Figure 3 ( A ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the TAL1 locus ( hg38 ; chr1 : 47 , 194 , 268 - 47 , 242 , 880 ; ( Kent et al . , 2002 ) ) in the leukemia cell lines Jurkat and K562 . For better visualization , TT - seq coverage is cut at 200 ( purple lines ) . eRNAs are shown between the plus and minus strand . Red line indicates the short 12 bp insertion in Jurkat cells , introducing novel binding sites for the transcription factor MYB ( B ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the 2 . 8 Mbp gene desert surrounding MYC ( hg38 ; chr8 : 127 , 042 , 406 - 129 , 779 , 109 ; ( Kent et al . , 2002 ) ) in fourteen cancer cell lines . eRNAs represent the union from all cell lines . Genome - wide association studies ( GWAS ) risk loci contain GWAS single nucleotide polymorphisms ( SNPs ) for prostate cancer ( PrCa ) , acute lymphocytic leukemia ( ALL ) , breast cancer ( BrCa ) , colorectal cancer ( CRC ) , and glioblastoma ( GBM ) including SNPs in linkage disequilibrium ( r2 ≥ 0 . 8 ) . TT - seq coverage is displayed between 8 - 250 to allow for better visualization of the eRNA signal surrounding MYC . Dashed rectangle is shown at full scale on the right . Note : TT - seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements ( e . g . translocations ) affecting the shown region in the used cancer cell line clones .

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

Figure 3(A) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the TAL1 locus (hg38; chr1:47,194,268-47,242,880; (Kent et al., 2002)) in the leukemia cell lines Jurkat and K562. For better visualization, TT-seq coverage is cut at 200 (purple lines). eRNAs are shown between the plus and minus strand. Red line indicates the short 12 bp insertion in Jurkat cells, introducing novel binding sites for the transcription factor MYB(B) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.


Figure 4 ( A ) Med1 - defined enhancers ranked by Med1 ChIP - seq signal ( Yan et al . , 2013 ) in LoVo colorectal cancer cells . Super - enhancers ( black rectangles ) were determined as enhancer regions lying above the inflection point of the curve ( B ) Increased eRNA synthesis from super - enhancers . Boxplots comparing eRNA synthesis ( TT - seq signal ) at Med1 - defined typical enhancers ( n = 2 , 111 ) and super - enhancers ( n = 130 ) in LoVo cells ( analysis was restricted to intergenic enhancers Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown . P - value by two - sided Mann - Whitney U - test . ( C ) Super - enhancers are associated with the production of long eRNAs . Boxplots showing length of TT - seq - defined eRNAs originating from Med1 - defined super - enhancers ( n = 552 ) or other enhancer regions ( n = 6 , 934 ) . Median , hinges , whiskers and notches are shown as in ( B ) . P - value by two - sided Mann - Whitney U - test . ( D ) Barplot showing the number of TT - seq enhancer regions per Med1 - defined super - enhancer region in LoVo cells . Super - enhancers spanning a protein - coding TSS ( n = 32 ) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT - seq enhancer calling . ( E ) UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the PHLDA1 locus ( hg38 ; chr12 : 75 , 612 , 350 - 76 , 042 , 552 ; in the colorectal cancer cell line HCT 116 . TT - seq coverage is cut at 250 ( purple lines ) to allow for better visualization . eRNAs are shown between the plus and minus strand . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) comprise ChIP - seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region . Binding regions for selected TFs ( TCF7l2 , YY1 , SP1 ) are shown below together with p300 and RNA polymerase II ( Pol II ) . TT - seq enhancer regions are shown at the bottom together with a H3K27ac - defined super - enhancer annotation , which was downloaded from SEdb 2019 ) . At date of publication no Med1 ChIP - seq data was available for HCT 116 .

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

Figure 4(A) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve(B) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.(C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test.(D) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling.(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.


Figure 5 ( A ) Unsupervised clustering of all pairwise Spearman correlations of normalized TT - seq signal for mRNAs ( left ) and eRNAs ( right ) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type , respectively . ( B ) Heatmaps showing mRNAs ( left ) and eRNAs ( right ) as being synthesized ( red , normalized TT - seq signal FPK ≥ 15 ) or not ( blue , normalized TT - seq signal FPK < 15 ) for all fourteen cancer cell lines ( columns ) . Heatmaps show only transcripts ( rows ) with normalized FPK ≥ 30 in at least one of the cell lines ( mRNAs , n = 14 , 040 ; eRNAs n = 48 , 105 ) . Heatmaps are ordered by increasing cell type specificity , as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in . ( D ) Boxplots showing normalized TT - seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed ( from left to right : n = 890 ; 500 ; 494 ; 214 ; 238 ; 239 ; 378 ; 62 ; 153 ; 879 ; 586 ; 173 ; 601 ; 232 ) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed . All pairwise comparisons are significantly different ( P - values < 1e - 9 , two - sided Mann - Whitney U - test ) . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Notches represent 95 % confidence intervals for the median values . Outliers not shown .

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

Figure 5(A) Unsupervised clustering of all pairwise Spearman correlations of normalized TT-seq signal for mRNAs (left) and eRNAs (right) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type, respectively.(B) Heatmaps showing mRNAs (left) and eRNAs (right) as being synthesized (red, normalized TT-seq signal FPK ≥ 15) or not (blue, normalized TT-seq signal FPK < 15) for all fourteen cancer cell lines (columns). Heatmaps show only transcripts (rows) with normalized FPK ≥ 30 in at least one of the cell lines (mRNAs, n=14,040; eRNAs n=48,105). Heatmaps are ordered by increasing cell type specificity, as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in.(D) Boxplots showing normalized TT-seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed (from left to right: n=890; 500; 494; 214; 238; 239; 378; 62; 153; 879; 586; 173; 601; 232) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed. All pairwise comparisons are significantly different (P-values < 1e-9, two-sided Mann-Whitney U-test). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown.


