A ) 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 .

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

A) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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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 ) .

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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).


A ) 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 ) .

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

A) 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 O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

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.


( 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 .

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

(A-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 .

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(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.


( 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

O O O O O O O O O O O O O O O O O O O O O O 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) 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

(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


Representative 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 .

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Representative 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.


( 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 .

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

(A) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 )

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

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 .

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

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 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 .

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

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 .

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

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

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.


( 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

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

(A) 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

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(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

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(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

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

(E-G) 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 - / -

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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 .

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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.


( 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

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(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

(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

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

(C-E) 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


( 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

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

(A, 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

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

(A, 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

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

(C) Ratio of IL-12- or TNF-producing cells in 5.2+ vs 5.1+ pDC of indicated MCMV-infected MBMC mice


Ratio 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 .

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

Ratio 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 .

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

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 .

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(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 ) .

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(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).


A , 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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A, 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.


( 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 .

(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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(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.


Average 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 ) .

Average 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).


A - 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 .

A-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.


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


( a ) DamID domainograms of NL interactions after activation of CCSER1 , GRID2 , or both . All data are from human RPE - 1 cells .

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(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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(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.


( 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 .

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(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 ) .

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(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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(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)


( 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 .

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(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 .

(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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(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).


( 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 .

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(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 .

(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 .

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(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 ) .

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(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 .

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(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 ) .

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(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 .

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(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.


( 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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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).


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 ) .

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(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 ) .

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(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).


( 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 ) .

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

(A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


( 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 ) .

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

(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 ) .

(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).


( 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 ) .

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

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

(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).


( 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 ) .

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

(F) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) 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).


( 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 ) .

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

(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 ) .

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

(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 .

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

(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 ) .

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

(E) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) 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).


Down regulation of SREBP target genes in Cideb - deficient liver . Microarray heatmap of fatty acids / cholesterol synthesis genes that were down - regulated in Cideb - / - mice and Scap liver - specific knockout mice , comparing to the respective WT controls ( WT VS Cideb - / - , WT VS L - Scap - / - , n = 2 per group ) . The expression level of each gene was linearly normalized to range from - 1 to 1 before plotting .

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

Down regulation of SREBP target genes in Cideb-deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down-regulated in Cideb-/- mice and Scap liver-specific knockout mice, comparing to the respective WT controls (WT VS Cideb-/-, WT VS L-Scap-/-, n = 2 per group). The expression level of each gene was linearly normalized to range from -1 to 1 before plotting.


Decreased SREBP processing and maturation in Cideb deficient liver . Immune blotting ( IB ) of the full length , precursor SREBP - 1 / 2 ( SREBP FL ) and the cleaved , active SREBP - 1 / 2 ( SREBP N ) from WT and Cideb - / - mice liver under ad - lib ( ND ) , fasting 12h ( F ) , and refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting .

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

Decreased SREBP processing and maturation in Cideb deficient liver. Immune blotting (IB) of the full length, precursor SREBP-1/2 (SREBP FL) and the cleaved, active SREBP-1/2 (SREBP N) from WT and Cideb-/- mice liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting.


Defective SREBP transcriptional response to refeeding in Cideb - deficient liver . Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb - / - liver under ad - lib ( ND ) , fasting 12h ( F ) , and refeeding ( R ) conditions . Mice were refed with high - carbohydrate low - fat diet for 12h after 12h fasting ( n = 3 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

Defective SREBP transcriptional response to refeeding in Cideb-deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb-/- liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting (n = 3 per group).


Re - introduction of Cideb rescues SREBP processing in Cideb - / - liver . WT and Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , and sacrificed for IB with the indicated antibodies 20 days after the injection .

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

Re-introduction of Cideb rescues SREBP processing in Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for IB with the indicated antibodies 20 days after the injection.


Re - introduction of Cideb rescues the expression of SREBP target genes in Cideb - / - liver . Transcript levels of lipogenic genes determined by qPCR from WT and Cideb - / - liver . WT and Cideb - / - mice were injected with AAV - GFP or AAV - Cideb , and sacrificed for qPCR 20 days after the injection ( n = 3 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

Re-introduction of Cideb rescues the expression of SREBP target genes in Cideb-/- liver. Transcript levels of lipogenic genes determined by qPCR from WT and Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for qPCR 20 days after the injection (n = 3 per group).


