TIGAR expression modulates autophagy independently of p53 . ( A ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were transfected with scrambled or TIGAR siRNAs , and 48 h after transfection , cells were left untreated ( t0 ) or treated with Bafilomycin A1 ( 100 nM ) for 1 or 2 h ( t1 and t2 ) . The percentage of cells with GFP - LC3 puncta was calculated at the indicated time points . Data are shown as the mean and standard deviation from three independent experiments .

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TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments.


( B ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were cotransfected with scrambled or TIGAR siRNAs , and scrambled or p53 siRNA . After 48 h transfection , cells were left untreated or exposed for 5 h to nutrient starvation . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments .

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(B) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.


( C ) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled or p53 siRNA , and 48 h later exposed to nutrient starvation for 5 h . Actin expression was examined as a loading control .

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(C) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA, and 48 h later exposed to nutrient starvation for 5 h. Actin expression was examined as a loading control.


( D ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were cotransfected with scrambled or TIGAR siRNAs , and scrambled or DRAM siRNA1 / 2 . After 48 h transfection , cells were left untreated or exposed for 5 h to nutrient starvation . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments ; * represents significant difference from starved control conditions ( P < 0 . 05 ) ; @ represents significant difference from untreated control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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(D) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or DRAM siRNA1/2. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments; * represents significant difference from starved control conditions (P<0.05); @ represents significant difference from untreated control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( A ) U2OS cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments .

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(A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.


( B ) ( Left panel ) ROS levels in U2OS cells left untreated or treated with 0 . 5 or 1 mM of H2O2 for 24 h . ROS levels were measured by flow cytometry after DCF treatment . The results are expressed as the mean DCF fluorescence ( and standard deviation ) from three independent experiments . ( Right panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells for cells treated as described above . Data are shown as the mean and standard deviation from three independent experiments .

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(B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation) from three independent experiments. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments.


( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably expressing GFP - LC3 were transfected with vector pCHER1A expressing the mCherry gene as control , or expression plasmids for Flag - tagged - TIGAR or HA - tagged - FBPase - 2 . After 48 h transfection , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments .

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(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.


( D ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 6 h with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) , AO2 ( glutathione ethyl ester ( 4 mM ) ) or AO3 ( ethyl pyruvate ( 4 mM ) ) for 24 h . U2OS cells stably expressing GFP - LC3 were transfected with vector pCHER1A expressing the mCherry gene as control , or expression plasmid for Flag - tagged - TIGAR . After 48 h transfection , cells were treated . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments .

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(D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.


( E ) ( Left panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . ( Right panel ) Basal or nutrient starvation - induced ( 5 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h , measured by flow cytometry after DCF treatment . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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(E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( A ) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA .

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(A) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA.


( B ) RT - PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA .

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(B) RT-PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA.


( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and either scrambled , ATG5 siRNA or ATG10 siRNA . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h .

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(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h.


( D ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and scrambled , ATG5 siRNA or ATG10 siRNA , measured by flow cytometry after DCF treatment . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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(D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( A ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) were left untreated or exposed to nutrient starvation for 6 h , with or without treatment with Z - VAD - FMK for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation of the mean from three independent experiments .

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(A) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) were left untreated or exposed to nutrient starvation for 6 h, with or without treatment with Z-VAD-FMK for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation of the mean from three independent experiments.


( B ) Apoptosis in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) , as measured by cells with a sub - G1 DNA content . Cells were transfected with either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . Cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h .

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(B) Apoptosis in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3), as measured by cells with a sub-G1 DNA content. Cells were transfected with either scrambled, TIGAR siRNA1 or TIGAR siRNA2. Cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h.


( C ) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled , ATG5 siRNA or ATG10 siRNA , as measured by cells with a sub - G1 DNA content . After 48 h , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . Data are shown as the mean and standard deviation from three independent experiments . In each case , the increase in apoptosis after knockdown of ATG5 or ATG10 , compared with the matched control , was statistically significant ; * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( G ) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group ( numbered 1 - 4 ) was detected by anti - IgG secondary antibody .

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(G) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group (numbered 1-4) was detected by anti-IgG secondary antibody .


Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . After 2 weeks , 7 μm spleen sections were stained with Red : Alexa - Fluor - 594 - CD4 and Green : FITC - B220 ( D ) or FITC - GL7 ( E ) . Scale bar represent 100 nm .

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Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm.


Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . Serum was assessed for level of IgG antibodies binding NP - 31 and NP - 4 ( F ) .

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Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).


( A ) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3 + CD4 + Th cells against CD127 ( left ) and intracellular Foxp3 ( right )

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(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)


NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . Representative staining is shown for CD4 + CD3 + Th cells ( B ) , and summary data for CD3 + CD4 + Th cells ( C ) Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test .

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NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.


NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . summary data for CD3 + CD4 + CD127hi conventional cells ( D ) . Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test .

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NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. summary data for CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.


( E ) Individuals were divided into NRP1 - high subjects ( NRP1 + cells > 1 . 3 % in Th cells ) and NRP1 - low subjects ( NRP1 + cells ≤ 1 . 35 % in Th cells ) . Numbers of HC and SLE falling into each group are shown . * * * p < 0 . 001 Chi squared test with Yates ' correction .

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(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.


( F , G ) NRP1 - expressing cells amongst CD3 + CD4 + CD45RO - cells were measured , representative staining ( F ) and summary data ( G ) . The plot shows individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * * p < 0 . 0001 Unpaired Student ' s T test .

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(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.


B . Bright - field picture of a 0B K8 - eYFP embryo ( Left ) and time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 3 ) . C . Bright - field picture of EB K8 - eYFP embryo ( Left ) and time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 4 ) . D . Bright - field pictures of K8 - eYFP LB embryos ( n = 30 ) , and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas . K8 - eYFP and DAPI appear in yellow and cyan , respectively .