Figure 7UCSC genome browser view of normalized TT - seq coverage on the plus and minus strand at the BMP4 locus ( hg38 ; chr14 : 53 , 935 , 697 - 54 , 264 , 430 ; in LoVo colorectal cancer cells . TT - seq coverage is cut at 500 ( purple lines ) to allow for better visualization . eRNAs and enhancers are shown above and below the minus strand , respectively . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) include ChIP - seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region . Significant promoter capture Hi - C ( CHi - C ) interactions are shown in

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

Figure 7UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; in LoVo colorectal cancer cells. TT-seq coverage is cut at 500 (purple lines) to allow for better visualization. eRNAs and enhancers are shown above and below the minus strand, respectively. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region. Significant promoter capture Hi-C (CHi-C) interactions are shown in purple


Figure 1Representative microscopic views of Ndrg4 + / + and Ndrg4 - / - murine intestinal sections ( n = 4 , 12 months of age ) reveal an even distribution of alkaline phosphatase along the enterocyte brush border , and a similar number and distribution of Paneth cells ( Lysozyme ) in the small intestine ; and neuroendocrine ( Chromogranin A ) , Goblet ( PAS + ) , and proliferating ( Ki67 ) cells in the colon . Scale bars , 50 µm . Representative microscopic views of the myenteric plexus of Ndrg4 + / + and Ndrg4 - / - murine colonic sections labeled with either TuJ1 and S100 ( B , n = 3 ) or HuC / D ( C , n = 6 ) did not reveal structural or organizational differences in the ganglionic network of Ndrg4 + / + and Ndrg4 - / - mice . Scale bars , 100 µm . Quantification of the enteric neuronal cell number ( HuC / D ) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4 + / + and Ndrg4 - / - mice ( n = 6 ) .

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

Figure 1Representative microscopic views of Ndrg4+/+ and Ndrg4-/- murine intestinal sections (n=4, 12 months of age) reveal an even distribution of alkaline phosphatase along the enterocyte brush border, and a similar number and distribution of Paneth cells (Lysozyme) in the small intestine; and neuroendocrine (Chromogranin A), Goblet (PAS+), and proliferating (Ki67) cells in the colon. Scale bars, 50 µm.Representative microscopic views of the myenteric plexus of Ndrg4+/+ and Ndrg4-/- murine colonic sections labeled with either TuJ1 and S100 (B, n=3) or HuC/D (C, n=6) did not reveal structural or organizational differences in the ganglionic network of Ndrg4+/+ and Ndrg4-/- mice. Scale bars, 100 µm. Quantification of the enteric neuronal cell number (HuC/D) shows a similar number of enteric neurons in the proximal and distal colon of Ndrg4+/+ and Ndrg4-/- mice (n=6).


Figure 2In contrast , Ndrg4fl / fl and Ndrg4fl / fl - VillinCre mice have a similar bodyweight ( I ) and develop an equal number of adenomas ( J ) with an equivalent diameter ( K , L ) . N = 13 vs . 12 in I , n = 13 vs . 11 in J and n = 10 vs . 8 in K .

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Figure 2In contrast, Ndrg4fl/fl and Ndrg4fl/fl-VillinCre mice have a similar bodyweight (I) and develop an equal number of adenomas (J) with an equivalent diameter (K,L). N=13 vs. 12 in I, n=13 vs. 11 in J and n=10 vs. 8 in K.


Figure 3Compared to Ndrg4 + / + ENS - derived medium , addition of medium derived from Ndrg4 - / - ENS cultures enhances the relative growth of intestinal epithelial organoids ( E , IEOs ) and accelerates the formation of new crypt buds per IEO in the course of five days ( F ) .

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Figure 3Compared to Ndrg4+/+ ENS-derived medium, addition of medium derived from Ndrg4-/- ENS cultures enhances the relative growth of intestinal epithelial organoids (E, IEOs) and accelerates the formation of new crypt buds per IEO in the course of five days (F).


Figure 4Box plot showing the quantitative protein expression results of nanoLC - MS / MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4 - / - compared to the Ndrg4 + / + ENS cell secretome ( n = 4 ; NSAF , normalized spectral abundance factor ) . Data are analyzed with R version 3 . 5 . 2 and the ibb R package . Each dot within the box plot represents the NSAF of an individual sample ; the inside band reflects the median , and the bottom and top of the box the first and third quartile , respectively . The whiskers reflect the minimum and maximal values within 1 . 5 × the interquartile range . NSAF values were compared using the Mann - Whitney U test . Representative immunohistochemistry ( C ) and more detailed immunofluorescence ( D , E ) labeling indicate that Nid1 ( C , D ) and Fbln2 ( C , E ) are expressed within the myenteric plexus and by primary ENS - cells ( ( C ) , black arrowheads , scale bars , 50 µm ; ( D , E ) , n = 3 , scale bars , 10 µm ) .

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

Figure 4Box plot showing the quantitative protein expression results of nanoLC-MS/MS analysis reveals the significantly higher presence of Nid1 and Fbln2 in the Ndrg4-/- compared to the Ndrg4+/+ ENS cell secretome (n=4; NSAF, normalized spectral abundance factor). Data are analyzed with R version 3.5.2 and the ibb R package. Each dot within the box plot represents the NSAF of an individual sample; the inside band reflects the median, and the bottom and top of the box the first and third quartile, respectively. The whiskers reflect the minimum and maximal values within 1.5× the interquartile range. NSAF values were compared using the Mann-Whitney U test.Representative immunohistochemistry (C) and more detailed immunofluorescence (D, E) labeling indicate that Nid1 (C, D) and Fbln2 (C, E) are expressed within the myenteric plexus and by primary ENS-cells ((C), black arrowheads, scale bars, 50 µm; (D, E), n=3, scale bars, 10 µm).


Figure 5Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia ( Black arrowheads ) and interconnecting nerve fibers , and in a limited level throughout the epithelium ( n = 3 ) . Scale bar , 50 µm . Addition of NID1 & FBLN2 to the Matrigel ® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition . Box plot reflecting the quantitative nanoLC - MS / MS analysis results shows the up - regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome ( n = 17 ) compared to the normal colon tissue secretome ( n = 17 ) . Data are analyzed with R version 3 . 5 . 2 and the ibb R package . Within the box plots , each dot represents the log10 transformed raw count of an individual sample ; the inside band is the median value ; the bottom and top of the box the first and third quartile , respectively ; and the whiskers reflect the minimum and maximal values within 1 . 5 × the interquartile range . Representative images of human colonic cancer epithelium ( n = 3 ) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium . Scale bar , 50 μm .

