows ) are represented by colors according to the scale shown above the heatmap . For the legend of the symbols depicting the classes of biological samples above each column please refer to the symbol key in Fig 1D .

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

Figure 2Representative genes were selected for modules identified on Venn diagram in Fig 1E and their expression pattern across all samples shown as a heatmap. Red font, pDC ISG (module A); green font, pDC MSG (module D); mixed red and green font, pDC ISG and MSG (module E); black font, non-ISG, non-MSG (module B). The relative mRNA expression levels for each gene (lanes) across biological samples (rows) are represented by colors according to the scale shown above the heatmap. For the legend of the symbols depicting the classes of biological samples above each column please refer to the symbol key in Fig 1D.


Figure 3 ( A ) Histogram representing CD86 expression in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - infected MBMC ; grey , 5 . 2 + and 5 . 1 + pDC from CTR MBMC ; red , 5 . 2 + Ifnar1 - / - pDC isolated from Ifnar1 - TST MBMC ; purple , 5 . 1 + WT pDC isolated from Ifnar1 - TST MBMC . Dotted line shows isotype control staining . One histogram representative of 6 animals from 3 independent experiments is shown . ( B ) Dot plots represent intracellular IFN - I staining in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - infected MBMC . Isotype control ( IC ) staining was performed on a mixture of cells isolated from both types of MCMV - infected MBMC . The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from MCMV - infected Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red ) The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from CpG - stimulated mice . Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red ) Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group . The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from MCMV - infected Black , CTR MBMC pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group . The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from CpG - stimulated mice . Black , CTR MBMC pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group . The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Ratio of IL - 12 - and TNF - producing cells in pDC isolated from indicated MCMV - infected MBMC . Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red ) Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group . The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Ratio of IL - 12 - and TNF - producing cells in pDC isolated from indicated MCMV - infected MBMC . Black , CTR MBMC ; pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group .

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

Figure 3(A) Histogram representing CD86 expression in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-infected MBMC; grey, 5.2+ and 5.1+ pDC from CTR MBMC; red, 5.2+ Ifnar1-/- pDC isolated from Ifnar1-TST MBMC; purple, 5.1+ WT pDC isolated from Ifnar1-TST MBMC. Dotted line shows isotype control staining. One histogram representative of 6 animals from 3 independent experiments is shown.(B) Dot plots represent intracellular IFN-I staining in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-infected MBMC. Isotype control (IC) staining was performed on a mixture of cells isolated from both types of MCMV-infected MBMC.The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from MCMV-infected Black, CTR MBMC; red, Ifnar1-TST MBMC (red)The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from CpG-stimulated mice. Black, CTR MBMC; red, Ifnar1-TST MBMC (red) Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from MCMV-infected Black, CTR MBMC pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from CpG-stimulated mice. Black, CTR MBMC pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Ratio of IL-12- and TNF-producing cells in pDC isolated from indicated MCMV-infected MBMC. Black, CTR MBMC; red, Ifnar1-TST MBMC (red) Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Ratio of IL-12- and TNF-producing cells in pDC isolated from indicated MCMV-infected MBMC. Black, CTR MBMC ; pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.


Figure 4 ( A ) Dot plots represent IFN - I vs GFP staining in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - GFP - infected MBMC . One staining representative of 6 animals from 2 independent experiments is shown ( B , C ) Percentages of GFP + cells within 5 . 2 + pDC ( B ) or of GFP + ( green ) vs GFP - ( white ) cells within 5 . 2 + IFN - I - producing pDC ( C ) isolated from indicated MCMV - GFP - infected MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group ( D ) qPCR for the expression of the indicated genes was performed on whole mRNA isolated from the spleen of indicated MCMV - infected MBMC . CTR MBMC ( black ) and Myd88 - / - TST MBMC ( dark green ) were treated with isotype control ( IC , square ) or blocking α - IFNAR1 mAbs ( triangle ) . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 2 - 3 mice per group ( E - G ) CD86 MFI in 5 . 2 + pDC of each indicated MBMC type ( E ) and 5 . 2 / 5 . 1 ratio of cytokine - producing pDC ( F , G ) . Data ( mean ± SEM ) are representative of 2 pooled independent experiments each with 2 - 3 mice per group CD86 MFI and percentages of cytokine - producing cells within pDC of each indicated mouse strain . Black , C57BL6 ; red , Ifnar1 - / - ; orange , Ifnar1 - / - x Myd88 - / - CD86 MFI and percentages of cytokine - producing cells within pDC of each indicated mouse strain . Black , C57BL6 green , Tlr9 - / - and pale green , Tlr7 - / - . In ( K - L ) indicated mice were treated with IC ( square ) or blocking α - IFNAR1 mAbs ( triangle ) . Data depicted ( mean ± SEM ) are from 2 pooled independent experiments each with 2 - 3 mice per group .