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B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3). C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4). D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively. Da


A - H . SEM images of an opened embryo ( A ) , the ExE cavity wall ( B ) where blood islands are indicated by white arrow , the allantois surface ( C - C ' ) , an opened allantois ( D ) and its zooms ( E - E ' ) where filaments are indicated by white arrows , the epiblast - derived side of the amnion ( F ) , the mesoderm - derived side of the amnion ( G ) and its zoom ( G ' ) .

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A-H. SEM images of an opened embryo (A), the ExE cavity wall (B) where blood islands are indicated by white arrow, the allantois surface (C-C'), an opened allantois (D) and its zooms (E-E') where filaments are indicated by white arrows, the epiblast-derived side of the amnion (F), the mesoderm-derived side of the amnion (G) and its zoom (G').


B . Sagittal sections of C57BL / 6J wild - type and KtyII - / - E7 . 5 embryos immunostained for Krt8 ( n = 15 and n = 5 , respectively ) , F - actin ( n = 11 and 2 ) , Vimentin ( n = 5 and 1 ) , E - cadherin ( n = 1 and 1 ) , T ( n = 4 and 3 ) , Dsg2 ( n = 15 and 5 ) and Pecam - 1 ( n = 4 and 3 ) . Data information : White arrows point toward allantois and scale bars represent 25 µm .

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B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm.


Quantification of embryo morphology at LB stage in C57BL / 6J wild - type ( white boxes / black dots , n = 22 including 16 in which allantois could be measured ) , and KtyII - / - embryos ( grey boxes / black triangles , n = 9 including 8 in which allantois could be measured ) . ( D ) Embryonic region length . Ratios of whole embryo and ExE cavity lengths ( E ) , over embryonic region length .

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Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E) , over embryonic region length.


I - I ' ' . SEM images of opened C57BL / 6J wild - type ( n = 3 ) and KtyII - / - ( n = 2 ) LB stage embryos with zooms on the allantois region ( I ' - I ' ' ) . Scale bars represent 200 ( I ) , 20 ( I ' ) and 10 ( I ' ' ) µm .

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I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.


F . Dot plot of specific gene expression in amnion clusters .

F. Dot plot of specific gene expression in amnion clusters.


G . Immunostaining for Cdx2 ( n = 20 ) . H . Immunostaining for Nrp1 ( n = 8 ) and Col1a1 ( n = 7 ) , primarily expressed in Amnion Mesenchyme . I . Immunostaining for Epcam ( n = 12 ) , Cldn7 ( n = 7 ) , and Lrp2 ( n = 6 ) , primarily expressed in Amnion Ectoderm .

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G. Immunostaining for Cdx2 (n=20). H. Immunostaining for Nrp1 (n=8) and Col1a1 (n=7), primarily expressed in Amnion Mesenchyme. I. Immunostaining for Epcam (n=12), Cldn7 (n=7), and Lrp2 (n=6), primarily expressed in Amnion Ectoderm.


J - K . Sections of C57BL / 6J wild - type and KtyII - / - E7 . 5 embryos immunostained for Nrp1 ( K ) ( n = 8 and 2 , respectively ) , and Lrp2 ( L ) ( n = 3 and 2 ) .

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J-K. Sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Nrp1 (K) (n=8 and 2, respectively), and Lrp2 (L) (n=3 and 2).


D . Dot plot of specific gene expression in chorion clusters .

D. Dot plot of specific gene expression in chorion clusters.


E . Immunostaining for Cdx2 ( n = 20 ) . F . Immunostaining for DAPI ( cyan , left ) , Sox2 ( middle ) , and Gata2 ( right ) . Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm . G . Immunostaining for Cebpb , detectable in most cells with variable intensity . H . Immunostaining for Fn1 ( n = 22 ) , Dlk1 ( n = 8 ) , and Acta2 ( n = 12 ) , primarily expressed in Chorion Mesenchyme . I . Sections from C57BL / 6J wild - type and KtyII - / - LB embryos immunostained for Dlk - 1 ( n = 8 and 4 , respectively ) and Acta2 ( n = 6 and 3 ) .

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E. Immunostaining for Cdx2 (n=20). F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm. G. Immunostaining for Cebpb, detectable in most cells with variable intensity. H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme. I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3). Data


D . Dot plot of specific gene expression in allantois clusters .

D. Dot plot of specific gene expression in allantois clusters.


F . Immunostaining for Ifitm3 ( n = 7 ) ( Allantois Base ) . G . Immunostaining for Cdh11 ( n = 9 ) , Stard8 ( n = 4 ) and Nrp1 ( n = 8 ) ( Allantois Cortex ) . H . Immunostaining for Pitx1 ( n = 4 ) ( Allantois Mesenchyme ) . I . Hhex - GFP ( n = 5 ) and immunostaining for Kdr ( n = 8 ) ( Endothelial Progenitors ) . J . Immunostaining for Runx1 ( n = 3 ) ( Erythroid Progenitors ) . K - L .

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F. Immunostaining for Ifitm3 (n=7) (Allantois Base). G. Immunostaining for Cdh11 (n=9), Stard8 (n=4) and Nrp1 (n=8) (Allantois Cortex). H. Immunostaining for Pitx1 (n=4) (Allantois Mesenchyme). I. Hhex-GFP (n=5) and immunostaining for Kdr (n=8) (Endothelial Progenitors). J. Immunostaining for Runx1 (n=3) (Erythroid Progenitors). K-L.


K - L . Sections from C57BL / 6J wild - type and KtyII - / - LB embryos immunostained for Cdh11 ( Top ) and Krt8 ( Bottom ) ( K , n = 8 and 4 , respectively ) and Ifitm3 ( L , n = 2 and 2 ) . Data information : White arrows point toward signal in allantois . Sagittal sections with anterior to the left .