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

Figure 5Representative microscopic views show that NID1 and FBLN2 are highly expressed in the human colonic ganglia (Black arrowheads) and interconnecting nerve fibers, and in a limited level throughout the epithelium (n=3). Scale bar, 50 µm.Addition of NID1&FBLN2 to the Matrigel® dome significantly enhances the relative growth rate of HIOs after five days of culture as compared to the PBS control condition.Box plot reflecting the quantitative nanoLC-MS/MS analysis results shows the up-regulation of NID1 and FBLN2 in the colorectal cancer tissue secretome (n=17) compared to the normal colon tissue secretome (n=17). Data are analyzed with R version 3.5.2 and the ibb R package. Within the box plots, each dot represents the log10 transformed raw count of an individual sample; the inside band is the median value; the bottom and top of the box the first and third quartile, respectively; and the whiskers reflect the minimum and maximal values within 1.5× the interquartile range.Representative images of human colonic cancer epithelium (n=3) shows a high scattered expression of NID1 and FBLN2 within the cancerous epithelium. Scale bar, 50 μm.


Figure 1 ( A , B ) t - SNE visualization of 20 , 009 human retinal cells colored by ( A ) annotation of 18 transcriptionally distinct clusters ( C0 - C17 ) and ( B ) their distribution in 3 donor retina samples . C ) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters . The size of each circle depicts the percentage of cells expressing the marker within the cluster . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) . D ) t - SNE plots showing expression of a set of selected marker genes for major retinal classes . E ) Correlation matrix for the identified 18 clusters . The upper triangle depicts the z - value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters . F ) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters . The rows correspond to top 10 genes most selectively upregulated in individual clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) and the columns show individual cells ordered by cluster ( C0 - C17 ) .

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

Figure 1 (A, B) t-SNE visualization of 20,009 human retinal cells colored by (A) annotation of 18 transcriptionally distinct clusters (C0-C17) and (B) their distribution in 3 donor retina samples.   C) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥10 nTrans (number of transcripts).  D) t-SNE plots showing expression of a set of selected marker genes for major retinal classes. E) Correlation matrix for the identified 18 clusters. The upper triangle depicts the z-value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters.  F) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered by cluster (C0-C17).


Figure 2 A ) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters . Brown colour indicates ≥ 100 nTrans ( number of transcripts ) . B ) Representation of the three donor retina samples in the six rod photoreceptor clusters . C ) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters . D ) Distribution of rod photoreceptor populations with high MALAT1 expression ( MALAT1 - hi ) or low MALAT1 expression ( MALAT1 - lo ) in three donor retina samples . E ) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer ( ONL ) . INL : inner nuclear layer ; OPL : outer plexiform layer . Scale bar = 20µm .

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Figure 2 A) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters. Brown colour indicates ≥100 nTrans (number of transcripts).  B) Representation of the three donor retina samples in the six rod photoreceptor clusters.  C) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters.  D) Distribution of rod photoreceptor populations with high MALAT1 expression (MALAT1-hi) or low MALAT1 expression (MALAT1-lo) in three donor retina samples.  E) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer (ONL). INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm.


Figure 3 A , B ) ( A ) t - SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters ( CCA0 - CCA12 ) and ( B ) their distribution in 3 donor retina samples . C ) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters . The size of each circle depicts the percentage of cells expressing the marker within the cluster . Brown colour indicates ≥ 50 nTrans ( number of transcripts ) . D ) t - SNE plots showing expression of MALAT1 . E ) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1 - hi ( CCA1 , CCA10 ) and MALAT1 - lo ( CCA0 ) subpopulations . x - axis depicts normalized transcript levels . F ) t - SNE plots showing expression of major ribosomal genes . G ) Heatmap of differentially expressed genes between the two MALAT1 - hi clusters CCA1 and CCA10 . The rows correspond to top 10 genes most selectively upregulated in individual clusters ( p < 0 . 01 , Benjamini - Hochberg correction ) and the columns show individual cells ordered in CCA1 and CCA10 .

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Figure 3 A, B) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples.  C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts).  D) t-SNE plots showing expression of MALAT1.  E) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels.  F) t-SNE plots showing expression of major ribosomal genes.  G) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.


Figure 4 Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post - mortem ( 7 and 13 hours , Retina 7 ) , showing decreases in MALAT1 - hi rod subpopulations in the outer nuclear layer ( ONL ) at later time point . INL : inner nuclear layer ; OPL : outer plexiform layer . Scale bar = 20µm . White arrows indicated MALAT1 - hi rod photoreceptors .

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Figure 4 Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.


Figure 5 A ) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters . Brown colour indicates ≥ 10 nTrans ( number of transcripts ) . C ) Scatter plots showing expression of OPN1LW / OPN1MW or OPN1SW in individual cone photoreceptors for population C10 . The colour depicts expression level for OPN1LW / OPN1MW in individual cells . D ) Heatmap of top 20 differentially expressed genes between L / M cones and S cones . The colour depicts normalised gene expression ( z - score capped at 2 . 5 ) . E ) Expression pattern of glial markers in Muller glia ( C9 ) and retinal astrocytes ( C16 ) . x - axis depicts normalized transcript levels .

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Figure 5 A) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts).  C) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells.  D) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5).  E) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.


Figure 6C ) t - SNE analysis of the human Müller glia cell line MIO - M1 with the retinal cell types identified in this human neural retina atlas .

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Figure 6C) t-SNE analysis of the human Müller glia cell line MIO-M1 with the retinal cell types identified in this human neural retina atlas.


Figure 1A ) GST pulldown used to analyse TBC1D14 interactors . Recombinant GST or GST - TBC1D14 was incubated with or without HEK 293A lysate . Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS - PAGE , with visible bands being excised from the gel and analysed by mass spectrometry . B ) Lysates of HEK293A cells expressing either GFP or GFP - TBC1D14 were subjected to immunoprecipitation ( IP ) with GFP - Trap , and then immunoblotted for GFP , TRAPPC12 and TRAPPC4 . C ) Lysates of cells expressing GFP or GFP - TRAPPC3 were subjected to IP with GFP - Trap , and then immunoblotted for TRAPPC4 , GFP and TBC1D14 . D ) Cells expressing GFP - TBC1D14 , generating a tubulated endosomal compartment , were fed Alexa647 labelled Transferrin ( white , blue in merge ) for 15 minutes in full medium , fixed , stained for endogenous TRAPPC4 ( red ) and analysed by confocal microscopy . White arrowheads indicate transfected cells , yellow arrowheads depict regions of colocalisation in inset . Scale bars = 10 μm . E ) Cells treated as in D were stained for endogenous TRAPPC12 ( red ) and analysed by confocal microscopy . Scale bars = 10 μmF ) Cells in complete medium were fixed , stained for endogenous TBC1D14 ( green ) , RAB1B ( red ) and TRAPPC4 ( white , blue in merge ) and analysed by confocal microscopy . Inset shows protein localisation in juxtanuclear area . Yellow arrowheads show regions of colocalisation Scale bars = 10 μm . Data shown are representative from 3 independent experiments .