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

Figure 4 (A) Dot plots represent IFN-I vs GFP staining in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-GFP-infected MBMC. One staining representative of 6 animals from 2 independent experiments is shown  (B, C) Percentages of GFP+ cells within 5.2+ pDC (B) or of GFP+ (green) vs GFP- (white) cells within 5.2+ IFN-I-producing pDC (C) isolated from indicated MCMV-GFP-infected MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group  (D) qPCR for the expression of the indicated genes was performed on whole mRNA isolated from the spleen of indicated MCMV-infected MBMC. CTR MBMC (black) and Myd88-/- TST MBMC (dark green) were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group  (E-G) CD86 MFI in 5.2+ pDC of each indicated MBMC type (E) and 5.2/ 5.1 ratio of cytokine-producing pDC (F, G). Data (mean±SEM) are representative of 2 pooled independent experiments each with 2-3 mice per group CD86 MFI and percentages of cytokine-producing cells within pDC of each indicated mouse strain. Black, C57BL6; red, Ifnar1-/-; orange, Ifnar1-/-x Myd88-/-CD86 MFI and percentages of cytokine-producing cells within pDC of each indicated mouse strain. Black, C57BL6 green, Tlr9-/- and pale green, Tlr7-/-. In (K-L) indicated mice were treated with IC (square) or blocking α-IFNAR1 mAbs (triangle). Data depicted (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group.


Figure 5 ( A ) IRF7 protein expression in pDC isolated from Irf7 - / - ( pink , bottom ) , C57BL / 6 ( WT , black , top ) and Ifnar1 - / - ( red , middle ) uninfected ( empty histograms , Ø , left ) vs MCMV - infected mice ( filled histograms , MCMV , right ) . For each condition histograms are representative of one out of 4 - 6 mice from 2 independent experiments ( B ) IRF7 protein expression in pDC ( left ) , cDC ( middle ) and conventional monocytes ( cMo , right ) isolated from uninfected ( Ø , empty ) or MCMV - infected ( MCMV , filled ) C57BL / 6 ( black ) or Ifnar1 - / - ( red ) mice . Data are shown as net IRF7 MFI , after subtraction of the MFI obtained using isotype control mAbs . Data shown ( mean ± SEM ) are pooled from 2 independent experiments with 5 - 6 mice for each group ( C - E ) The expression of CD86 ( C ) or of indicated cytokines ( D - E ) was analyzed , as described in Fig 2 . Black , CTR MBMC ; pink , Irf7 - TST MBMC . Mice were treated with isotype control ( IC , square ) or blocking α - IFNAR1 mAbs ( triangle ) . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 2 - 3 mice per

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Figure 5 (A) IRF7 protein expression in pDC isolated from Irf7-/- (pink, bottom), C57BL/6 (WT, black, top) and Ifnar1-/- (red, middle) uninfected (empty histograms, Ø, left) vs MCMV-infected mice (filled histograms, MCMV, right). For each condition histograms are representative of one out of 4-6 mice from 2 independent experiments  (B) IRF7 protein expression in pDC (left), cDC (middle) and conventional monocytes (cMo, right) isolated from uninfected (Ø, empty) or MCMV-infected (MCMV, filled) C57BL/6 (black) or Ifnar1-/- (red) mice. Data are shown as net IRF7 MFI, after subtraction of the MFI obtained using isotype control mAbs. Data shown (mean±SEM) are pooled from 2 independent experiments with 5-6 mice for each group  (C-E) The expression of CD86 (C) or of indicated cytokines (D-E) was analyzed, as described in Fig 2. Black, CTR MBMC; pink, Irf7-TST MBMC. Mice were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group


Figure 6 ( A , B ) Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from MCMV - infected ( A MBMC of each indicated type ( A , B ) Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated fro CpG - stimulated ( B ) MBMC of each indicated type ( C ) Ratio of IL - 12 - or TNF - producing cells in 5 . 2 + vs 5 . 1 + pDC of indicated MCMV - infected MBMC mice

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Figure 6 (A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected (A MBMC of each indicated type (A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated fro CpG-stimulated (B) MBMC of each indicated type (C) Ratio of IL-12- or TNF-producing cells in 5.2+ vs 5.1+ pDC of indicated MCMV-infected MBMC mice


Figure 7Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from MCMV - infected MBMC of each indicated type . Black , CTR MBMC ; pale pink , Itgal - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group . Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from CpG - stimulated ( B ) MBMC of each indicated type . Black , CTR MBMC ; pale pink , Itgal - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group . ( C ) EYFP expression in splenocytes isolated from uninfected ( NI ) vs MCMV - infected ( MCMV ) IFNbtm1 ( EYFP ) reporter mice . The expression of indicated markers was analyzed on YFP + cells . Plots are representative of one out of 6 mice from 2 independent experiments . ( D - F ) Immunohistological analysis of splenic sections stained with mAb against MOMA1 / CD169 ( blue ) , GFP / EYFP ( green ) , whose expression correlates with that of IFN - β , and MCMV IE - 1 ( red ) . Scale bars , 100µm . The image shown is one representative of 15 obtained from 5 different mice . Optical magnification was 20x , and images were scanned on the microscope at zoom x2 for ( E , F ) . White dotted lines delimit marginal zone . Yellow squares in ( D ) defines zoomed region in ( E , F ) .