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K-L. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Cdh11 (Top) and Krt8 (Bottom) (K, n=8 and 4, respectively) and Ifitm3 (L, n=2 and 2). Data information: White arrows point toward signal in allantois. Sagittal sections with anterior to the left.


( A ) Srcap expression in murine different tissues was examined by Northern blot . A 277 nt probe of Srcap ( 81 - 358nt ) was labeled for Northern blot analysis . 18S RNA was used as a loading control .

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(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control.


( B ) Two - week - old SRCAP - C - HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining . A global look of the section was shown . Scale bar : 500 μm . Green : EpCAM , Red : HA tag , Nuclei were counterstained by DAPI .

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(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.


( C ) Murine intestine tissues were fixed and stained according to iDISCO staining protocol . SRCAP was highly expressed in vast majority of crypts in mouse intestine . Scale bar : 50 μm . Green : EpCAM , Red : SRCAP , Nuclei were counterstained by DAPI .

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(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.


( D ) Duodenum region of intestinal tissues were obtained from Lgr5GFP - CreERT2 ( Lgr5GFP ) mice for immunofluorescence staining by indicated antibodies with OpalTM 7 - color fIHC kit . Nuclei were counterstained with DAPI . Scale bar : 50 μm .

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(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.


( E ) ISCs from Lgr5GFP mice were collected for organoid formation . Organoids were fixed for immunofluorescence staining with indicated antibodies . Scale bar : 50 μm . Data information : Green : EpCAM , Red : SRCAP , Purple : PCNA , Nuclei were counterstained by DAPI .

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(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.


( A ) Representative images of intestines from 8 - week - old Srcap + / + and Srcap - / - mice ( n = 6 per group ) . Length of 120 villi and 120 crypts ( 20 villi and 20 crypts per mouse ) was measured and shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 100 μm . Dotted lines indicate the boundary between crypts and villi . Arrows indicate the length of crypts or villi .

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(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.


( B ) Intestinal tissues from 8 - week - old Srcap + / + and Srcap - / - mice ( n = 6 per group ) were collected for immunofluorescence staining with indicated antibodies . Typical images were shown in left panel and statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 50 μm .

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(B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.


( C ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test .

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(C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.


( D ) Srcap + / + and Srcap - / - mice were treated with 10 Gy ' s radiation and sacrificed at different time points . Intestinal tissues were collected for H & E staining . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 100 μm .

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(D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm.


( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - YFP ( LRYFP ) were crossed with Srcapflox / flox mice , followed by administration of tamoxifen ( TAM ) for lineage tracing analysis . Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained . Numbers of traced crypt - villus units were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 50 μm .

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(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.


( F ) Proliferation of Srcap + / + and Srcap - / - ISCs were examined by Ki67 staining followed by flow cytometry analysis . Percentages of proliferated cells were counted as means ± S . D ( lower panel ) . * * P < 0 . 01 by two - tailed Student ' s t - test . n = 5 per group .

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(F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group.


( G ) Multiple color staining was performed in Srcap + / + and Srcap - / - intestine sections . AP - Red , DAB and AP - Blue were used for immunohistochemistray coloration . Numbers of each kind of epithelial cells from 50 crypts or 50 villi ( 10 crypts and 10 villi per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bars , 50 μm .

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(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.


( A ) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti - SRCAP antibody or rabbit IgG followed by ADASDS - PAGE , sliver staining and mass spectrometry .

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(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.


( B ) SRCAP and REST were stained in murine intestinal crypts . Scale bar , 10 μm .

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(B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm.


( D ) Flag - tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti - Flag antibody . REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting .

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(D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting.


( E ) C - terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay .

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(E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay.


( F ) Intestinal crypt lysates were incubated with anti - REST antibody , followed by immunoblotting with indicated antibodies .

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(F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies.


( G ) Intestinal crypt lysates were incubated with anti - REST antibody and protein A / G beads in 4 ℃ overnight . Recombinant SRCAP protein was added with different concentrations , followed by immunoblotting .

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(G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.


( B ) Transcriptome microarray analysis between Srcap - / - ISCs and WT ISCs . Top ten downregulated TFs in Srcap - / - ISCs were listed .

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(B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed.


( C ) Prdm16 expression was tested in Srcap + / + and Srcap - / - ISCs by Western blotting .

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(C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting.


( D ) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining . Green : GFP ; Red : SRCAP ; Blue : Prdm16 . Scale bar , 10 μm .

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(D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm.


( E ) Prdm16 promoter ( - 875 ~ - 854 ) region was used as probe to perform DNA - pulldown assay with murine intestinal crypt lysates followed by immunoblotting .

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(E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting.


( F , G ) Indicated promoter activation was analyzed with luciferase assay . DKK1 was used as a control . Results of relative fold changes are shown as means ± S . D . Data represent five independent replicates . . * * p < 0 . 01 by two - tailed Student ' s t - test . .

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(F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. .


( H ) 1 × 104 ISCs from indicated mice were collected for organoid formation . For gene depletion or restoration , ISCs were infected with lentivirus . Scale bar , 200 μm . Data information : All data represent five independent experiments .

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(H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.


( A ) WT and Srcap - / - ISCs were conducted for transcriptome microarray , followed by Gene Set Enrichment Analysis ( GSEA ) analysis for PPARδ target genes .

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(A) WT and Srcap-/- ISCs were conducted for transcriptome microarray, followed by Gene Set Enrichment Analysis (GSEA) analysis for PPARδ target genes.


( B ) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

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(B) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 and *** p<0.001 by two-tailed Student's t-test.


( C ) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining . Scale bar , 50 μm .

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(C) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining. Scale bar, 50 μm.


( D ) ISCs were collected for organoid formation followed by quantitative RT - PCR . For Prdm16 overexpression , ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 by two - tailed Student ' s t - test .