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Figure 1A) GST pulldown used to analyse TBC1D14 interactors. Recombinant GST or GST-TBC1D14 was incubated with or without HEK 293A lysate. Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS-PAGE, with visible bands being excised from the gel and analysed by mass spectrometry.B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4.C) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14.D) Cells expressing GFP-TBC1D14, generating a tubulated endosomal compartment, were fed Alexa647 labelled Transferrin (white, blue in merge) for 15 minutes in full medium, fixed, stained for endogenous TRAPPC4 (red) and analysed by confocal microscopy. White arrowheads indicate transfected cells, yellow arrowheads depict regions of colocalisation in inset. Scale bars = 10 μm.E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μmF) Cells in complete medium were fixed, stained for endogenous TBC1D14 (green), RAB1B (red) and TRAPPC4 (white, blue in merge) and analysed by confocal microscopy. Inset shows protein localisation in juxtanuclear area. Yellow arrowheads show regions of colocalisation Scale bars = 10 μm. Data shown are representative from 3 independent experiments.


Figure 2A ) HEK293A cells transfected with GFP ( upper panels ) or GFP - TBC1D14 ( lower panels ) ( green ) were labelled with anti - RAB11A ( red ) and anti - RAB1B ( white , blue in merge ) antibodies . Arrows depict transfected cells . Inset : GFP - TBC1D14 transfected cell showing triple co - localisation between GFP - TBC1D14 , RAB11A and RAB1B ( white arrows ) and double co - localisation between GFP - TBC1D14 and RAB11A ( yellow arrows ) . B ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB11A ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation . C ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB1B ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation , yellow arrowheads regions of double co - localisation between GFP - TBC1D14 and TRAPPC4 . All scale bars = 20 μm .

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Figure 2A) HEK293A cells transfected with GFP (upper panels) or GFP-TBC1D14 (lower panels) (green) were labelled with anti-RAB11A (red) and anti-RAB1B (white, blue in merge) antibodies. Arrows depict transfected cells. Inset: GFP-TBC1D14 transfected cell showing triple co-localisation between GFP-TBC1D14, RAB11A and RAB1B (white arrows) and double co-localisation between GFP-TBC1D14 and RAB11A (yellow arrows).B) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB11A (white, blue in merge). White arrowheads indicate regions of triple co-localisation.C) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB1B (white, blue in merge). White arrowheads indicate regions of triple co-localisation, yellow arrowheads regions of double co-localisation between GFP-TBC1D14 and TRAPPC4. All scale bars = 20 μm.


Figure 3B ) HEK 293A cell lysates ( from approximately 8 × 106 cells per construct ) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP , TRAPPC12 and TRAPPC4 . Data are representative of 3 independent experiments .

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Figure 3B) HEK 293A cell lysates (from approximately 8 × 106 cells per construct) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP, TRAPPC12 and TRAPPC4. Data are representative of 3 independent experiments.


Figure 4A ) Cells expressing the indicated GFP - fusions ( green ) were loaded with Alexa647 - Transferrin ( red ; arrowheads indicate transfected cells ) for 15 minutes , fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy . Scale bars = 10 μm . Arrows in inset indicate the juxtanuclear ERCB ) Cells transfected as in ( A ) were loaded with Alexa - 647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods . At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry . Results are expressed as a percentage of Tfn fluorescence at t = 0 and are the mean of 3 independent experiments , ± s . e . m . C ) Cells transfected as in ( A ) were stained for the cis - Golgi marker GM130 . Arrowheads depict transfected cells . Scale bars = 10 μm . Bar chart is quantification of fragmented Golgi complex ( > 100 cells per condition from three independent experiments , ± s . e . m . * * p < 0 . 01 , one way ANOVA with Sidak ' s post - test ) . D ) HeLa C1 cells transfected with CFP or CFP - TBR were treated with D / D solubiliser ( Clontech ) or D / D solubiliser plus 2 . 5 μg / ml Brefeldin A ( BFA ) for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . Results are expressed as a percentage of GFP levels at t = 0 , and the mean of 3 independent experiments ± s . e . m .

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Figure 4A) Cells expressing the indicated GFP-fusions (green) were loaded with Alexa647-Transferrin (red; arrowheads indicate transfected cells) for 15 minutes, fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy. Scale bars = 10 μm. Arrows in inset indicate the juxtanuclear ERCB) Cells transfected as in (A) were loaded with Alexa-647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods. At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry. Results are expressed as a percentage of Tfn fluorescence at t=0 and are the mean of 3 independent experiments, ± s.e.m.C) Cells transfected as in (A) were stained for the cis-Golgi marker GM130. Arrowheads depict transfected cells. Scale bars = 10 μm. Bar chart is quantification of fragmented Golgi complex (> 100 cells per condition from three independent experiments, ± s.e.m. ** p < 0.01, one way ANOVA with Sidak's post-test).D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.


Figure 5A ) HEK293A cells expressing GFP , GFP - TBC1D14 224 - 669 or GFP - TBR were treated in duplicate with EBSS , EBSS plus 100 nM BafA1 or not for two hours , lysed and subjected to immunoblotting for LC3B , tubulin and GFP . The amount of LC3B / tubulin for each condition from three independent experiments is shown on the bar graph , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test . B ) HEK293A GFP - LC3B ( 2GL9 ) cells were transfected with mCherry or mCherry - TBR , treated with EBSS for two hours , fixed and analysed by confocal microscopy . C ) HEK293A cells were treated as in ( B ) , fixed , stained for WIPI2 and analysed by confocal microscopy . D ) HEK293A cells stably expressing GFP - DFCP1 were treated as in ( B ) , fixed and analysed by confocal microscopy . Scale bars = 20 μm . For quantification of ( B ) , ( C ) and ( D ) , 10 fields of view containing transfected cells were imaged for each of three experiments , and the number of GFP - LC3B , WIPI2 or GFP - DFCP1 puncta per cell enumerated using Imaris software ( Bitplane ) .