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

Figure 7Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from CpG-stimulated (B) MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.(C) EYFP expression in splenocytes isolated from uninfected (NI) vs MCMV-infected (MCMV) IFNbtm1(EYFP) reporter mice. The expression of indicated markers was analyzed on YFP+ cells. Plots are representative of one out of 6 mice from 2 independent experiments.(D-F) Immunohistological analysis of splenic sections stained with mAb against MOMA1/CD169 (blue), GFP/EYFP (green), whose expression correlates with that of IFN-β, and MCMV IE-1 (red). Scale bars, 100µm. The image shown is one representative of 15 obtained from 5 different mice. Optical magnification was 20x, and images were scanned on the microscope at zoom x2 for (E, F). White dotted lines delimit marginal zone. Yellow squares in (D) defines zoomed region in (E, F).


Figure 1A , B Bottom panels : gene annotation track ( mm10 ) ; the activated gene is marked in green and the location of the TALE - VP64 target sequence is shown by the vertical dashed green line . Middle panels : DamID tracks of NL interactions in control cells ( " control " , blue line ) and cells expressing TALE - VP64 ( " experimental " , red line ) . n indicates the number of independent biological replicates that were combined . Noise was suppressed by a running mean filter of indicated window size . Shading between the lines corresponds to the color of the sample with the highest value . Arrowheads on the right - hand side mark the 5th and 95th percentiles of genome - wide DamID values . Top panels : domainograms ; for every window of indicated size ( vertical axis ) and centered on a genomic position ( horizontal axis ) the pixel shade indicates the ranking of the change in DamID score ( experimental minus control ) in this window compared to the genome - wide changes in DamID scores across all possible windows of the same size . Blue : DamID score is highest in control samples ; red : DamID score is highest in experimental samples ( color key on the right of panel ( a ) ) . In ( a ) activation of Sox6 was the experimental perturbation , activation of Nrp1 ( which is located on a different chromosome ) served as control ; in ( b ) activation of Nrp1 was the experimental condition and activation of Sox6 served as control .

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Figure 1A, B Bottom panels: gene annotation track (mm10); the activated gene is marked in green and the location of the TALE-VP64 target sequence is shown by the vertical dashed green line. Middle panels: DamID tracks of NL interactions in control cells ("control", blue line) and cells expressing TALE-VP64 ("experimental", red line). n indicates the number of independent biological replicates that were combined. Noise was suppressed by a running mean filter of indicated window size. Shading between the lines corresponds to the color of the sample with the highest value. Arrowheads on the right-hand side mark the 5th and 95th percentiles of genome-wide DamID values. Top panels: domainograms; for every window of indicated size (vertical axis) and centered on a genomic position (horizontal axis) the pixel shade indicates the ranking of the change in DamID score (experimental minus control) in this window compared to the genome-wide changes in DamID scores across all possible windows of the same size. Blue: DamID score is highest in control samples; red: DamID score is highest in experimental samples (color key on the right of panel (a)). In (a) activation of Sox6 was the experimental perturbation, activation of Nrp1 (which is located on a different chromosome) served as control; in (b) activation of Nrp1 was the experimental condition and activation of Sox6 served as control.


Figure 2 ( a - b ) Plots as in Figure 1 , showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 ( a ) and SOX6 ( b ) . Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes . Vertical green dotted lines mark the position of the sgRNA target sequence . ( c ) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa . Genomic regions are aligned by the respective sgRNA target positions , and oriented so that the activated genes are all transcribed from left to right .

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Figure 2(a-b) Plots as in Figure 1, showing changes in Lamin B1 DamID signals upon CRISPRa activation of NLGN1 (a) and SOX6 (b). Control cells were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes. Vertical green dotted lines mark the position of the sgRNA target sequence.(c) Domainograms showing regions with reduced NL interactions around 12 genes individually activated by CRISPRa. Genomic regions are aligned by the respective sgRNA target positions, and oriented so that the activated genes are all transcribed from left to right.


Figure 3Average DamID values plotted against average expression levels of eight genes activated by CRISPRa ( red ; n = 2 ) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes ( blue ; n ranging from 19 to 27 ) .

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Figure 3Average DamID values plotted against average expression levels of eight genes activated by CRISPRa (red; n = 2) or in control cells that were treated either without sgRNA or with one of various sgRNAs targeting promoters on different chromosomes (blue; n ranging from 19 to 27).