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(D) ISCs were collected for organoid formation followed by quantitative RT-PCR. For Prdm16 overexpression, ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 by two-tailed Student's t-test.


( E ) mRNA levels of ISC signature genes were examined by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates .

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(E) mRNA levels of ISC signature genes were examined by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates.


( F ) Representative images of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test . ( lower panel ) . n = 6 for each group . GW501516 ( 10 μM ) were added directly into organoid culture medium .

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(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.


( A ) Representative images of intestines from 8 - week - old Rest + / + and Rest - / - mice ( n = 6 per group ) . Length of 120 villi and 120 crypts ( 20 villi and 20 crypts per mouse ) were measured in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 100 μm . Dotted lines indicate the boundary between crypts and villi . Arrows indicate the length of crypts or villi .

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(A) Representative images of intestines from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) were measured in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.


( B ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test .

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(B) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.


( C ) Intestine tissues from 8 - week - old Rest + / + and Rest - / - mice ( n = 6 per group ) were collected for immunohistochemistry staining . Typical images were shown in left panel and statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 50 μm .

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(C) Intestine tissues from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group) were collected for immunohistochemistry staining. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.


( D ) Immunofluorescence staining of intestine tissues from Srcapfl / fl ; Lgr5GFP - CreERT2 and Restfl / fl ; Lgr5GFP - CreERT2 mice . Scale bar , 50 μm .

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(D) Immunofluorescence staining of intestine tissues from Srcapfl/fl; Lgr5GFP-CreERT2 and Restfl/fl; Lgr5GFP-CreERT2 mice. Scale bar, 50 μm.


( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - lacZ ( LRlacZ ) were crossed with Rest - / - mice , followed by TAM administration for lineage tracing for intestinal whole - mount staining for β - gal ( upper panel ) . Mice were sacrificed after one week and typical sections were shown ( middle panel ) . Scale bar , 200 μm . Numbers of traced crypt - villus units were calculated as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . 50 fields ( 10 fields per mouse ) were calculated for each group ( n = 5 ) ( lower panel ) .

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(E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel).


( F ) Rest + / + and Rest - / - mice were treated with 10 Gy ' s radiation and sacrificed at the indicated time points . Intestinal tissues were collected for H & E staining . Scale bars , 100 μm . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) .

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(F) Rest+/+ and Rest-/- mice were treated with 10 Gy's radiation and sacrificed at the indicated time points. Intestinal tissues were collected for H&E staining. Scale bars, 100 μm. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5).


( G ) Representative images of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . n = 6 for each group . Organoid number per well was counted as means ± S . D . ( lower panel ) . Lentivirus - carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM - treated LRCas9 mice to obtain Prdm16 - / - and Ppard - / - ISCs . 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 5 for each group .

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(G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.


( B - D ) Immunofluorescent images of mouse liver ( B ) , testis ( C ) and heart ( D ) . H3K27ac , PolII - S5P , and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti - mouse ChIL - probe ( red : H3K27ac and PolIIS5P ) and anti - rabbit IgG ( green : laminin ) . DNA was counterstained with Hoechst 33342 . Scale Bar : 20 µm ( left images ) , 10 µm ( right images ) .

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(B-D) Immunofluorescent images of mouse liver (B), testis (C) and heart (D). H3K27ac, PolII-S5P, and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti-mouse ChIL-probe (red: H3K27ac and PolIIS5P) and anti-rabbit IgG (green: laminin). DNA was counterstained with Hoechst 33342. Scale Bar: 20 µm (left images), 10 µm (right images).


( E ) Genome browser images of ChIL - seq for H3K27ac and PolII - S5P and bulk tissue RNA - seq data at the Alb locus in liver tissues .

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(E) Genome browser images of ChIL-seq for H3K27ac and PolII-S5P and bulk tissue RNA-seq data at the Alb locus in liver tissues.


( B ) The IGV tracks of H3K27ac ChIL - seq at identified tissue - specific enhancers of Rxra , Gnat3 , Eps8 , and a house - keeping gene of Actb loci are shown with the replicates .

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(B) The IGV tracks of H3K27ac ChIL-seq at identified tissue-specific enhancers of Rxra, Gnat3, Eps8, and a house-keeping gene of Actb loci are shown with the replicates.


( C ) Specific motif enrichment analysis was conducted using chromVAR ( Schep et al , 2017 ) . Hierarchical clustering of deviation - Z scores of three replicates of each tissue is shown .

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(C) Specific motif enrichment analysis was conducted using chromVAR(Schep et al, 2017). Hierarchical clustering of deviation-Z scores of three replicates of each tissue is shown.


( D ) Super - enhancer identification . Tissue - specific enhancers are identified , so that they are listed more than twice ( twice : blue , all : red ) in the top 5 % in all enhancer candidates and are not in the SEs of other tissues . Grey shades indicate the top 5 % of tag count in enhancer candidates .

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(D) Super-enhancer identification. Tissue-specific enhancers are identified, so that they are listed more than twice (twice: blue, all: red) in the top 5% in all enhancer candidates and are not in the SEs of other tissues. Grey shades indicate the top 5% of tag count in enhancer candidates.


( F ) Hnf4ɑ binds to the SE at Alb gene loci .

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(F) Hnf4ɑ binds to the SE at Alb gene loci.


( B ) Signal intensities of ChIL - seq correlated with the expression levels of genes . The lines indicate the average CPM of each expression group at TSS . The expression groups were assigned with respect to the expression levels ( TPM ) of genes .

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(B) Signal intensities of ChIL-seq correlated with the expression levels of genes. The lines indicate the average CPM of each expression group at TSS. The expression groups were assigned with respect to the expression levels (TPM) of genes.


( E ) The tissue - specific genes identified by PolII S5P ChIL - seq . The IGV tracks of all replicates of PolII S5P ChIL - seq are shown at each specific gene ( Trf , Myh6 , and Meig for the liver , heart , and testis , respectively ) . Actb is also shown as the ubiquitously expressed gene in the three tissues .