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Figure 5A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.B) HEK293A GFP-LC3B (2GL9) cells were transfected with mCherry or mCherry-TBR, treated with EBSS for two hours, fixed and analysed by confocal microscopy.C) HEK293A cells were treated as in (B), fixed, stained for WIPI2 and analysed by confocal microscopy.D) HEK293A cells stably expressing GFP-DFCP1 were treated as in (B), fixed and analysed by confocal microscopy. Scale bars = 20 μm. For quantification of (B), (C) and (D), 10 fields of view containing transfected cells were imaged for each of three experiments, and the number of GFP-LC3B, WIPI2 or GFP-DFCP1 puncta per cell enumerated using Imaris software (Bitplane).


Figure 6B ) Samples prepared according to ( A ) ( 8 × 107 HEK293A cells per transfection ) were lysed in TNTE , split equally and denatured with 1 % SDS ( D ) or not ( ND ) and subjected to 1 hour streptavidin resin pulldown at room temperature . The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins . C ) siRNA transfected control cells ( RISC Free ) or TRAPPC8 knockdown ( TRAPPC8 ) ( 1x10cm plate per condition ) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST - TBR . Bound proteins were subjected to immunoblot analysis using TRAPPC8 , TRAPPC4 and tubulin antibodies . Ponceau staining was used to verify loading of the GST fusion proteins ( GST - TBR indicated with arrowhead ) . The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments , error bars are ± s . e . m . * * p < 0 . 01 , unpaired T - testD ) HEK293T cells stably depleted of TRAPPC8 ( shC8 ) or not ( shCtrl ) were transfected with constructs encoding GFP or GFP - TRAPPC3 and subjected to GFP Trap IP . Precipitated proteins were immunoblotted for GFP , TRAPPC4 and TRAPPC8 . Bar chart : the amount of TRAPPC4 isolated was normalised to the amount of GFP - TRAPPC3 precipitated from three independent experiments and plotted ± s . e . m . , ns = non - significant , unpaired T - test .

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Figure 6B) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins.C) siRNA transfected control cells (RISC Free) or TRAPPC8 knockdown (TRAPPC8) (1x10cm plate per condition) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST-TBR. Bound proteins were subjected to immunoblot analysis using TRAPPC8, TRAPPC4 and tubulin antibodies. Ponceau staining was used to verify loading of the GST fusion proteins (GST-TBR indicated with arrowhead). The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments, error bars are ± s.e.m. ** p<0.01, unpaired T-testD) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.


Figure 7A ) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B ( knockdown confirmed by immunoblot analysis ) were fed with Alexa - 647 Transferrin for 15 minutes in full medium , and the fluorescent transferrin chased out for the indicated time periods . At the end of the time course , cells were trypsinised , fixed and the Alexa - 647 fluorescence analysed by flow cytometry . Results are plotted as percentage of transferrin fluorescence at t = 0 for the RISC free control cells , expressed as the mean of three independent experiments ± s . e . m . B ) Cells transiently transfected with RISC Free control siRNA , or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis - Golgi markers GM130 or RAB1B and analysed by confocal microscopy . Scale bars = 20 μm in main panels , 10 μm in inset panels . Bar graph : > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments . Error bars ± s . e . m . , * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test . C ) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B ( knockdown confirmed by immunoblot analysis ) , were treated with D / D solubiliser for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . The line graph shows the GFP fluorescence as a percentage of t = 0 , ± s . e . m . from three independent experiments .

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Figure 7A) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B (knockdown confirmed by immunoblot analysis) were fed with Alexa-647 Transferrin for 15 minutes in full medium, and the fluorescent transferrin chased out for the indicated time periods. At the end of the time course, cells were trypsinised, fixed and the Alexa-647 fluorescence analysed by flow cytometry. Results are plotted as percentage of transferrin fluorescence at t=0 for the RISC free control cells, expressed as the mean of three independent experiments ± s.e.m.B) Cells transiently transfected with RISC Free control siRNA, or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis-Golgi markers GM130 or RAB1B and analysed by confocal microscopy. Scale bars = 20 μm in main panels, 10 μm in inset panels. Bar graph: > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments. Error bars ± s.e.m., * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.


Figure 8A ) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed ( F ) or starved ( S ) HEK293A cells using a monoclonal hamster anti - ATG9 ( ATG9 ) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody , TRAPPC8 , TRAPPC4 and actin . B ) HEK293A cells transfected with GFP or GFP - TBR ( green ) were stained for ATG9 using hamster anti - ATG9 ( red ) and analysed by confocal microscopy . Scale bar = 20 μmC ) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B ( green ) and ATG9 using hamster anti - ATG9 ( red ) and imaged using confocal microscopy . D ) Stills at t = 30 minutes from time lapse imaging of 9B9 cells ( stably expressing GFP - LC3 and mRFP - ATG9 ) expressing either CFP or CFP - TBR ( blue ) . Scale bars = 20 μm . Inset panels show mRFP - ATG9 and GFP - LC3 contact events at the indicated time points . Bar chart shows number of times a GFP - LC3 spot was within 1 μm of an mRFP - ATG9 spot ( a contact event ) , expressed as total mRFP - ATG9 contacts per GFP - LC3 spot , n = 18 cells per condition pooled from 4 independent experiments .

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Figure 8A) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed (F) or starved (S) HEK293A cells using a monoclonal hamster anti-ATG9 (ATG9) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody, TRAPPC8, TRAPPC4 and actin.B) HEK293A cells transfected with GFP or GFP-TBR (green) were stained for ATG9 using hamster anti-ATG9 (red) and analysed by confocal microscopy. Scale bar = 20 μmC) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B (green) and ATG9 using hamster anti-ATG9 (red) and imaged using confocal microscopy.D) Stills at t = 30 minutes from time lapse imaging of 9B9 cells (stably expressing GFP-LC3 and mRFP-ATG9) expressing either CFP or CFP-TBR (blue). Scale bars = 20 μm. Inset panels show mRFP-ATG9 and GFP-LC3 contact events at the indicated time points. Bar chart shows number of times a GFP-LC3 spot was within 1 μm of an mRFP-ATG9 spot (a contact event), expressed as total mRFP-ATG9 contacts per GFP-LC3 spot, n = 18 cells per condition pooled from 4 independent experiments.