Figure 4A - C CRISPRa activation of ADAM22 ( a ) , ABCB1 ( b ) and PTN ( c ) in human RPE - 1 cells . Top panels visualize DamID data similar to Figure 2a - b . Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels , except that different colors are used as indicated . Bottom panels show gene track , with activated gene highlighted in green .

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Figure 4A-C CRISPRa activation of ADAM22 (a), ABCB1 (b) and PTN (c) in human RPE-1 cells. Top panels visualize DamID data similar to Figure 2a-b. Middle panels show maps of replication timing at the same resolution and in the same plotting style as panels, except that different colors are used as indicated. Bottom panels show gene track, with activated gene highlighted in green.


Figure 5Changes in NL interactions after CRISPRa activation of MLK4 in human RPE - 1 cells . Visualization of DamID data as in Figure 2a - b .

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Figure 5Changes in NL interactions after CRISPRa activation of MLK4 in human RPE-1 cells. Visualization of DamID data as in Figure 2a-b.


Figure 6 ( a ) DamID domainograms of NL interactions after activation of CCSER1 , GRID2 , or both . All data are from human RPE - 1 cells . ( b ) DamID domainograms after activation of ADAM22 , ABCB1 , or the genes ABCB4 , ABCB1 , ADAM22 and RUNC3B simultaneously . All data are from human RPE - 1 cells . DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a , b .

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Figure 6(a) DamID domainograms of NL interactions after activation of CCSER1, GRID2, or both. All data are from human RPE-1 cells.(b) DamID domainograms after activation of ADAM22, ABCB1, or the genes ABCB4, ABCB1, ADAM22 and RUNC3B simultaneously. All data are from human RPE-1 cells. DamID data of activation of ADAM22 and ABCB1 alone are same as in Figure 4a, b.


Figure 7 ( a ) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc , Morc1 and DppA2 ( deletions marked by yellow vertical boxes ) , and in control cells . ( b ) Same as ( a ) , but for the non - mutated Cast alleles . ( c - d ) Same design as ( a - b ) , but with only a single mono - allelic deletion of the Morc promoter ( vertical yellow box ) . ( e - f ) Effect of PAS insertion on NL interactions of Cobl gene locus . Same design as ( a - b ) but with insertion of a PAS ( located at red triangle and vertical dotted line ) that truncates the 129Sv allele of the Cobl transcription unit . 129Sv allele is shown in ( e ) , non - mutated Cast allele in ( f ) . Clones with Morc1 locus mutations ( each assayed in four independent biological replicates ) served collectively as control in ( e - f )

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Figure 7(a) DamID profiles of the 129Sv allele of the Morc1 locus with deletions of the promoters of Morc, Morc1 and DppA2 (deletions marked by yellow vertical boxes), and in control cells. (b) Same as (a), but for the non-mutated Cast alleles. (c-d) Same design as (a-b), but with only a single mono-allelic deletion of the Morc promoter (vertical yellow box).(e-f) Effect of PAS insertion on NL interactions of Cobl gene locus. Same design as (a-b) but with insertion of a PAS (located at red triangle and vertical dotted line) that truncates the 129Sv allele of the Cobl transcription unit. 129Sv allele is shown in (e), non-mutated Cast allele in (f). Clones with Morc1 locus mutations (each assayed in four independent biological replicates) served collectively as control in (e-f)


Figure 8 ( c ) Average DamID profiles across 17 transgene integration sites inside LADs . Blue curve shows alleles without integrations , red curve shows the corresponding alleles with integrations . Shading between the lines shows which curve has the highest value . ( d ) Relative expression levels of individual barcoded transgenes in LADs and iLADs . ( e ) Estimated expression levels of integrated transgenes in LADs ( vertical orange lines ; median value indicated by arrowhead ) , compared to the distribution of expression levels of all active endogenous genes ( black curve ) .

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Figure 8(c) Average DamID profiles across 17 transgene integration sites inside LADs. Blue curve shows alleles without integrations, red curve shows the corresponding alleles with integrations. Shading between the lines shows which curve has the highest value.(d) Relative expression levels of individual barcoded transgenes in LADs and iLADs.(e) Estimated expression levels of integrated transgenes in LADs (vertical orange lines; median value indicated by arrowhead), compared to the distribution of expression levels of all active endogenous genes (black curve).