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(E) The tissue-specific genes identified by PolII S5P ChIL-seq. The IGV tracks of all replicates of PolII S5P ChIL-seq are shown at each specific gene (Trf, Myh6, and Meig for the liver, heart, and testis, respectively). Actb is also shown as the ubiquitously expressed gene in the three tissues.


( B ) PolII S5P ChIL - seq signal of the marker genes of mature skeletal muscle ( Acta1 ) and macrophages ( Cd68 ) .

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(B) PolII S5P ChIL-seq signal of the marker genes of mature skeletal muscle (Acta1) and macrophages (Cd68).


( F ) Bulk - tissue expression levels ( TPM ) of the representative genes .

(F) Bulk-tissue expression levels (TPM) of the representative genes.


( H ) Activities of major cell types in muscle regeneration . The colors of the heatmap show the log2FC to day 0 ( uninjured ) of TR and TSS levels . Representative genes with significant changes in TR are shown .

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(H) Activities of major cell types in muscle regeneration. The colors of the heatmap show the log2FC to day 0 (uninjured) of TR and TSS levels. Representative genes with significant changes in TR are shown.


( A ) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues . For each lane , individual samples were selected from each progressive stage of breast cancer .

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(A) IGV screen shots of PolII S5P ChIL signals are shown for representative genes in breast cancer tissues. For each lane, individual samples were selected from each progressive stage of breast cancer.


( C ) Expression , TSS level , and TR of genes . EEF1A1 is shown as a control housekeeping gene . The horizontal dotted line indicates the median level of all genes . Error bars indicate 95 % confidence intervals for each estimate indicated by dots ( N = 1 for each ) .

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(C) Expression, TSS level, and TR of genes. EEF1A1 is shown as a control housekeeping gene. The horizontal dotted line indicates the median level of all genes. Error bars indicate 95% confidence intervals for each estimate indicated by dots (N=1 for each).


A . qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice ; n = 4 / group ; * P < 0 . 05 , Unpaired t - test , two - tailed . Note that the transgene is not detected in the brain .

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A. qPCR data comparing the expression of the CamkIIδc transgene in the brain and heart of 3 month old CamkIIδc TG mice; n=4/group; *P <0.05, Unpaired t-test, two-tailed. Note that the transgene is not detected in the brain.


C . Ejection fraction , cardiac output and index are significantly decreased in CamkIIδc TG mice ( n = 8 ) when compared to control mice ( n = 5 ; * P < 0 . 05 , , * * P < 0 . 01 , * * * P < 0 . 001 , Unpaired t - test , two - tailed ) . Heart to body ratio , left ventricle to body ratio , and lung to body weight ratio are increased in CamkIIδc TG ( n = 8 ) compared to control ( n = 5 ; * P < 0 . 05 , * * P < 0 . 01 , Unpaired t - test ; two - tailed ) .

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C. Ejection fraction, cardiac output and index are significantly decreased in CamkIIδc TG mice (n =8) when compared to control mice (n=5; *P <0.05, , **P<0.01, ***P<0.001, Unpaired t-test, two-tailed). Heart to body ratio, left ventricle to body ratio, and lung to body weight ratio are increased in CamkIIδc TG (n=8) compared to control (n=5; *P <0.05, **P<0.01, Unpaired t-test; two-tailed).


H . qPCR quantification of selected genes reflecting ER - stress or protein methylation - related processes in CamkIIδc TG mice ( n = 4 ) and control mice ( n = 5 ) . * p < 0 . 05 , ns = non - significant , Unpaired t - test ; two - tailed . Data is normalized to Hprt1 expression .

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H. qPCR quantification of selected genes reflecting ER-stress or protein methylation-related processes in CamkIIδc TG mice (n=4) and control mice (n=5). *p<0.05, ns = non-significant, Unpaired t-test; two-tailed. Data is normalized to Hprt1 expression.


D . The cumulative score of hippocampus - dependent search strategies during Barnes maze training is impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - tailed , unpaired t - test , * p < 0 . 05 ) . E . Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG ( n = 16 ) when compared to control mice ( n = 13 ; two - tailed , unpaired t - test , * p < 0 . 05 ) . Data information : Bars and error bar indicates mean ± SEM .

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D. The cumulative score of hippocampus-dependent search strategies during Barnes maze training is impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). E. Number of visits to escape hole during probe test to assay memory retrieval was impaired in CamkIIδc TG (n = 16) when compared to control mice (n = 13; two-tailed, unpaired t-test, *p<0.05). Data information: Bars and error bar indicates mean ± SEM.


A . Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A , Kmt2A and Kmt2b knock out mice . Fisher ' s hypergeometric test , Benjamini - Hochberg ( BH ) correction .

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A. Hypergeometric overlap analysis comparing genes deregulated in CamkIIδc TG mice to genes differentially expressed in the hippocampal CA1 region of Kat2A, Kmt2A and Kmt2b knock out mice. Fisher's hypergeometric test, Benjamini-Hochberg (BH) correction.


E . Cumulative hippocampus - dependent strategy scores during the Barnes maze training ( One - way ANOVA ) . n = 10 per group . F . Number of visits to the escape hole during probe test ( n = 10 / group , One - Way ANOVA ) . Data information : Bars and error bars indicate mean ± SEM .

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E. Cumulative hippocampus-dependent strategy scores during the Barnes maze training (One-way ANOVA). n = 10 per group. F. Number of visits to the escape hole during probe test (n = 10/group, One-Way ANOVA). Data information: Bars and error bars indicate mean ± SEM.


E . H3K4me3 at TSS region of genes from RNA module1 . Inset shows statistical comparisons among groups ( One - way ANOVA , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) . Bars and error bars indicate mean ± SEM . Number of replicates analyzed for H3K4me3 profile at TSS ( Wildtype vehicle : 3 ; CamkIIδc TG - vehicle n = 4 ; CamkIIδc TG - Vorinostat n = 4 ) .