Liver morphology of WT and Cideb - / - liver under chow diet and HFLF diet . H / E staining , oil red - O staining , immunohistochemistry and ultra - structure ( Electron Microscope ) were performed . Scale bar represented 50 µm in the upper three rows of images , and 2 µm in the bottom row of images . LD : lipid droplet , ER : endoplasmic reticulum , M : mitochondrial , N : nucleus .

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

Liver morphology of WT and Cideb-/- liver under chow diet and HFLF diet. H/E staining, oil red-O staining, immunohistochemistry and ultra-structure (Electron Microscope) were performed. Scale bar represented 50 µm in the upper three rows of images, and 2 µm in the bottom row of images. LD: lipid droplet, ER: endoplasmic reticulum, M: mitochondrial, N: nucleus.


Total liver TAG levels ( B ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Total liver TAG levels (B) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Liver cholesterol ester levels ( C ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Liver cholesterol ester levels (C) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


D Loss of Cideb inhibits diet - induced SREBP activation . IB of hepatic SREBP processing and lipogenic enzyme levels ( Fasn , Scd1 ) of WT and Cideb - / - mice under chow diet and HFLF diet .

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

D Loss of Cideb inhibits diet-induced SREBP activation. IB of hepatic SREBP processing and lipogenic enzyme levels (Fasn, Scd1) of WT and Cideb-/- mice under chow diet and HFLF diet.


E Serum AST and ALT levels of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

E Serum AST and ALT levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


F Cideb deficiency alleviates the whole body inflammation response . Serum TNFα , MCP1 and IL6 levels of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

F Cideb deficiency alleviates the whole body inflammation response. Serum TNFα, MCP1 and IL6 levels of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Serum fed glucose levels ( G ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

Serum fed glucose levels (G) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Serum fed insulin levels ( H ) of WT and Cideb - / - mice under chow diet and HFLF diet ( n = 8 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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

Serum fed insulin levels (H) of WT and Cideb-/- mice under chow diet and HFLF diet (n = 8 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


I Glucose tolerance test ( GTT ) of WT and Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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I Glucose tolerance test (GTT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


J Insulin tolerance test ( ITT ) of WT and Cideb - / - mice under HFLF diet ( n = 5 per group ) . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

J Insulin tolerance test (ITT) of WT and Cideb-/- mice under HFLF diet (n = 5 per group). Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Decreased SREBP - 1 / 2 and SCAP levels in Golgi apparatus in Cideb - / - mice . IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

Decreased SREBP-1/2 and SCAP levels in Golgi apparatus in Cideb-/- mice. IB of organelle fractions of the indicated proteins and quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb deficiency impairs the packaging of SCAP / SREBP into COPII vesicles . Protein present in the vesicle or microsome fractions obtained from WT or Cideb - / - liver were detected by IB . Quantification of IB data from 3 independent experiments . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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Cideb deficiency impairs the packaging of SCAP/SREBP into COPII vesicles. Protein present in the vesicle or microsome fractions obtained from WT or Cideb-/- liver were detected by IB. Quantification of IB data from 3 independent experiments. Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells . Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A - Flag - BirA * . Cells were cultured in sterol - depleted medium and treated with 80 µM biotin , prior to pulldown with streptavidin - conjugated beads . The biotinylated proteins were detected by IB with the indicated antibodies following SDS - PAGE .

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Cideb depletion decreases the recruitment of SCAP to the COPII machinery in cells. Primary hepatocytes transfected with control or Cideb siRNA were infected with adenovirus expressing Sar1A-Flag-BirA*. Cells were cultured in sterol-depleted medium and treated with 80 µM biotin, prior to pulldown with streptavidin-conjugated beads. The biotinylated proteins were detected by IB with the indicated antibodies following SDS-PAGE.