Figure 1 ( A ) ARPE - 19 cells were incubated in the presence of DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved ( EBSS ) for 16 h . Cells were fixed , permeabilized and stained with an antibody against TFE3 . Scale bar , 10 µm . Data are representative of three independent experiments . ( B ) Immunoblot of protein lysates of ARPE - 19 cells treated as shown in ( A ) . Data are representative of three independent experiments . ( C ) ARPE - 19 cells incubated with Tunicamycin as indicated in ( A ) were immunostained with antibody against CHOP . Scale bar , 10 µm . Data are representative of three independent experiments . ( D ) Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or starvation ( Strv . ) treatments as indicated in ( A ) ( mean ± SD of three independent experiments , one - way ANOVA analysis versus Control treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) . ( E ) Wild - type mouse embryonic fibroblast ( MEF ) cells were incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for the indicated times . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( F ) Wild - type MEF cells were incubated in the presence of DMSO ( Control ) or BFA ( 2 . 5 µg / ml ) for 16 h . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( G ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) or BFA 16 h treatment ( mean ± SD of two independent experiments , student ' s t - test analysis versus DMSO treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) . ( H ) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO ( Ctrl . ) or Tunicamycin ( Tun . ) for 16 h using TFE3 , TFEB , Lamp1 ( as a lysosomal membrane marker ) , and H3b ( as a nuclear marker ) . Data are representative of three independent experiments .

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

Figure 1(A) ARPE-19 cells were incubated in the presence of DMSO (Control), Tunicamycin (5 µg/ml), or starved (EBSS) for 16 h. Cells were fixed, permeabilized and stained with an antibody against TFE3. Scale bar, 10 µm. Data are representative of three independent experiments.(B) Immunoblot of protein lysates of ARPE-19 cells treated as shown in (A). Data are representative of three independent experiments.(C) ARPE-19 cells incubated with Tunicamycin as indicated in (A) were immunostained with antibody against CHOP. Scale bar, 10 µm. Data are representative of three independent experiments.(D) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or starvation (Strv.) treatments as indicated in (A) (mean ± SD of three independent experiments, one-way ANOVA analysis versus Control treated cells, ***p < 0.001; n>400 cells per condition).(E) Wild-type mouse embryonic fibroblast (MEF) cells were incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(F) Wild-type MEF cells were incubated in the presence of DMSO (Control) or BFA (2.5 µg/ml) for 16 h. Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(G) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or BFA 16 h treatment (mean ± SD of two independent experiments, student's t-test analysis versus DMSO treated cells, ***p < 0.001; n>400 cells per condition).(H) Immunoblots of the subcellular distribution of TFE3 and TFEB in MEF cells incubated with DMSO (Ctrl.) or Tunicamycin (Tun.) for 16 h using TFE3, TFEB, Lamp1 (as a lysosomal membrane marker), and H3b (as a nuclear marker). Data are representative of three independent experiments.


Figure 2 ( A ) Immunoblot of protein lysates from wild - type , PERK - KO and GCN2 - KO MEF cells incubated in the presence of DMSO ( Control ) , Tunicamycin ( 0 . 1 µg / ml ) , or starved in EBSS for 16 h . Data are representative of three independent experiments . ( B - D ) Wild - type ( B ) , GCN2 - KO ( C ) , and PERK - KO ( D ) MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) as indicated in ( A ) . Cells were fixed , permeabilized and stained with antibodies against TFE3 ( green ) and CHOP ( red ) . Scale bar , 10 µm . Data are representative of three independent experiments . ( E - F ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) , Tunicamycin treatments for 16 h ( E ) , or starvation for 2 h ( F ) ( mean ± SD of three independent experiments , one - way ANOVA analysis versus DMSO treated cells , ns = not significant , * * * p < 0 . 001 ; n > 400 cells per condition ) . ( G ) Quantification of phospho - 4EBP1 protein levels in wild - type and GCN2 - KO MEF cells upon starvation for 2 h ( the mean ± SD of the fold increase of the phospho - 4EBP1 to total 4EBP1 ratio from three independent experiments , one - way ANOVA analysis versus starvation treated wild - type cells , * * * p < 0 . 001 ) . ( H ) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO ( Control ) or Torin - 1 ( 250 nM ) treatments for 1 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus DMSO treated wild - type cells , * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

Figure 2(A) Immunoblot of protein lysates from wild-type, PERK-KO and GCN2-KO MEF cells incubated in the presence of DMSO (Control), Tunicamycin (0.1 µg/ml), or starved in EBSS for 16 h. Data are representative of three independent experiments.(B-D) Wild-type (B), GCN2-KO (C), and PERK-KO (D) MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) as indicated in (A). Cells were fixed, permeabilized and stained with antibodies against TFE3 (green) and CHOP (red). Scale bar, 10 µm. Data are representative of three independent experiments.(E-F) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control), Tunicamycin treatments for 16 h (E), or starvation for 2 h (F) (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated cells, ns= not significant, ***p < 0.001; n>400 cells per condition).(G) Quantification of phospho-4EBP1 protein levels in wild-type and GCN2-KO MEF cells upon starvation for 2 h (the mean ± SD of the fold increase of the phospho-4EBP1 to total 4EBP1 ratio from three independent experiments, one-way ANOVA analysis versus starvation treated wild-type cells, ***p < 0.001).(H) Quantification of the percentage of MEF cells with nuclear TFE3 upon DMSO (Control) or Torin-1 (250 nM) treatments for 1 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus DMSO treated wild-type cells, ***p < 0.001; n>400 cells per condition).