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E. H3K4me3 at TSS region of genes from RNA module1. Inset shows statistical comparisons among groups (One-way ANOVA, ***P<0.001, ****P<0.0001). Bars and error bars indicate mean ± SEM. Number of replicates analyzed for H3K4me3 profile at TSS (Wildtype vehicle: 3; CamkIIδc TG-vehicle n = 4; CamkIIδc TG-Vorinostat n = 4).


dHepaRG - TR - A3A cells were infected with HBV at an MOI of 300 virions / cell . Cells were treated with tetracycline ( Tet ( A3A ) ) or / and 300 U / ml IFNα from day 9 to 18 p . i . cccDNA amplicons were analyzed by 3D ‑

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dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell. Cells were treated with tetracycline (Tet (A3A)) or/and 300 U/ml IFNα from day 9 to 18 p.i. cccDNA amplicons were analyzed by 3D‑PCR


dHepaRG cells were infected with HBV at an MOI of 100 virions / cell and treated 9 days later with 300 U / ml IFNα ( B ) or 200 U / ml IFNγ ( C ) for indicated times ( h : hours ) . Expression of indicated genes was analyzed by qRT - PCR relative to TATA - box binding protein ( TBP ) mRNA ( n = 6 biological replicates of two independent experiments ) .

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dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated 9 days later with 300 U/ml IFNα (B) or 200 U/ml IFNγ (C) for indicated times (h: hours). Expression of indicated genes was analyzed by qRT-PCR relative to TATA-box binding protein (TBP) mRNA (n=6 biological replicates of two independent experiments).


Liver tissue samples obtained from HBV - negative patients undergoing metastasis resection ( Control ) , or patients with acute , self - limiting HBV infection or chronic hepatitis B , or chronic coinfection with HBV and hepatitis D virus ( HDV ) were stained for ISG20 by immunohistochemistry . For each clinical entity , tissue sections from three different patients are shown ; scale bar : 50 µm . ISG20 positive area of each sample ( n = 3 biological replicates ) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned .

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Liver tissue samples obtained from HBV-negative patients undergoing metastasis resection (Control), or patients with acute, self-limiting HBV infection or chronic hepatitis B, or chronic coinfection with HBV and hepatitis D virus (HDV) were stained for ISG20 by immunohistochemistry. For each clinical entity, tissue sections from three different patients are shown; scale bar: 50 µm. ISG20 positive area of each sample (n=3 biological replicates) was determined by Tissue IA image analysis software and is given in % of total tissue area scanned.


dHepaRG cells were infected with HBV at an MOI of 100 virions / cell and treated with 600 U / ml IFNα or 400 U / ml IFNγ from day 10 to 14 p . i . and ISG20 was stained by immunofluorescence . Primary human hepatocytes were infected with HBV at an MOI of 100 virions / cell and treated with 500 U / ml IFNα or 200 U / ml IFNγ from day 4 to 6 p . i . and ISG20 was stained by immunofluorescence .

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dHepaRG cells were infected with HBV at an MOI of 100 virions/cell and treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 10 to 14 p.i. and ISG20 was stained by immunofluorescence. Primary human hepatocytes were infected with HBV at an MOI of 100 virions/cell and treated with 500 U/ml IFNα or 200 U/ml IFNγ from day 4 to 6 p.i. and ISG20 was stained by immunofluorescence.


dHepaRG cells were infected with HBV at an MOI of 500 virions / cell , treated with 600 U / ml IFNα or 400 U / ml IFNγ from day 7 to 9 p . i . and stained by immunofluorescence for ISG20 and PML ( C ) or ISG20 and nucleophosmin ( D ) .

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dHepaRG cells were infected with HBV at an MOI of 500 virions/cell, treated with 600 U/ml IFNα or 400 U/ml IFNγ from day 7 to 9 p.i. and stained by immunofluorescence for ISG20 and PML (C) or ISG20 and nucleophosmin (D).


dHepaRG cells were infected at an MOI of 100 virions / cell and transduced at day 7 p . i . with an adenoviral vector for expression of shRNA targeting ISG20 ( AdV - shISG20 ) . After 2 days , cells were treated with 300 U / ml IFNα or 200 U / ml IFNγ . ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment , respectively .

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dHepaRG cells were infected at an MOI of 100 virions/cell and transduced at day 7 p.i. with an adenoviral vector for expression of shRNA targeting ISG20 (AdV-shISG20). After 2 days, cells were treated with 300 U/ml IFNα or 200 U/ml IFNγ. ISG20 expression was analyzed by western blot after 7 days of IFNα or 12 days of IFNγ treatment, respectively.


A subsequence of the 3D - PCR amplicon was used as ( - ) ssDNA oligomer , complementary ( + ) ssDNA and a dU - containing ( ‑ ) ssDNA , where all cytosines were replaced by uracils . These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated .

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A subsequence of the 3D-PCR amplicon was used as (-) ssDNA oligomer, complementary (+) ssDNA and a dU-containing (‑) ssDNA, where all cytosines were replaced by uracils. These oligomers were digested in vitro with recombinant ISG20 with or without EDTA as indicated.


dHepaRG - TR - A3A cells were infected with HBV at an MOI of 300 virions / cell and at day 7 p . i . transduced with AdV - ISG20 and treated with tetracycline to induce A3A expression as indicated ( ± Tet ( A3A ) ) for 7 days . Cell lysates were analyzed for cccDNA after T5 digest ( B ) and total intracellular HBV DNA ( C ) by qPCR relative to Prnp ( n = 5 - 6 biological replicates from two independent experiments ) .

