Top 20 protein hits from the ChIRP - MS of Xist FL ; The ranking was based on fold - enrichment of Xist FL DOX versus Xist FL noDOX ; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 ( out of 5 ) were excluded ; Fold - enrichment for Xist ΔB + C is also displayed for comparison ; Light green boxes correspond to proteins as Xist interactors [ 13 ] ; protein in red ( RNF2 / RING1B ) represents a protein not found in the Xist ΔB + C interactome ; Protein in light brown ( TRIM71 ) is less enriched in Xist ΔB + C than in Xist FL .

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

Top 20 protein hits from the ChIRP-MS of Xist FL; The ranking was based on fold-enrichment of Xist FL DOX versus Xist FL noDOX; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 (out of 5) were excluded; Fold-enrichment for Xist ΔB+C is also displayed for comparison; Light green boxes correspond to proteins as Xist interactors [13]; protein in red (RNF2/RING1B) represents a protein not found in the Xist ΔB+C interactome; Protein in light brown (TRIM71) is less enriched in Xist ΔB+C than in Xist FL.


Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB + C for the 74 out of 81 Xist - interactors with a minimum of fold - change of 2 . 5 in Xist FL or Xist ΔB + C ; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions ; proteins retrieved by both Xist FL and Xist ΔB + C ChIRPs with a proposed role in XCI such as SPEN , RBM15 , WTAP , YTHDC1 and hnRNPU are indicated ; light brown dots mark proteins more represented in Xist FL than in Xist ΔB + C ChIRPs , while red dots display proteins which are only retrieved by Xist FL ChIRP .

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

Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB+C for the 74 out of 81 Xist-interactors with a minimum of fold-change of 2.5 in Xist FL or Xist ΔB+C; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions; proteins retrieved by both Xist FL and Xist ΔB+C ChIRPs with a proposed role in XCI such as SPEN, RBM15, WTAP, YTHDC1 and hnRNPU are indicated; light brown dots mark proteins more represented in Xist FL than in Xist ΔB+C ChIRPs, while red dots display proteins which are only retrieved by Xist FL ChIRP.


Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB + C with upon DOX induction at day 2 ( D2 ) of differentiation ; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition ; Black line is a loess regression on all windows ; Xist locus is represented by a blue long line , active genes by green lines .

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

Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB+C with upon DOX induction at day 2 (D2) of differentiation; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition; Black line is a loess regression on all windows; Xist locus is represented by a blue long line, active genes by green lines.


Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions , active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB + C cell lines at D2 upon DOX induction ; Shown is the log2 fold change of DOX vs noDOX conditions ; violin plots represent the distribution of the values , the horizontal band is the median , the lower and upper hinges correspond to the 25th and 75th percentiles ; n = indicates the number of regions / genes analyzed ; p - values were calculated using paired Wilcoxon test , comparing Xist FL and Xist ΔB + C cell lines .

O O O O O O O O O O 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 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

Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions, active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB+C cell lines at D2 upon DOX induction; Shown is the log2 fold change of DOX vs noDOX conditions; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; n = indicates the number of regions/genes analyzed; p-values were calculated using paired Wilcoxon test, comparing Xist FL and Xist ΔB+C cell lines.


Average plots showing the mean enrichment of H3K27me3 ( top ) and H2AK119ub ( bottom ) over all X - linked initially active transcriptional start sites ( TSS ) ; Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB + C cell lines .

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

Average plots showing the mean enrichment of H3K27me3 (top) and H2AK119ub (bottom) over all X-linked initially active transcriptional start sites (TSS); Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB+C cell lines.


Genome browser plots showing H3K27me3 ( top ) and H2AK119ub ( bottom ) enrichments in a region encompassing the inactive Atp1b4 and the initially active Lamp2 genes within the XqA3 . 3 region and the initially active Rlim / Rnf12 gene at the XqD region ; Region around the promoter of Rlim / Rnf12 is highlighted in yellow .

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

Genome browser plots showing H3K27me3 (top) and H2AK119ub (bottom) enrichments in a region encompassing the inactive Atp1b4 and the initially active Lamp2 genes within the XqA3.3 region and the initially active Rlim/Rnf12 gene at the XqD region; Region around the promoter of Rlim/Rnf12 is highlighted in yellow.