Figure 3 ( A ) ARPE - 19 cells infected with either adenovirus expressing TFE3 - WT - Myc or TFE3 - S321A - Myc for 16h . Cells were fixed , permeabilized and stained with antibodies against Myc . Scale bar , 10 µm . Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 - Myc ( means ± SD of 3 independent experiments , students t - test , * * * * p < 0 . 0001 ; n > 400 cells per condition ) . ( B ) Immunoblots of protein lysates from ARPE - 19 cells infected as indicated in ( A ) . Data are representative of three independent experiments . ( C ) Immunoblots of TFE3 - Ser321 phosphorylation state in nuclear fractions of ARPE - 19 cells incubated with DMSO ( Ctrl . ) , Tunicamycin ( Tun . ) , or Torin - 1 . Data are representative of three independent experiments . ( D ) Immunoblots of protein of a GST pull - down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin ( 5 µg / ml ) ( Tun . ) for 16 h . Data are representative of three independent experiments . ( E ) Immunoblots of protein lysates from ARPE - 19 cells incubated in the presence of DMSO ( Ctrl . ) , Tunicamycin ( 5 µg / ml ) ( Tun . ) , or starved in EBSS ( Strv . ) for 16 h . Data are representative of three independent experiments . ( F ) Quantification of the percentage of ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or Tunicamycin + FK506 treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus Tunicamycin treated cells , * * * p < 0 . 001 ; n > 400 cells per condition ) . ( G ) Quantification of the percentage of calcineurin depleted ARPE - 19 cells with nuclear TFE3 upon Tunicamycin ( Tun . ) or Starvation ( Strv . ) treatments for 16 h ( mean ± SD of three independent experiments , one - way ANOVA analysis versus siNon - target treated cells within the indicated treatment , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; n > 400 cells per condition ) . ( H ) Immunoblots of protein lysates from ARPE - 19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB . Data are representative of three independent experiments . ( I ) Relative Quantitative Real - Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE - 19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analsysis versus the siNon - Target ( siNT ) treated cells , * * * p < 0 . 001 ) . ( J ) Quantification of the percentage of ATF4 depleted ARPE - 19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h ( mean ± SD of two independent experiments , one - way ANOVA analysis versus siNon - target treated cells within the indicated treatment * * * p < 0 . 001 ; n > 400 cells per condition ) .

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

Figure 3(A) ARPE-19 cells infected with either adenovirus expressing TFE3-WT-Myc or TFE3-S321A-Myc for 16h. Cells were fixed, permeabilized and stained with antibodies against Myc. Scale bar, 10 µm. Quantification of the percentage of ARPE-19 cells with nuclear TFE3-Myc (means ± SD of 3 independent experiments, students t-test, ****p < 0.0001; n>400 cells per condition).(B) Immunoblots of protein lysates from ARPE-19 cells infected as indicated in (A). Data are representative of three independent experiments.(C) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear fractions of ARPE-19 cells incubated with DMSO (Ctrl.), Tunicamycin (Tun.), or Torin-1. Data are representative of three independent experiments.(D) Immunoblots of protein of a GST pull-down from nuclear fractions of MEF cells treated with DMSO or Tunicamycin (5 µg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.(E) Immunoblots of protein lysates from ARPE-19 cells incubated in the presence of DMSO (Ctrl.), Tunicamycin (5 µg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.(F) Quantification of the percentage of ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus Tunicamycin treated cells, ***p < 0.001; n>400 cells per condition).(G) Quantification of the percentage of calcineurin depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin (Tun.) or Starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment, ns= not significant, *p < 0.05, **p < 0.01, ***p < 0.001; n>400 cells per condition).(H) Immunoblots of protein lysates from ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB. Data are representative of three independent experiments.(I) Relative Quantitative Real-Time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE-19 cells depleted of Calcineurin catalytic subunits PPP3CA and PPP3CB (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analsysis versus the siNon-Target (siNT) treated cells, ***p < 0.001).(J) Quantification of the percentage of ATF4 depleted ARPE-19 cells with nuclear TFE3 upon Tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one-way ANOVA analysis versus siNon-target treated cells within the indicated treatment***p < 0.001; n>400 cells per condition).