Figure 9A ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with GFP or GFP - TBR ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) . Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm . B ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with myc - RAB1B or myc - RAB1B ( S22N ) ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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Figure 9A) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with GFP or GFP-TBR (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red). Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.B) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with myc-RAB1B or myc-RAB1B (S22N) (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red) Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


Figure 1C Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP . Conserved histidines in octarepeats 2 - 5 are coloured blue and missense mutations are shown in red . Overproduction of C2 fragment is indicated by + + signs to the right of the panel .

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Figure 1C Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP. Conserved histidines in octarepeats 2-5 are coloured blue and missense mutations are shown in red. Overproduction of C2 fragment is indicated by ++ signs to the right of the panel.


Figure 2A Lysates from RK13 cells transfected with WT , S1 , S3 and S3 . F88W PrP were probed with antibody Sha31 , 9A2 and 12B2 after digestion with PNGaseF . B , C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates . A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of ( C ) . B , C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates . A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of ( C ) . D Lysates of N2a , HEK , SMB - PS and SH - SY5Y cells not transfected ( − ) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31 . E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP , S1 PrP and S3 PrP . Lysates were PNGaseF - digested before Western blot using the Sha31 antibody . F1 : A114F , F2 : G118F , F3 : G122F , F4 :

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Figure 2A Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).D Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F


Figure 3A Brain homogenates of TgPrP ( WT ) , TgPrP ( S1 ) - 17 and TgPrP ( S3 . F88W ) - 35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31 , 12B2 and 9A2 . Prnp0 / 0 , and WT brain homogenates were used as controls . B The ratio of C2 to full - length PrP in TgPrP ( S3 . F88W ) - 35 and WT mice ( left panel , n = 7 , P = 1 . 46E - 06 ) and in RK13 cells expressing WT and S3 PrP ( right panel , n = 5 , P = 4 . 01E - 05 ) is shown . Values are presented as mean ± SEM . Unpaired , two - tailed t - test , * * * * P < 0 . 0001 . C Different tissue homogenates from a TgPrP ( S3 . F88W ) - 35 mouse were PNGaseF - digested and analysed for PrP using the antibody 1A6 . FL , full - length PrP ; ns , non - specific signal detected in the immunoblotting procedure .

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Figure 3A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.B The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001.C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.


Figure 4A Sections of sciatic nerve from TgPrP ( S3 . F88W ) - 14 female mice ( all 610 days old ) and TgPrP ( S3 . F88W ) - 35 female mice ( 614 , 570 and 614 days old , respectively ) were analysed using toluidine blue staining ( upper row ) , with the third panels for each line showing a hypermyelinated phenotype . Scale bar in left panel represents 50 μm . The lower rows show EM analyses of mice of the indicated genotypes . Scale bar is 10 μm for panels 1 - 4 , with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3 . F88W PrP ( scale bar , 5 μm ) . B Western blot analysis of sciatic nerve protein extracts ( 10 μg ) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain ( see Fig3 ) . The asterisk indicates a greater than full - length fragment present in TgPrP ( S3 . F88W ) mice , possibly corresponding to incomplete removal of the N - terminal signal peptide . C Quantification of fibre morphology in aged animals ( 492 - 608 days old ) , representing the percentage of the nerve occupied by fibres versus the total cross - sectional area of the nerve . The S1 and S3 . F88W alleles rescue Prnp0 / 0 nerves to a similar percentage as the WT allele , but the variance and P - value is greater for the two Tg lines expressing the TgPrP ( S3 . F88W ) allele . Unpaired , two - tailed t - test , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Sample sizes and exact P - values compared with Prnp0 / 0 ( n = 9 ) were as follows : TgPrP ( WT ) n = 6 , P = 3 . 63E - 05 ; Prnp0 / 0 , n = 9 ; TgPrP ( S1 ) - 17 n = 5 , P = 3 . 54E - 05 ; TgPrP ( S1 ) - 19 n = 3 , P = 6 . 35E - 06 ; TgPrP ( S3 . F88W ) - 14 n = 3 , P = 3 . 70E - 03 ; and TgPrP ( S3 . F88W ) - 35 n = 7 , P = 7 . 18E - 03 .

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Figure 4A Sections of sciatic nerve from TgPrP(S3.F88W)-14 female mice (all 610 days old) and TgPrP(S3.F88W)-35 female mice (614, 570 and 614 days old, respectively) were analysed using toluidine blue staining (upper row), with the third panels for each line showing a hypermyelinated phenotype. Scale bar in left panel represents 50 μm. The lower rows show EM analyses of mice of the indicated genotypes. Scale bar is 10 μm for panels 1-4, with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3.F88W PrP (scale bar, 5 μm).B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.C Quantification of fibre morphology in aged animals (492-608 days old), representing the percentage of the nerve occupied by fibres versus the total cross-sectional area of the nerve. The S1 and S3.F88W alleles rescue Prnp0/0 nerves to a similar percentage as the WT allele, but the variance and P-value is greater for the two Tg lines expressing the TgPrP(S3.F88W) allele. Unpaired, two-tailed t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sample sizes and exact P-values compared with Prnp0/0 (n = 9) were as follows: TgPrP(WT) n = 6, P = 3.63E-05; Prnp0/0, n = 9; TgPrP(S1)-17 n = 5, P = 3.54E-05; TgPrP(S1)-19 n = 3, P = 6.35E-06; TgPrP(S3.F88W)-14 n = 3, P = 3.70E-03; and TgPrP(S3.F88W)-35 n = 7, P = 7.18E-03.


Figure 5A , B Infected brain homogenates were PK - digested and analysed by Western blot using ( A ) Sha31 antibody and ( B ) after PK and PNGaseF digestion using Sha31 . C Conformational stability assay on RML - infected brain homogenates incubated with increasing concentrations of GdnHCl . The best - fit alignments of the data are presented . Genotypes are noted on the individual panels . Source data are available online for this figure .