Graph represents the mean % + S . E . M . of Xist - coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH ( as represented in B ) at D2 in the presence of DOX ( Xist FL was also used in noDOX conditions ) in the different Xist - TetOP mutants ; each bar represents the mean from to 2 - to - 4 independent experiments ; A minimum of 59 Xist - coated chromosomes were counted per experiment ; For Xist FL noDOX a minimum of 100 cells ( which do not have Xist - coated chromosome ) were counted ; Only p - values corresponding to significant differences comparing mutants ( or Xist FL noDOX ) to Xist FL DOX are indicated as * ( p - value < 0 . 05 ) or * * * ( p - value < 0 . 01 ) , unpaired Student ' s t - test ; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX .

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

Graph represents the mean % + S.E.M. of Xist-coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH (as represented in B) at D2 in the presence of DOX (Xist FL was also used in noDOX conditions) in the different Xist-TetOP mutants; each bar represents the mean from to 2-to-4 independent experiments; A minimum of 59 Xist-coated chromosomes were counted per experiment; For Xist FL noDOX a minimum of 100 cells (which do not have Xist-coated chromosome) were counted; Only p-values corresponding to significant differences comparing mutants (or Xist FL noDOX) to Xist FL DOX are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX.


Representative RNA FISH images for Xist ( red ) and nascent - transcript of Pgk1 ( green ) in Xist - TetOP lines at day 4 of differentiation in the presence of DOX ( Xist FL is also shown in noDOX conditions ) ; DNA stained in blue by DAPI ; Numbers represent % of Xist - coated X - chromosomes ± S . E . M . with active Pgk1 gene ( except for Xist FL noDOX , where numbers represent % of cells with Pgk1 active gene ) ; The values represent 2 - to - 4 independent experiments , where a minimum of 50 Xist - coated chromosomes were counted per experiment ; Significant differences compared with Xist FL ( DOX ) are indicated as * ( p - value < 0 . 05 ) or * * * ( p - value < 0 . 01 ) , unpaired 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 B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Representative RNA FISH images for Xist (red) and nascent-transcript of Pgk1 (green) in Xist-TetOP lines at day 4 of differentiation in the presence of DOX (Xist FL is also shown in noDOX conditions); DNA stained in blue by DAPI; Numbers represent % of Xist-coated X-chromosomes ± S.E.M. with active Pgk1 gene (except for Xist FL noDOX, where numbers represent % of cells with Pgk1 active gene); The values represent 2-to-4 independent experiments, where a minimum of 50 Xist-coated chromosomes were counted per experiment; Significant differences compared with Xist FL (DOX) are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test.


Clustering analysis of the normalized RNA - seq counts on the X chromosome ( chrX ) for all the duplicates of Xist FL , Xist ΔA and Xist ΔB + C in DOX and noDOX conditions .

O O O O O O O O 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

Clustering analysis of the normalized RNA-seq counts on the X chromosome (chrX) for all the duplicates of Xist FL, Xist ΔA and Xist ΔB+C in DOX and noDOX conditions.


Plots displays the log2 ( fold - change ) in the expression of X - linked genes along the chrX comparing DOX versus noDOX samples for Xist FL , Xist ΔA and Xist ΔB + C at day 2 of differentiation ; red dots correspond to genes which are differently expressed in DOX vs noDOX ( p < 0 . 05 , Limma t - test ) , while black dots represent genes which are not differentially expressed between the two conditions ( p ≥ 0 . 05 ) .

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 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

Plots displays the log2(fold-change) in the expression of X-linked genes along the chrX comparing DOX versus noDOX samples for Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; red dots correspond to genes which are differently expressed in DOX vs noDOX (p < 0.05, Limma t-test), while black dots represent genes which are not differentially expressed between the two conditions (p ≥ 0.05).


Violin plots displaying the average log2 ( fold - change ) in gene expression between DOX and noDOX conditions on the chrX in Xist FL , Xist ΔA and Xist ΔB + C at day 2 of differentiation ; violin plots represent the distribution of the values , the horizontal band is the median , the lower and upper hinges correspond to the 25th and 75th percentiles ; p - values were calculated using paired Wilcoxon test ; n = indicates the number of genes analyzed .

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

Violin plots displaying the average log2(fold-change) in gene expression between DOX and noDOX conditions on the chrX in Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; p-values were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.