Figure 4 ( A ) Relative Quantitative Real - Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE - 19 cells depleted of TFEB and TFE3 ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , student ' s t - test versus siNon - Target ( siNT ) treated cells , * * * p < 0 . 001 ) . ( B ) Relative Quantitative Real - Time PCR analysis of MCOLN1 , ATP6V1C1 , HEXA and UVRAG mRNA transcript levels in ARPE - 19 cells depleted of TFEB and TFE3 upon incubation with DMSO ( Control ) or Tunicamycin ( 5 µg / ml ) for 16 h ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the same treatment condition in the siNon - Target ( siNT ) treated cells or versus the DMSO condition in siNon - Target ( siNT ) treated cells , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

Figure 4(A) Relative Quantitative Real-Time PCR analysis of TFEB and TFE3 mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, student's t-test versus siNon-Target (siNT) treated cells, ***p < 0.001).(B) Relative Quantitative Real-Time PCR analysis of MCOLN1, ATP6V1C1, HEXA and UVRAG mRNA transcript levels in ARPE-19 cells depleted of TFEB and TFE3 upon incubation with DMSO (Control) or Tunicamycin (5 µg/ml) for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the same treatment condition in the siNon-Target (siNT) treated cells or versus the DMSO condition in siNon-Target (siNT) treated cells, *p < 0.05, **p < 0.01, ***p < 0.001).


Figure 5 ( A ) Immunoblots of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null , TFEB - S211A , or TFE3 upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) or starved in EBSS for 16 h . Data are representative of five independent experiments . ( B ) Quantification of ATF4 , CHOP and GADD34 protein levels in ARPE - 19 cells treated as indicated in ( A ) ( mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected cells , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) . ( C ) Relative Quantitative Real - Time PCR analysis of ATF4 , CHOP , GADD34 and MCOLN1 mRNA transcript levels in ARPE - 19 cells treated as indicated in ( A ) ( mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected cells , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

Figure 5(A) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A, or TFE3 upon incubation with DMSO (Control), Tunicamycin (5 µg/ml) or starved in EBSS for 16 h. Data are representative of five independent experiments.(B) Quantification of ATF4, CHOP and GADD34 protein levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, *p < 0.05, **p < 0.01, ***p < 0.001).(C) Relative Quantitative Real-Time PCR analysis of ATF4, CHOP, GADD34 and MCOLN1 mRNA transcript levels in ARPE-19 cells treated as indicated in (A) (mean ± SD of the RNA fold change of indicated genes normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected cells, **p < 0.01, ***p < 0.001).


Figure 6 ( A - D ) Relative Quantitative Real - Time PCR analysis of ATF4 ( A ) , ASNS ( B ) , CAT1 ( C ) and xCT ( D ) mRNA transcript levels in ATF4 - depleted ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A upon incubation with DMSO ( Control ) , Tunicamycin ( 5 µg / ml ) , or starved in EBSS for 16 h ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , one - way ANOVA analysis versus the corresponding treatment condition in the adenovirus - Null infected and siRNA Non - Target ( siNT ) treated cells , * p < 0 . 05 , * * * p < 0 . 001 ) . ( E ) ChIP - qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest ( ATF4 - Control ) from MEF cells that were untreated ( control ) , starved for 2 h , or treated with Tunicamycin ( 0 . 1 µg / ml ) for 16 h . Amplification regions are indicated by arrows . Chromatin DNA was immunoprecipitated with antibodies for anti - TFE3 . Bar graphs show the amount of immunoprecipitated DNA detected by the real - time PCR assay . Values were normalized to the input and plotted as relative enrichment compared to untreated conditions ( means ± SD of three independent experiments , students t - test , * p < 0 . 05 , * * p < 0 . 01 ) .

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

Figure 6(A-D) Relative Quantitative Real-Time PCR analysis of ATF4 (A), ASNS (B), CAT1 (C) and xCT (D) mRNA transcript levels in ATF4-depleted ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A upon incubation with DMSO (Control), Tunicamycin (5 µg/ml), or starved in EBSS for 16 h (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, one-way ANOVA analysis versus the corresponding treatment condition in the adenovirus-Null infected and siRNA Non-Target (siNT) treated cells, *p < 0.05, ***p < 0.001).(E) ChIP-qPCR analysis of the ATF4 promoter and 2000bp upstream of the region of interest (ATF4-Control) from MEF cells that were untreated (control), starved for 2 h, or treated with Tunicamycin (0.1 µg/ml) for 16 h. Amplification regions are indicated by arrows. Chromatin DNA was immunoprecipitated with antibodies for anti-TFE3. Bar graphs show the amount of immunoprecipitated DNA detected by the real-time PCR assay. Values were normalized to the input and plotted as relative enrichment compared to untreated conditions (means ± SD of three independent experiments, students t-test, *p < 0.05, **p < 0.01).


Figure 7 ( F ) Relative Quantitative Real - Time PCR analysis of SUMF1 , DERL1 , ATF6 , APEX1 and GPX1 mRNA transcript levels in ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A ( mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments , students t - test , * * p < 0 . 01 , * * * p < 0 . 001 ) . ( G ) Immunoblots of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null or TFEB - S211A . Data are representative of three independent experiments . ( H ) Quantification of IRE1α and APEX1 protein levels in ARPE - 19 cells infected as indicated in ( G ) ( mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments , students t - test , * * * p < 0 . 001 ) .