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Figure 5A, B Infected brain homogenates were PK-digested and analysed by Western blot using (A) Sha31 antibody and (B) after PK and PNGaseF digestion using Sha31.C Conformational stability assay on RML-infected brain homogenates incubated with increasing concentrations of GdnHCl. The best-fit alignments of the data are presented. Genotypes are noted on the individual panels.Source data are available online for this figure.


Figure 6Pathology of RML - infected miceA - H The pattern of PrPSc deposition ( A - D ) and GFAP immunostaining ( E - H ) is presented . I - P A higher power view of H & amp ; amp ; E stained hippocampus ( I - L ) and thalamus ( M - P ) to reveal vacuolation is presented . Data information : Animals of the same genotype are shown within a column , and incubation times of the presented animals with the RML prion isolate are shown ( days ) . Mice expressing PrP S1 and S3 alleles ( central columns ) exhibit attenuated PrPSc deposition , gliosis and spongy change . Pathological features of the RML isolate are not altered upon sequential passage through TgPrP ( S3 . 88W ) - 35 mice ( see schematic at bottom of the diagram indicating sequential passages and finally , passage back into WT mice , compare the data in first and fourth columns ) .

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Figure 6Pathology of RML-infected miceA-H The pattern of PrPSc deposition (A-D) and GFAP immunostaining (E-H) is presented.I-P A higher power view of H&amp;E stained hippocampus (I-L) and thalamus (M-P) to reveal vacuolation is presented.Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).


Figure 7A Spot counts in the scrapie cell assay using WT , TgPrP ( S1 ) - 17 and TgPrP ( S3 . F88W ) - 14 brain homogenates ( 0 . 1 % or 0 . 01 % ) from mice infected with RML are shown in the upper panel ( WT vs S1 P = 2 . 09E - 05 and vs S3 P = 4 . 62E - 08 ) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel ( WT vs S1 P = 1 . 04E - 13 and vs S3 P = 3 . 39E - 20 ; two - tailed t - test , n = 3 ) . B A comparison of incubation time vs PrPC : PrPSc ratio for mice expressing different levels of WT PrP ( WT , TgPrP ( WT ) , tga20 ) , S1 PrP ( TgPrP ( S1 ) - 17 , TgPrP ( S1 ) - 19 , TgPrP ( S1 ) - 39 ) and S3 PrP ( TgPrP ( S3 . F88W ) - 14 , TgPrP ( S3 . F88W ) - 35 ) .

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Figure 7A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3).B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35).


Figure 1BATs were isolated from cold - exposed mice ( 4 ℃ , 72 h ) or control mice ( 30 ℃ , 72 h ) , then lysed and subjected to western blotting for analysis of the indicated proteins ( A ) . Relative protein expression levels , normalized to Actin , are shown in ( B ) . mRNA expression levels of the indicated genes were assayed by real - time PCR and normalized to Actb ( C ) . Mitochondrial DNA copy numbers were determined by real - time PCR and normalized to nuclear DNA standards ( D ) . Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted ( E ) . ( F - G ) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30 ℃ or 4 ℃ for 72 h . Scale bar , 50 μm . The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining .

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Figure 1BATs were isolated from cold-exposed mice (4℃, 72 h) or control mice (30℃, 72 h), then lysed and subjected to western blotting for analysis of the indicated proteins (A). Relative protein expression levels, normalized to Actin, are shown in (B).mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).Mitochondrial DNA copy numbers were determined by real-time PCR and normalized to nuclear DNA standards (D).Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted (E).(F-G) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30℃ or 4℃ for 72 h. Scale bar, 50 μm. The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining.


Figure 2Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30 ℃ ( Normal ) or following 72 h at 4 ℃ ( Cold ) ( A ) . Relative protein expression levels were normalized to Actin ( B ) . mRNA expression levels of the indicated genes were assayed by real - time PCR and normalized to Actb ( C ) . Flag - PGC - 1α was introduced into PGC - 1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins ( D ) . Expression levels of the indicated proteins were quantified ( E ) ( mean ± SEM ; n = 3 ) . Scramble , scramble shRNA . Samples were collected for real - time PCR analysis . The expression of Fundc1 was normalized ( F ) . ( G - H ) Scramble and PGC - 1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses ( G ) and quantification of the indicated proteins ( H )

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Figure 2Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B).mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).Flag-PGC-1α was introduced into PGC-1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins (D). Expression levels of the indicated proteins were quantified (E) (mean ± SEM; n=3). Scramble, scramble shRNA.Samples were collected for real-time PCR analysis. The expression of Fundc1 was normalized (F).(G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)


Figure 3 ( B ) Luciferase activity stimulated by the human Fundc1 promoter . The activity of Fundc1 promoter fragments , either wild type ( WT ) or with a mutation in the potential NRF1 binding site , was assayed using a dual luciferase reporter system . ( C , D ) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 ( F1 probe or mut F1 probe ) , the confirmed NRF1 binding site in the promoter of VSNL1 ( positive probe ) . The probes were incubated with HeLa nuclear extracts , then run on a gel ( C ) . A super - shifted band ( asterisk ) is detected in the presence of an anti - NRF1 antibody ( D ) . Labeled probe : biotin labeled wild type Fundc1 probe ; Cold probe : wild type Fundc1 probe ; Mut probe : NRF1 binding site mutant Fundc1 probe ; asterisk indicates the supershifted band generated with antibody to NRF1 . ( E , F ) ChIP assay to analyze the interaction of NRF1 ( E ) or PGC - 1α ( F ) proteins with the Fundc1 promoter . ( G ) NRF1 - myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies . ( H ) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC - 1α - Flag for 24 h and samples were collected for western blotting with the indicated antibodies . ( I ) ChIP analysis of the interaction of PGC - 1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells .

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

Figure 3(B) Luciferase activity stimulated by the human Fundc1 promoter. The activity of Fundc1 promoter fragments, either wild type (WT) or with a mutation in the potential NRF1 binding site, was assayed using a dual luciferase reporter system.(C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D). Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1.(E,F) ChIP assay to analyze the interaction of NRF1 (E) or PGC-1α (F) proteins with the Fundc1 promoter.(G) NRF1-myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies. (H) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC-1α-Flag for 24 h and samples were collected for western blotting with the indicated antibodies. (I) ChIP analysis of the interaction of PGC-1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells.