Box plots displaying the log2 ( DOX / noDOX ) fold - change in expression of X - linked genes in Xist FL and Xist ΔB + C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB + C upon DOX induction ( with no or little accumulation vs accumulation ) ; the horizontal band of the boxplot is the median of the values , the lower and upper hinges correspond to the 25th and 75th percentiles , the upper whisker extends from the hinge to the largest value not further than 1 . 5 interquartile range from the hinge , the lower whisker extends from the hinge to the smallest value at most 1 . 5 interquartile range of the hinge ; p - values between samples were calculated using paired Wilcoxon test ; n = indicates the number of genes analyzed .

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 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

Box plots displaying the log2(DOX/noDOX) fold-change in expression of X-linked genes in Xist FL and Xist ΔB+C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB+C upon DOX induction (with no or little accumulation vs accumulation); the horizontal band of the boxplot is the median of the values, the lower and upper hinges correspond to the 25th and 75th percentiles, the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; p-values between samples were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.


Genome browser plots showing RNA - seq reads , H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL ( left ) and Xist ΔB + C ( right ) at day 2 of differentiation in both DOX and noDOX conditions .

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

Genome browser plots showing RNA-seq reads, H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL (left) and Xist ΔB+C (right) at day 2 of differentiation in both DOX and noDOX conditions.


Boxplots showing differences of the APTT time ( left ) and ME1 - enriched protein expressions ( right ) between mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Mann - Whitney - Wilcoxon test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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

Boxplots showing differences of the APTT time (left) and ME1-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Boxplots showing differences of IL - 6 level , IL - 10 level ( left ) and ME15 - enriched protein expressions ( right ) between mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Mann - Whitney - Wilcoxon test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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

Boxplots showing differences of IL-6 level, IL-10 level (left) and ME15-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Rose plots indicating the number of all detected and differently expressed tissues - enhanced proteins in mild ( left ) and severe patients ( right ) compared with healthy controls . Tissues labeled in orange represented tissues with the largest proportions ( 100 % of all detected tissue - enhanced proteins ) of altered tissues - enhanced proteins .

Rose plots indicating the number of all detected and differently expressed tissues-enhanced proteins in mild (left) and severe patients (right) compared with healthy controls. Tissues labeled in orange represented tissues with the largest proportions (100% of all detected tissue-enhanced proteins) of altered tissues-enhanced proteins.


Boxplots indicating the expression level of known brain dysfunctional biomarkers in control ( n = 12 ) , mild ( n = 13 ) and severe ( n = 18 ) patients . Differences between groups were estimated using Kruskal - Wallis test . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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

Boxplots indicating the expression level of known brain dysfunctional biomarkers in control (n=12), mild (n=13) and severe (n=18) patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Boxplot showing the selected cell type specific proteins among control ( n = 12 ) , mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Kruskal - Wallis test . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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

Boxplot showing the selected cell type specific proteins among control (n=12), mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Heatmap of IFNG , GZMB and PRF1 gene expression in COVID - 19 patients . Average expression values were centered and scaled . Red indicates a higher expression and blue indicates a lower expression .

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

Heatmap of IFNG, GZMB and PRF1 gene expression in COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.


Heatmap of the expression of genes enriched in IL - 17 signaling pathway between healthy control and COVID - 19 patients . Average expression values were centered and scaled . Red indicates a higher expression and blue indicates a lower expression .

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

Heatmap of the expression of genes enriched in IL-17 signaling pathway between healthy control and COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.


COVID - 19 severity is associated with significant changes in lipoprotein subclasses including high - density lipoprotein subclass - 1 ( HDL1 ) , HDL4 , low - density lipoprotein subclasses ( LDL1 , LDL4 , LDL5 ) , very low - density lipoprotein subclass - 5 ( VLDL5 ) and their compositional components ( ApoA1 , triglycerides , cholesterol ) . TG : triglycerides ; FC : free cholesterol ; CE : cholesteryl esters ; CH : total cholesterol ( i . e . , FC + CE ) ; PL : total phospholipids ; A1 : ApoA1 ; A2 : ApoA2 ; L1TG : TG in LDL1 ; L1TG % : percentages of L1TG in total lipids of LDL1 ; L1 % : percentage of LDL1 in all LDL ; L - TG % : percentages of L - TG ( TG in LDL ) in total lipids of LDL ; V5FC % , V5CE % : percentages of V5FC ( FC in VLDL5 ) and V5CE ( CE in VLDL5 ) in total lipids of VLDL5 ; L5CE % , L5CH % : percentages of L5CE ( CE in LDL5 ) and L5CH ( CH in LDL5 ) in total lipids of LDL5 ; H1FC % : percentages of H1FC ( FC in HDL1 ) in total lipids of HDL1 ; H - A2 : ApoA2 in both HDL and nascent HDL ; H4A1 , H4A2 , H4CE , H4CH , H4FC , H4PL : ApoA1 , ApoA2 , CE , CH , FC and PL in HDL4 ;