Cleaved SREBP bypasses Cideb deficiency . WT and Cideb - / - mice were injected with adenovirus expressing GFP , SREBP - 1c full length ( FL ) or SREBP - 1c active form ( N ) . Mice were sacrificed 7 days after injection , and liver TAG levels and hepatic expression SREBP - 1 target genes in the indicated groups were determined ( n = 5 per group ) . Data information : Data represent the Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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Cleaved SREBP bypasses Cideb deficiency. WT and Cideb-/- mice were injected with adenovirus expressing GFP, SREBP-1c full length (FL) or SREBP-1c active form (N). Mice were sacrificed 7 days after injection, and liver TAG levels and hepatic expression SREBP-1 target genes in the indicated groups were determined (n=5 per group). Data information: Data represent the Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb co - precipitates with SCAP . Flag - tagged Cideb was co - expressed with different HA - tagged proteins ( PLIN2 , Insig , SCAP , SREBP - 1 or SREBP - 2 ) in 293T cells and immuno - precipitated with an anti - Flag antibody , and the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE .

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Cideb co-precipitates with SCAP. Flag-tagged Cideb was co-expressed with different HA-tagged proteins (PLIN2, Insig, SCAP, SREBP-1 or SREBP-2) in 293T cells and immuno-precipitated with an anti-Flag antibody, and the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Interaction between the endogenous SCAP and Cideb . Endogenous SCAP was immuno - precipitated from primary hepatocytes under normal medium , 25 - HC depletion or addition conditions . And the presence of Cideb in the immune - complex was detected by IB . SREBP - 1 was detected as the positive control . Grp94 was detected as the negative control .

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Interaction between the endogenous SCAP and Cideb. Endogenous SCAP was immuno-precipitated from primary hepatocytes under normal medium, 25-HC depletion or addition conditions. And the presence of Cideb in the immune-complex was detected by IB. SREBP-1 was detected as the positive control. Grp94 was detected as the negative control.


Cideb interacts with the 416 - 800 portion of SCAP . Flag - tagged Cideb was co - expressed with the indicated HA - tagged SCAP truncation mutants in 293T cells and immuno - precipitated with an anti - Flag antibody . Levels of the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE .

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Cideb interacts with the 416-800 portion of SCAP. Flag-tagged Cideb was co-expressed with the indicated HA-tagged SCAP truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Sterol - dependent interaction switch of SCAP . SCAP interacts with Insig in the presence of sterol , while switches to Cideb binding upon sterol deprivation . Flag - tagged Insig - 1 or Cideb was co - expressed with HA - tagged SREBP - 2 and SCAP or SCAP - Y298C in CHO - K1 cells in sterol - depleted medium . When indicated , 10 µg / ml 25 - HC were supplemented before cells were immuno - precipitated with an anti - Flag antibody , and the levels of co - immunoprecipitated proteins were detected by IB following SDS - PAGE . The dotted blue vertical line was drawn to separate the result between SCAP - WT and SCAP - Y298C .

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Sterol-dependent interaction switch of SCAP. SCAP interacts with Insig in the presence of sterol, while switches to Cideb binding upon sterol deprivation. Flag-tagged Insig-1 or Cideb was co-expressed with HA-tagged SREBP-2 and SCAP or SCAP-Y298C in CHO-K1 cells in sterol-depleted medium. When indicated, 10 µg/ml 25-HC were supplemented before cells were immuno-precipitated with an anti-Flag antibody, and the levels of co-immunoprecipitated proteins were detected by IB following SDS-PAGE. The dotted blue vertical line was drawn to separate the result between SCAP-WT and SCAP-Y298C.


Cideb relieves sterol - dependent suppression of SREBP processing . Hepatocytes infected with adenovirus expressing GFP or Flag - Cideb were cultured in sterol deprived condition . When indicated , cells were treated with different doses of 25 - HC before subjected to IB with the indicated antibodies following SDS - PAGE . Quantification of IB data from 3 independent experiments . Data represents Mean ± SEM .