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

Figure 7(F) Relative Quantitative Real-Time PCR analysis of SUMF1, DERL1, ATF6, APEX1 and GPX1 mRNA transcript levels in ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A (mean ± SD of the RNA fold change of indicated gene mRNAs normalized to actin mRNA from three independent experiments, students t-test, **p < 0.01, ***p < 0.001).(G) Immunoblots of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null or TFEB-S211A. Data are representative of three independent experiments.(H) Quantification of IRE1α and APEX1 protein levels in ARPE-19 cells infected as indicated in (G) (mean ± SD of the fold increase of the indicated protein to actin ratio from three independent experiments, students t-test, ***p < 0.001).


Figure 8 ( A ) Immunoblots of protein lysates from Null - or TFEB / 3 knockout - MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( Tun . ) ( 0 . 1 µg / ml ) for the indicated times . Data are representative of three independent experiments . ( B ) Quantification of ATF4 protein levels in Null - or TFEB / 3 knockout - MEF cells treated as indicated in ( A ) ( mean ± SD of the ATF4 to actin protein ratio from three independent experiments , student ' s t - test versus the corresponding time point in Null - MEF cells , ns = not significant , * * p < 0 . 01 ) . ( C ) Cell viability was determined in Null - or TFEB / 3 knockout - MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( Tun . ) ( 0 . 1 µg / ml ) for the indicated times ( mean ± SD of the percentage of viable cells compared to control from three independent experiments , one - way ANOVA analysis versus the corresponding time point in Null - MEF cells * * * p < 0 . 001 ) . ( D ) Chromatin condensation , an indication of cells undergoing apoptosis , was assessed in Null - or TFEB / 3 knockout - MEF cells incubated in the presence of DMSO ( Control ) or Tunicamycin ( 0 . 1 µg / ml ) for the indicated times . Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342 . Scale bar , 20 µm . Data are representative of three independent experiments . ( E ) Immunoblots of protein lysates from Null - or TFEB / 3 knockout - MEF cells treated as described in ( A ) . Data are representative of three independent experiments . ( F - H ) Quantification of CHOP ( F ) , PUMA ( G ) and NOXA ( H ) protein levels in Null - or TFEB / 3 knockout - MEF cells treated as indicated in ( E ) ( mean ± SD of the indicated protein to actin ratio from three independent experiments , student ' s t - test analysis versus the corresponding time point in Null - MEF cells , ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ) . ( I ) adenovirus of protein lysates from ARPE - 19 cells infected with either adenovirus expressing Null , TFEB - S211A or TFE3 . Data are representative of three independent experiments . ( J ) Quantification of PUMA protein levels in ARPE - 19 cells infected as indicated in ( I ) ( mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments , one - way ANOVA analysis versus the adenovirus - Null infected cells , * p < 0 . 05 , * * * p < 0 . 001 ) .

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

Figure 8(A) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times. Data are representative of three independent experiments.(B) Quantification of ATF4 protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (A) (mean ± SD of the ATF4 to actin protein ratio from three independent experiments, student's t-test versus the corresponding time point in Null-MEF cells, ns= not significant, **p < 0.01).(C) Cell viability was determined in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (Tun.) (0.1 µg/ml) for the indicated times (mean ± SD of the percentage of viable cells compared to control from three independent experiments, one-way ANOVA analysis versus the corresponding time point in Null-MEF cells ***p < 0.001).(D) Chromatin condensation, an indication of cells undergoing apoptosis, was assessed in Null- or TFEB/3 knockout-MEF cells incubated in the presence of DMSO (Control) or Tunicamycin (0.1 µg/ml) for the indicated times. Confocal microscopy images were obtained from fixed cells stained with Hoechst 33342. Scale bar, 20 µm. Data are representative of three independent experiments.(E) Immunoblots of protein lysates from Null- or TFEB/3 knockout-MEF cells treated as described in (A). Data are representative of three independent experiments.(F-H) Quantification of CHOP (F), PUMA (G) and NOXA (H) protein levels in Null- or TFEB/3 knockout-MEF cells treated as indicated in (E) (mean ± SD of the indicated protein to actin ratio from three independent experiments, student's t-test analysis versus the corresponding time point in Null-MEF cells, ns= not significant, *p < 0.05, **p < 0.01).(I) adenovirus of protein lysates from ARPE-19 cells infected with either adenovirus expressing Null, TFEB-S211A or TFE3. Data are representative of three independent experiments.(J) Quantification of PUMA protein levels in ARPE-19 cells infected as indicated in (I) (mean ± SD of the fold change of the indicated protein to actin ratio from three independent experiments, one-way ANOVA analysis versus the adenovirus-Null infected cells, *p < 0.05, ***p < 0.001).