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COVID-19 severity is associated with significant changes in lipoprotein subclasses including high-density lipoprotein subclass-1 (HDL1), HDL4, low-density lipoprotein subclasses (LDL1, LDL4, LDL5), very low-density lipoprotein subclass-5 (VLDL5) and their compositional components (ApoA1, triglycerides, cholesterol). TG: triglycerides; FC: free cholesterol; CE: cholesteryl esters; CH: total cholesterol (i.e., FC + CE); PL: total phospholipids; A1: ApoA1; A2: ApoA2; L1TG: TG in LDL1; L1TG%: percentages of L1TG in total lipids of LDL1; L1%: percentage of LDL1 in all LDL; L-TG%: percentages of L-TG(TG in LDL) in total lipids of LDL; V5FC%, V5CE%: percentages of V5FC(FC in VLDL5) and V5CE(CE in VLDL5) in total lipids of VLDL5; L5CE%, L5CH%: percentages of L5CE(CE in LDL5) and L5CH(CH in LDL5) in total lipids of LDL5; H1FC%: percentages of H1FC (FC in HDL1) in total lipids of HDL1; H-A2: ApoA2 in both HDL and nascent HDL; H4A1, H4A2, H4CE, H4CH, H4FC, H4PL: ApoA1, ApoA2, CE, CH, FC and PL in HDL4;


Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID - 19 severity . Data were represented as means ± SD and differences between groups were estimated using a Student ' s t test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Control , n = 12 ; mild , n = 29 ; severe , n = 17 ; discharge , n = 16 ; sLDLR : soluble low - density lipoprotein receptor ; LCAT : lecithin - cholesterol acyltransferase ; CEPT : cholesteryl - ester transfer protein .

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

Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID-19 severity. Data were represented as means ± SD and differences between groups were estimated using a Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Control, n=12; mild, n=29; severe, n=17; discharge, n=16; sLDLR: soluble low-density lipoprotein receptor; LCAT: lecithin-cholesterol acyltransferase; CEPT: cholesteryl-ester transfer protein.


TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress . ( A ) ROS levels in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) left untreated , after 6 h of nutrient starvation or 24 h of metabolic stress . 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 .

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

TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. 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.


TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress . ( B ) Basal , nutrient starvation - induced ( 5 h ) or metabolic stress - induced ( 18 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , 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 ) .

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

TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.(B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, 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).


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( A ) ( Left panel ) confocal microscopy images of the fluorescence in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clone TIGAR # 7 ) or control cells ( clone Cont # 1 ) and infected with an adenovirus expressing GFP - LC3 for 16 h . Cells were then left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . ( Right panel ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta from three independent experiments . The mean values with standard deviation are presented .

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 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

TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( B ) ( Left panel ) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP - LC3 and transfected with scrambled or TIGAR siRNAs . After 48 h transfection , cells were then left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h . ( Right panel ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta from three independent experiments . The mean values with standard deviation are presented .

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 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

TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(B) (Left panel) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( C ) ( Left panel ) Western blot showing the expression levels of endogenous LC3 - I , LC3 - II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs , and 48 h later exposed to nutrient starvation for 0 , 2 . 5 and 6 h . ( Middle panel ) Western blot showing the expression levels of endogenous LC3 - I , LC3 - II and TIGAR in U2OS stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) and left untreated . ( Right panel ) Western blot showing the expression levels of p62 , COX - IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated . Actin expression was examined as a loading control .

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

TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control.


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( D ) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0 , 1 , 3 , 5 and 8 h ; * 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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TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-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

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 .

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

(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 .

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

(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 ) .

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

(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 .

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

(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 .

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 B-CONTROLLED_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

(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 .

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

(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 .

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

(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 ) .

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

(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 .

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


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

(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 .

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

(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 ) .

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 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

(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 .

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

(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 .

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

(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 ) .

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

(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 .

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

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

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 ) .

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

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 )

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

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

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 .

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

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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ' ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

Quantification of 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 .

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 .

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 ) .

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

J-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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

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 .

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

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

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

(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 .

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

(A) Representative 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 .

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

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

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

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

(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 .

(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 .

(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 .

(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 .

(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 .

(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.