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dHepaRG-TR-A3A cells were infected with HBV at an MOI of 300 virions/cell and at day 7 p.i. transduced with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. Cell lysates were analyzed for cccDNA after T5 digest (B) and total intracellular HBV DNA (C) by qPCR relative to Prnp (n=5-6 biological replicates from two independent experiments).


dHepaRG - TR - A3A cells were infected with wildtype HBV ( HBVwt ) or HBV ∆ x at an MOI of 300 virions / cell and transduced on day 7 p . i . with AdV - ISG20 and treated with tetracycline to induce A3A expression as indicated ( ± Tet ( A3A ) ) for 7 days . HBeAg ( E ) was measured by ELISA and cccDNA after T5 digest ( F ) by qPCR relative to Prnp ( n = 6 biological replicates from two independent experiments ) .

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dHepaRG-TR-A3A cells were infected with wildtype HBV (HBVwt) or HBV∆x at an MOI of 300 virions/cell and transduced on day 7 p.i. with AdV-ISG20 and treated with tetracycline to induce A3A expression as indicated (± Tet (A3A)) for 7 days. HBeAg (E) was measured by ELISA and cccDNA after T5 digest (F) by qPCR relative to Prnp (n=6 biological replicates from two independent experiments).


( E ) Normalized expression levels of the native core promoters . Their activities spanned over a broad range of three orders of magnitude ( promoter constructs contained block 1 . 11 and block 7 . 11 combination as nucleosomal sequences ) . Each color represents a different class of core promoter architecture . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements . ( including new cell transfection procedures and measurements of promoter activities ) .

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(E) Normalized expression levels of the native core promoters. Their activities spanned over a broad range of three orders of magnitude (promoter constructs contained block 1.11 and block 7.11 combination as nucleosomal sequences). Each color represents a different class of core promoter architecture. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (including new cell transfection procedures and measurements of promoter activities).


( I ) Scatter plot of expression levels obtained in D . mel . S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D . mel . embryos , revealing a high correlation ( Pearson coefficient 0 . 91 ) between the two datasets . Error bars represent standard deviations of 3 - 4 biological replicate measurements .

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(I) Scatter plot of expression levels obtained in D. mel. S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D. mel. embryos, revealing a high correlation (Pearson coefficient 0.91) between the two datasets. Error bars represent standard deviations of 3-4 biological replicate measurements.


( C ) Boxplots depicting block 1 effects for tested core promoters . Effects of different block 7s were merged in each column ( within the same block 1 ) : the median SD is 0 . 66 for developmental promoters compared to 1 . 23 for constitutive promoters ( lower right corner ) ; Wilcoxon rank - sum test * * * p = 3 . 1 × 10 - 4 , significant . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements . ( D ) Boxplots depicting block 7 effects for tested core promoters . Effects of different block 1s were merged in each column ( within the same block 7 ) : the median SD is 0 . 54 for developmental promoters compared to 0 . 64 for constitutive promoters ( lower right corner ) ; Wilcoxon rank - sum test p = 0 . 3 , not significant . Block 7 with column " w / o B7 " represents the results obtained from promoters without block 7 sequence . Developmental and constitutive promoters are highlighted in green and red , respectively . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements .

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(C) Boxplots depicting block 1 effects for tested core promoters. Effects of different block 7s were merged in each column (within the same block 1): the median SD is 0.66 for developmental promoters compared to 1.23 for constitutive promoters (lower right corner); Wilcoxon rank-sum test ***p = 3.1×10-4, significant. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (D) Boxplots depicting block 7 effects for tested core promoters. Effects of different block 1s were merged in each column (within the same block 7): the median SD is 0.54 for developmental promoters compared to 0.64 for constitutive promoters (lower right corner); Wilcoxon rank-sum test p = 0.3, not significant. Block 7 with column "w/o B7" represents the results obtained from promoters without block 7 sequence. Developmental and constitutive promoters are highlighted in green and red, respectively. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements.


( B - C ) Comparison of normalized expression levels between wild - type configuration and motif knockouts for two types of core promoters ( developmental : CG8157 ( B ) ; constitutive : RpL5 ( C ) ) . Upper panels : schematic depiction of the wild - type motif compositions ( TTGTT motif in RpL5 is ignored due to its strong overlap with R - INR ) . Two - sample t - test : ns , not significant , * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . 3 - 4 biological replicate measurements .

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(B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements.


( A ) Consensus replacement . Heatmap depicting the mean expression fold changes compared to wild - type expressions after replacing with motif consensus sequences derived by XXmotif . Constitutive and developmental promoters are highlighted in red and green , respectively .

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(A) Consensus replacement. Heatmap depicting the mean expression fold changes compared to wild-type expressions after replacing with motif consensus sequences derived by XXmotif. Constitutive and developmental promoters are highlighted in red and green, respectively.


( B ) Expression fold changes ( ecdysone inducibility ) versus measurements without ecdysone and grouped by native core promoter sequences . The colors refer to different core promoter architectures . Three types of line are used to indicate the expression fold change with no increase ( y = x ; solid line ) , 2 - fold increase ( y = x + 1 ; dotted line ) and 4 - fold increase ( y = x + 2 ; dashed line ) . Red vertical dashed line : log2 basal expressions = 2 .

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(B) Expression fold changes (ecdysone inducibility) versus measurements without ecdysone and grouped by native core promoter sequences. The colors refer to different core promoter architectures. Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2.


( C ) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters . Disrupted INR ( highlighted with the black dotted line rectangle ) had a slightly negative effect on changing the core promoter responsiveness to ecdysone . ( ~ 2 . 3 - fold reduction on average , Wilcoxon rank - sum test p = 2 . 1 × 10 - 5 ) . Constitutive and developmental promoters highlighted in red and green , respectively .