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Cideb relieves sterol-dependent suppression of SREBP processing. Hepatocytes infected with adenovirus expressing GFP or Flag-Cideb were cultured in sterol deprived condition. When indicated, cells were treated with different doses of 25-HC before subjected to IB with the indicated antibodies following SDS-PAGE. Quantification of IB data from 3 independent experiments. Data represents Mean ± SEM.


Cideb promotes Golgi localization of SCAP . HepG2 cells transfected with GFP - SCAP alone or with HA - Cideb were cultured in sterol deprived conditions or treated with 1 µg / ml 25 - HC . Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA . Scale bars represent 10 µm ( Merge ) , and 2 µm ( Inlay ) . Bottom : quantification of the percentage of SCAP localized in Golgi - positive region per cell ( left ) and quantification of the percentage of cells containing Golgi - localized GFP - SCAP ( right ) under indicated conditions , from 3 independent experiments ; Data represent Mean ± SEM ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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Cideb promotes Golgi localization of SCAP. HepG2 cells transfected with GFP-SCAP alone or with HA-Cideb were cultured in sterol deprived conditions or treated with 1 µg/ml 25-HC. Cells were fixed and subjected to confocal microscopy following staining with antibodies against GM130 or HA. Scale bars represent 10 µm (Merge), and 2 µm (Inlay). Bottom: quantification of the percentage of SCAP localized in Golgi-positive region per cell (left) and quantification of the percentage of cells containing Golgi-localized GFP-SCAP (right) under indicated conditions, from 3 independent experiments; Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb interacts with Sec12 . 293T cells transfected with Cideb and GFP - tagged Sec12 , Sec23 , Sec16S , or Sec16L were subjected to immuno - precipitation with an anti - Cideb antibody , and levels of the co - immunoprecipitated protein were detected by an anti - GFP antibody .

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Cideb interacts with Sec12. 293T cells transfected with Cideb and GFP-tagged Sec12, Sec23, Sec16S, or Sec16L were subjected to immuno-precipitation with an anti-Cideb antibody, and levels of the co-immunoprecipitated protein were detected by an anti-GFP antibody.


Co - localization of SCAP and Cideb at the ER exit sites . HepG2 cells transfected with GFP - SCAP and HA - Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours , then fixed with methanol and stained with antibodies against Sec12 or HA . 3D reconstruction was obtained by processing 3D - original image with Imaris . Scale bars represent 10 µm ( Merge ) , and 1 µm ( Inlay and 3D reconstruction ) .

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Co-localization of SCAP and Cideb at the ER exit sites. HepG2 cells transfected with GFP-SCAP and HA-Cideb were arrested with 10 µM BFA and 1 µM Nocodazole for 2 hours, then fixed with methanol and stained with antibodies against Sec12 or HA. 3D reconstruction was obtained by processing 3D-original image with Imaris. Scale bars represent 10 µm (Merge), and 1 µm (Inlay and 3D reconstruction).


Localization of Cideb at the ER exit sites . AML12 cells knocked - in with a GFP tag at the C - terminus of Cideb genome was treated with 10 µM BFA for 1 hour , then fixed with methanol and stained with antibodies against Sec12 . Scale bars represent 10 µm ( Merge ) , and 1 µm ( Inlay ) .

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Localization of Cideb at the ER exit sites. AML12 cells knocked-in with a GFP tag at the C-terminus of Cideb genome was treated with 10 µM BFA for 1 hour, then fixed with methanol and stained with antibodies against Sec12. Scale bars represent 10 µm (Merge), and 1 µm (Inlay).


Sec12 interacts with the 118 - 165 portion of Cideb . HA - tagged Sec12 was co - expressed with the indicated Flag - tagged Cideb truncation mutants in 293T cells and immuno - precipitated with an anti - Flag antibody . Levels of the co - immunoprecipitated protein were detected by an anti - HA antibody following SDS - PAGE .

Sec12 interacts with the 118-165 portion of Cideb. HA-tagged Sec12 was co-expressed with the indicated Flag-tagged Cideb truncation mutants in 293T cells and immuno-precipitated with an anti-Flag antibody. Levels of the co-immunoprecipitated protein were detected by an anti-HA antibody following SDS-PAGE.