Figure 4 ( A , B ) Western blotting analyses of the levels of autophagy - related proteins in BAT from mice at the normal temperature ( 30 ℃ ) or at 4 ℃ for 72 h ( A ) . Expression of the indicated proteins was analyzed before and after 72 h cold exposure ( 4 ℃ ) ( B ) . ( C ) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h . ( D Representative transmission electron microscope ( TEM ) images of BAT cells isolated from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 1 μm ( D ) . The number of mitophagosomes per 100 mitochondria was counted and normalized ( E ) . ( F , G ) BATs from mice exposed to normal conditions ( 30 ℃ ) or cold ( 4 ℃ ) were lysed and immunoprecipitated with antibodies against FUNDC1 , LC3B or IgG , then immunoblotted with the indicated antibodies . The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted ( G ) .

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

Figure 4(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A). Expression of the indicated proteins was analyzed before and after 72 h cold exposure (4℃) (B).(C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h.(D Representative transmission electron microscope (TEM) images of BAT cells isolated from mice at the normal temperature (30℃) or 4℃ for 72 h. Scale bar, 1 μm(D).The number of mitophagosomes per 100 mitochondria was counted and normalized (E).(F,G) BATs from mice exposed to normal conditions (30℃) or cold (4℃) were lysed and immunoprecipitated with antibodies against FUNDC1, LC3B or IgG, then immunoblotted with the indicated antibodies. The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted (G).


Figure 5 ( A , B ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) mice at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . Representative images are shown , scale bar , 1μm ( A ) . The number of mitophagosomes per 100 mitochondria were counted and normalized ( B ) . ( C , D ) . Representative fluorescence images of BAT sections from Fundc1fl / fl / Adiponectincre - ( Cre - ) and Fundc1fl / fl / Adiponectincre + ( Cre + ) mito - Keima mice at normal conditions ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 50 μm ( C ) . Quantification of the ratio between red and green signals ( D ) . ( E ) Fundcfl / fl / Ucp1cre - ( Cre - ) and Fundcfl / fl / Ucpcre + ( Cre + ) mice were maintained at 30 ℃ ( Normal ) or exposed to 4 ℃ for 72 h ( Cold ) . Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards . ( F ) The relative total mito - keima signal ( green signal ) in Fundc1 deficiency and control mito - keima mice under normal condition or cold exposure were quantified and plotted . ( G ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . The percent of mitochondria without cristae is shown in the histogram . Oxygen consumption rate ( OCR ) ( H ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h . mitochondrial superoxide ( I ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h .

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

Figure 5(A,B) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) mice at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. Representative images are shown, scale bar, 1μm (A). The number of mitophagosomes per 100 mitochondria were counted and normalized (B).(C,D).Representative fluorescence images of BAT sections from Fundc1fl/fl/Adiponectincre- (Cre-) and Fundc1fl/fl/Adiponectincre+ (Cre+) mito-Keima mice at normal conditions (30℃) or 4℃ for 72 h. Scale bar, 50 μm (C). Quantification of the ratio between red and green signals (D).(E) Fundcfl/fl/Ucp1cre- (Cre-) and Fundcfl/fl/Ucpcre+ (Cre+) mice were maintained at 30℃ (Normal) or exposed to 4℃ for 72 h (Cold). Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards.(F) The relative total mito-keima signal (green signal ) in Fundc1 deficiency and control mito-keima mice under normal condition or cold exposure were quantified and plotted.(G) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. The percent of mitochondria without cristae is shown in the histogram.Oxygen consumption rate (OCR) (H) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.mitochondrial superoxide (I) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.


Figure 6 ( A ) Body temperature measurements in Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) fasting male mice before and during cold exposure ( 4 ° C ) for the indicated time . Body temperature ( B ) before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice . BAT tissue weight ( C ) measurements before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice . Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Western blotting analysis of BAT protein levels ( D ) . Protein expression levels were normalized to Actin ( E ) . Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real - time PCR and normalized to Actb ( F ) . ( G ) Representative images of UCP1 immunohistochemistry of BAT sections from Cre - or Cre + mice in normal conditions ( 30 ℃ ) or following 72 h at 4 ℃ . Scale bar , 50 μm .

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

Figure 6(A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time. Body temperature (B) before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.BAT tissue weight (C) measurements before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Western blotting analysis of BAT protein levels (D). Protein expression levels were normalized to Actin (E).Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real-time PCR and normalized to Actb (F).(G) Representative images of UCP1 immunohistochemistry of BAT sections from Cre- or Cre+ mice in normal conditions (30℃) or following 72 h at 4℃. Scale bar, 50 μm.


Figure 1 ( A ) Amylose pulldown from benzonase - treated HEK293T cell lysates expressing 2xMBP - BRCA2NT in untreated or irradiated cells ( 6Gy ; + IR ) . DDX5 and BRCA2NT ( MBP ) detected by immunoblot . StainFree images of the gels before transfer were used as loading control ( B ) Immunoprecipitation ( IP ) of endogenous BRCA2 from benzonase - treated HEK293T cell lysates left untreated or treated with IR ( 6Gy ) and harvested 4h post - IR , as indicated . Mouse IgG was used as negative control . Immunoblot of DDX5 and BRCA2 . StainFree image of the gels before transfer were used as loading control ( cropped image is shown ) . Asterisk ( * ) indicate a non - specific band detected by anti - DDX5 antibody . ( C ) Left : Representative images of in situ proximity ligation assay ( PLA ) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated ( - ) or irradiated ( 4h post - IR ; 6 Gy ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from untreated siC cells are shown . Scale bar indicates 10 µm . Right : Quantification of the number of PLA spots per nucleus . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot . ( D ) Diagram showing the BRCA2 N - terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N - terminal truncations ( BRCA2T1 , BRCA2LT2 , BRCA2LT3 ) or the 2xMBP tag . DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP , respectively . StainFree images of the gels before transfer were used as loading control ( E ) Left : GST pulldown assay using purified BRCA2T1 and DDX5 ; MBP antibody was used for the detection of both proteins . UB : unbound ; E : eluate . Right : SDS - PAGE showing 300 ng of purified MBP - DDX5 - GST and of 2xMBP - tagged BRCA2T1 used in the pulldown assay .

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

Figure 1(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control(B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody.(C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control(E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.