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(C) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters. Disrupted INR (highlighted with the black dotted line rectangle) had a slightly negative effect on changing the core promoter responsiveness to ecdysone. (~ 2.3-fold reduction on average, Wilcoxon rank-sum test p = 2.1×10-5). Constitutive and developmental promoters highlighted in red and green, respectively.


( B ) Violin plots displaying RNA - seq readings by FPKM ( normalized Fragments Per Kilobase Million ) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls ( n = 49 , 42 ; unpaired Kolmogorov - Smirnov test ; continuous lines : median ; dashed lines : quartiles ) .

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(B) Violin plots displaying RNA-seq readings by FPKM (normalized Fragments Per Kilobase Million) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls (n = 49,42; unpaired Kolmogorov-Smirnov test; continuous lines: median; dashed lines: quartiles).


( D ) Mean and SEM of mRNA levels of Xbp1s , Bip / Hspa5 and Chop / Ddit3 were determined in dissected hippocampus of i . p . treated mice by quantitative RT - PCR . ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups ) .

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(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).


( E ) Animals received bilateral intra - hippocampal injections of 2 μl tunicamcyin ( 5 mg / mL ) or vehicle for 24 h . Graphs indicate mean and s . e . m . of Xbp1s mRNA levels in the hippocampus ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups .

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(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.


( F - G ) Mean and SEM of relative levels of S - nitrosylated IRE1 ( f ) and total IRE1 ( g ) in brains from young and aged mice . Histograms show relative levels of SNO - IRE1 / input IRE1α measured by biotin - switch assay and total IRE1 / GAPDH quantified by densitometry on standard immunoblots for young ( 3 - month - old ) versus aged ( 24 month - old ) mouse brains ( n = 3 , unpaired Student ' s t test ) . Data information :

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(F-G) Mean and SEM of relative levels of S-nitrosylated IRE1 (f) and total IRE1 (g) in brains from young and aged mice. Histograms show relative levels of SNO-IRE1/input IRE1α measured by biotin-switch assay and total IRE1/GAPDH quantified by densitometry on standard immunoblots for young (3-month-old) versus aged (24 month-old) mouse brains (n = 3, unpaired Student's t test). Data information:


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( C ) Representative photomicrographs of β - galactosidase staining of hippocampal slices derived from young ( 3 months ) , middle - aged ( 12 months ) and aged ( 22 - 24 months ) of IRE1WT or IRE1cKO animals . Magnification : 20x ; scale bar : 200 μm . Arrows indicate β - galactosidase staining .

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Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (C) Representative photomicrographs of β-galactosidase staining of hippocampal slices derived from young (3 months), middle-aged (12 months) and aged (22-24 months) of IRE1WT or IRE1cKO animals. Magnification: 20x; scale bar: 200 μm. Arrows indicate β-galactosidase staining.


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( D ) Mean and SEM of percentage of β - galactosidase staining in CA3 region ( n = 3 - 5 animals / group ) .

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Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (D) Mean and SEM of percentage of β-galactosidase staining in CA3 region (n = 3-5 animals/group).


( F ) Representative confocal images of dendritic spines ( arrows ) in the CA1 region of middle - aged IRE1WT animals injected with AAV - Mock or AAV - Cre . 60x magnification , ( G ) Mean and SEM of spine density per μm ( n = 3 , 3 ; 4 , 4 animals ) . P computed by unpaired Student ' s t test within each age - matched group .

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(F) Representative confocal images of dendritic spines (arrows) in the CA1 region of middle-aged IRE1WT animals injected with AAV-Mock or AAV-Cre. 60x magnification, (G) Mean and SEM of spine density per μm (n = 3, 3; 4, 4 animals). P computed by unpaired Student's t test within each age-matched group.


( E ) Representative images of β - galactosidase staining of hippocampal slices derived from aged ( 22 - 24 months ) TgXBP1s and WT animals . ( F ) Mean and SEM of percentage of area stained by β - galactosidase in the hippocampus of aged ( 20 - 24 months ) TgXBP1s compared to littermate controls .

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(E) Representative images of β-galactosidase staining of hippocampal slices derived from aged (22-24 months) TgXBP1s and WT animals. (F) Mean and SEM of percentage of area stained by β-galactosidase in the hippocampus of aged (20-24 months) TgXBP1s compared to littermate controls.


( D ) Left panel : Representative images showing dendritic spines of aged ( 22 - 24 months ) animals injected with AAV2 - eGFP ( mock ) or with AAV2 - XBP1s ( arrows indicate dendritic spines ) . Young animal dendrite is shown for reference . 60x magnification . Scale bar : 5μm . Right panel : Mean and s . e . m . of spine density per μm for indicated experimental groups ( n = 24 , 53 and 35 dendrites from 3 , 5 and 6 animals ,

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(D) Left panel: Representative images showing dendritic spines of aged (22-24 months) animals injected with AAV2-eGFP (mock) or with AAV2-XBP1s (arrows indicate dendritic spines). Young animal dendrite is shown for reference. 60x magnification. Scale bar: 5μm. Right panel: Mean and s.e.m. of spine density per μm for indicated experimental groups (n = 24, 53 and 35 dendrites from 3, 5 and 6 animals, respectively


( E ) Left panel : Representative images of β - galactosidase staining of hippocampal slices derived from aged mice injected with AAV2 - Mock or AAV2 - XBP1s ( arrow indicate β - galactosidase staining ) . Magnification 20x ; scale bar , 200 μm . Right panel : Histograms show mean and s . e . m . of percentage of β - galactosidase - stained area . n = 6 , 6 ; unpaired Student ' s t test .

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(E) Left panel: Representative images of β-galactosidase staining of hippocampal slices derived from aged mice injected with AAV2-Mock or AAV2-XBP1s (arrow indicate β-galactosidase staining). Magnification 20x; scale bar, 200 μm. Right panel: Histograms show mean and s.e.m. of percentage of β-galactosidase-stained area. n = 6, 6; unpaired Student's t test.