Lysine 128 of Cideb is required for the interaction with Sec12 . 293T cells transfected with HA - Sec12 and the indicated Flag - tagged Cideb constructs were subjected to immunoprecipitation with an anti - Flag antibody , and levels of the co - immunoprecipitated HA - Sec12 were detected with an anti - HA antibody .

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Lysine 128 of Cideb is required for the interaction with Sec12. 293T cells transfected with HA-Sec12 and the indicated Flag-tagged Cideb constructs were subjected to immunoprecipitation with an anti-Flag antibody, and levels of the co-immunoprecipitated HA-Sec12 were detected with an anti-HA antibody.


Recombinant Cideb interacts with Sec12 . GST - Sec12 and MBP - tagged Cideb or Cideb - K128A were purified from E . coli and subjected to in vitro binding .

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Recombinant Cideb interacts with Sec12. GST-Sec12 and MBP-tagged Cideb or Cideb-K128A were purified from E.coli and subjected to in vitro binding.


Cideb - K128A fails to promote SREBP processing . Cideb - / - mice were injected with adenovirus expressing GFP , Flag - Cideb , or Flag - Cideb - K128A , and sacrificed 7 days after injection . Liver lysates were subjected to IB with the indicated antibodies following SDS - PAGE and quantification of IB data from 3 independent experiments . Data represent Mean ± SEM ; * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test .

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Cideb-K128A fails to promote SREBP processing. Cideb-/- mice were injected with adenovirus expressing GFP, Flag-Cideb, or Flag-Cideb-K128A, and sacrificed 7 days after injection. Liver lysates were subjected to IB with the indicated antibodies following SDS-PAGE and quantification of IB data from 3 independent experiments. Data represent Mean ± SEM; **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test.


Cideb , but not Cideb - K128A , promotes SCAP / SREBP incorporation into COPII vesicles . Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay . Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS - PAGE .

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Cideb, but not Cideb-K128A, promotes SCAP/SREBP incorporation into COPII vesicles. Liver microsomes were isolated from mice expressing the indicated proteins and subjected to in vitro budding assay. Resulting vesicles and the donor microsomes were subject to IB with the indicated antibodies following SDS-PAGE.


Cideb enriches SCAP to ERESs via interacting with Sec12 . CHO - K1 cells transfected with GFP - SCAP , HA - Cideb or HA - Cideb - K128A were arrested with 5 µM Nocodazole for 30 min , then fixed with methanol and stained with antibodies against Sec12 and HA . Scale bars represent 10 µm ( left ) , and 2 . 5 µm ( Inlay ) .

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Cideb enriches SCAP to ERESs via interacting with Sec12. CHO-K1 cells transfected with GFP-SCAP, HA-Cideb or HA-Cideb-K128A were arrested with 5 µM Nocodazole for 30 min, then fixed with methanol and stained with antibodies against Sec12 and HA. Scale bars represent 10 µm (left), and 2.5 µm (Inlay).


Number of SCAP - positive ER exit sites per cell . Normalized fluorescent intensity of SCAP at each individual ER exit site . Total fluorescent intensity of SCAP at ER exit sites per cell . Number of Sec12 - positive ER exit sites per cell . Data information : Data represent Mean ± SEM ; NS : not significant , * : p < 0 . 05 , * * : p < 0 . 01 , * * * : p < 0 . 001 , by 2 - tailed Student ' s t test . In panel C - F , quantification of images from 3 independent experiments .

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Number of SCAP-positive ER exit sites per cell.   Normalized fluorescent intensity of SCAP at each individual ER exit site.   Total fluorescent intensity of SCAP at ER exit sites per cell.   Number of Sec12-positive ER exit sites per   cell. Data information: Data represent Mean ± SEM; NS: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, by 2-tailed Student's t test. In panel C-F, quantification of images from 3 independent experiments.


A . Confocal microscopic image showing HeLa cells immunostained for Nup358 ( green , upper panel ) or Nup214 ( green , lower panel ) and RanGAP1 ( red ) using specific antibodies . DNA was stained with Hoechst 33342 ( blue ) . Scale bars , 10 μm .

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A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm.